EFS Applications

Thermo Scientific LC-MS Applications for Food, Beverage, and Water Testing
Includes sample preparation, chromatography, and mass spectrometry conditions
Contaminants Drug Residues Natural Compounds Pesticides
Part of Thermo Fisher Scientic
Table of Contents
t t t t t
Drug Residues Antibiotics Antibiotics & Antimicrobials Pharmaceuticals Pharmaceuticals, Personal Care Products, and Pesticides
Click the Search button to search for any word or phrase.
t t t t
t t
t t
Click on a compound class at right to go to related notes.
Contaminants Acrylamide Melamine & Cyanuric Acid Mycotoxins Phenolic Pollutants Toxins
Natural Compounds Flavanoids Flavonoids Pesticides Pesticides Pesticides, Mycotoxins, Plant toxins Index
Contaminants Acrylamide
319: Quantitation of Acrylamide in Food Samples on the Thermo Scientific TSQ Quantum Discovery by LC/APCI-MS/MS
Application Note: 319
Quantitation of Acrylamide in Food Samples on the TSQ Quantum Discovery by LC/APCI-MS/MS

Kevin J. McHale, Witold Winnik and Gary Paul; Thermo Fisher Scientic, Somerset, NJ, USA
Introduction
Key Words TSQ Quantum Discovery Hypercarb Acrylamide Quantitation Triple Quadrupole MS Acrylamide has been identied as a potential human carcinogen. This is important not only because acrylamide is a common industrial chemical, but acrylamide has been shown to be present at signicant levels in food samples,1 particularly cooked foods high in carbohydrates. This has led many government health agencies around the world to assess the risk of short- and long-term exposure to acrylamide in humans. This has led to the development of LC-MS/MS methodology for the quantitative analysis of acrylamide in foodstuffs.1-5 While a GC/MS protocol for the analysis of acrylamide exists, this method requires extensive sample cleanup and chemical derivatization.6 The advantage of LC-MS/MS is that chemical derivatization is not necessary prior to acrylamide analysis. To date, most LC-MS/MS methods for the assay of acrylamide have utilized an electrospray ionization (ESI) source for the production of acrylamide ions.1-4 Yet it is well-known that ESI-MS is problematic when highly aqueous solutions, such as those required for the reversedphase LC separation of acrylamide, are used.7 On the other hand, water does not pose a problem for the formation of a stable corona discharge used in APCI. One published report has demonstrated that APCI is a viable ion source for the production of acrylamide ions for LC-MS/MS detection.5 Furthermore, a study comparing ESI and APCI ion sources for the LC-MS/MS analysis of acrylamide showed that under the same chromatographic conditions, APCI-MS/MS yielded an improved detection limit.8 This report presents data acquired on the Thermo Scientic TSQ Quantum Discovery for the analysis of acrylamide. A simple LC-MS/MS method using the APCI source is used to measure acrylamide, via selective reaction monitoring (SRM), over a wide concentration range. A small selection of food samples was analyzed for acrylamide content following extraction with water. To preclude the need for a time-consuming solid-phase extraction procedure, a column-switching method was employed to selectively fractionate acrylamide from polar matrix interferences prior to LC-MS/MS detection.
Goals
1. DevelopmentA sensitive and rugged LC/APCI-MS/MS assay for the analysis of acrylamide 2. ApplicationAn on-line column-switching technique to aqueous food extracts as an alternative to solid-phase extraction (SPE) cleanup 3. MeasurementAcrylamide content in selected food samples
Experimental
Chemicals and Reagents: Acrylamide (>99.0%) was purchased from Fluka (Buchs SG, Switzerland). 2,3,3-d3acrylamide (98%) was obtained from Cambridge Isotope Laboratories (Andover, MA, USA). HPLC grade water was acquired from Burdick and Jackson (Muskegon, MI, USA). All chemicals were used as received without further purication. Sample Preparation: Standards were prepared by dilution of a stock solution of 1.0 mg/mL acrylamide or 1.0 mg/mL d3-acrylamide in water. The stock solutions were stored at 4 C for a period of no longer than two weeks. Two brands of potato chips and two brands of breakfast cereals were purchased and stored at room temperature until processed. After homogenizing approximately 50 grams of a food sample, two grams were weighed into a 35 mL polypropylene centrifuge tube. Aqueous extraction of acrylamide was initiated by the addition of 20 mL water containing 1000 ng d3-acrylamide as the internal standard (nal concentration = 50 ng/mL). The sample was vortexed for 30 s then subsequently centrifuged at 18,000 g for 15 minutes. Ten milliliters of the supernatant was decanted into a clean 35 mL centrifuge tube and centrifuged at 18,000 g for 10 minutes. Prior to analysis, 0.49 mL of the aqueous extract was ltered through a 0.45 m centrifuge lter (Millipore Corp., Bedford, MA, USA) at 9,000 g for 5 minutes. Sample Analysis: LC experiments were conducted with the Thermo Scientic Surveyor HPLC system. A Thermo Scientic Hypercarb 2.1 50 mm column was utilized as the analytical LC column. Separations of acrylamide were achieved under isocratic conditions using 100% water as the mobile phase at a ow rate of 0.4 mL/min. The injection volume for all LC experiments was 10 L.
Search | Contents | Index
To eliminate the need for solid phase extraction (SPE) purication prior to the analysis of the food sample extracts, a column-switching LC method was employed. Briey, the sample extract was loaded onto a 2.1 50 mm Thermo Scientic Aquasil C18 column, which was positioned before a 6-port switching valve. The eluent from the C18 column was diverted to waste except for the period when acrylamide eluted from the C18 column, whereby the valve was switched to the Hypercarb column for MS/MS detection. This column-switching method required a second Thermo Scientic Surveyor MS pump, which also delivered 100% water at 0.4 mL/min. Both Surveyor MS pumps and the 6-port switching valve were controlled using Thermo Scientic Xcalibur version 1.3 software. The experimental conditions for the TSQ Quantum Discovery were as follows: Source: APCI Ion polarity: Positive Vaporizer Temperature: 375 C Discharge Current: 5 A Ion Transfer Capillary Temperature: 250 C Source CID Offset: 6 V Scan Mode: Selective Reaction Monitoring Q2 Pressure: 1.0 mTorr argon SRM Transitions: m/z 72 55 for acrylamide; m/z 75 58 for d3-acrylamide Collision Energy: 13 eV Scan Width: 1.0 u Scan Time: 0.3 s (each SRM transition) Q1, Q3 Resolution: Unit (0.7 u FWHM)
2100 2000
Acrylamide 72 55
Intentisy
1900 1800 1700 1600 1500
160000 140000
RT: 1.95
Intentisy
120000 100000 80000 60000 40000 20000 0.0 0.5 1.0 1.5 2.0 2.5
d3-Acrylamide 75 58 Area = 1507019
3.0
Time (min)
Figure 1: SRM chromatograms for 50 ng/mL d3-acrylamide
RT: 2.95
6500 6000
Intentisy
5500 5000 4500 4000 3500
Acrylamide 75 55 Area = 26998 S/N = 17
RT: 2.91
500000
Intentisy
400000
300000
Results and Discussion

Prior to the acquisition of acrylamide standards, it was important to determine if there was any detectable native acrylamide contribution originating from the deuterated internal standard. As shown in Figure 1, there is no acrylamide signal observed for the m/z 72 55 SRM transition at the same retention time as the 50 ng/mL d3-acrylamide standard. The limit of quantitation (LOQ) for acrylamide on the TSQ Quantum Discovery was 0.25 ng/mL acrylamide or 2.5 pg on column (Figure 2). This compares favorably to LOQs previously reported by other research groups, including an 8-fold improvement over the mass LOQ by LC/ESI-MS/MS (20 pg)1 and a 40-fold improvement over the concentration LOQ on the TSQ 7000 (10 ng/mL),5 which used an LC/APCI-MS/MS method. The calibration curve for acrylamide from 0.25 ng/mL to 2500 ng/mL is displayed in Figure 3. This calibration curve was generated using the column-switching LC method just prior to the acquisition of the food extracts data. A linear regression t to these data using 1/x weighting yielded the following equation: y = 5.5997 104 + 0.0206125x. The correlation coefcient for this curve was r2 = 0.9999, indicating excellent linearity across the four orders of magnitude dynamic range. Table 1 summarizes the statistical results for the acrylamide calibration curve. At the LOQ,
200000
100000 1.5 2.0 2.5 3.0 3.5 4.0
Time (min)
Figure 2: SRM chromatograms for 0.25 ng/mL acrylamide (LOQ) with 50 ng/mL d3-acrylamide
0.25 0.20
Area Ratio
50 40
0.15 0.10 0.05 0.00
Area Ratio
30 20 10 0 0
10
ng/mL
500
1000
1500
2000
2500
ng/mL
Figure 3: Calibration curve for acrylamide using column-switching LC method with APCI-MS/MS detection
Nominal (ng/mL) 0.250 0.500 1.00 5.00 10.0 100 500 1000 2500
Mean Conc. (ng/mL) 0.253 0.485 1.00(4) 4.86 10.2 101 512 1006 2481
% Rel. Error 1.1 -2.9 0.4 -2.7 2.1 0.7 2.4 0.6 -0.8
% CV 12.1 6.7 4.6 0.9 0.7 0.5 0.8 0.6 0.6
Number of Replicates 5 5 5 5 5 5 3 3 3
Table 1: Statistical data for the calibration curve of acrylamide
RT: 2.97 250000 200000
Intentisy
Acrylamide 72 55 Area = 3268462
150000 100000 50000
RT: 2.93 400000 350000 300000
Intentisy
250000 200000 150000 100000 50000 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Time (min)
Figure 4: SRM chromatograms of the Potato Chip 2 sample aqueous extract
the accuracy, as a percent relative error, is 1.1% and the precision, as a percent coefcient of variance (%CV), is 12.1% for ve replicate injections. Above the LOQ, the relative error varied from -2.9 to +2.4% and the %CV ranged from 0.5 to 6.7%. Results obtained from the aqueous extract of Potato Chip 2 are presented in Figure 4. By utilizing a C18 column positioned before a switching valve to selectively elute acrylamide onto the Hypercarb column, background interferences are reduced. Unlike most of the other acrylamide reports where SPE cleanup was used following extraction of the sample with water,1-4 the columnswitching LC method employed here provides an on-line means of acrylamide fractionation. This has the advantage of minimizing sample losses during SPE and greatly reduces sample preparation time. To monitor the consistency and reproducibility of the column-switching LC-MS/MS method, a 1 ng/mL acrylamide standard was analyzed immediately following each food sample. An example of this quality control standard analyzed after the Potato Chip 2 sample is shown in Figure 5. Although the baseline for the m/z 72 55 SRM transition is somewhat elevated near the retention time for acrylamide, the calculated concentration for this standard is 0.99 ng/mL, equating to a relative error of -1.0%. Table 2 reports the results for four different food samples that were assayed for acrylamide using the column-switching LC method and MS/MS detection. The acrylamide concentrations in each food sample were calculated by multiplying the measured solution concentration from duplicate injections by the extraction volume and dividing by the food sample mass that was extracted. The determined acrylamide concentrations correlated well to those reported elsewhere for these classes of food.1-5
Cereal 1 Injection 1 Injection 2 Mean Extraction Vol. Mass Sample Acrylamide Conc. 17.17 ng/mL 17.00 ng/mL 17.09 ng/mL 20.0 mL 2.003 g 171 ng/g Cereal 2 55.93 ng/mL 56.18 ng/mL 56.06 ng/mL 20.0 mL 2.007 g 559 ng/g Potato Chip 1 57.11 ng/mL 56.52 ng/mL 56.82 ng/mL 20.0 mL 2.021 562 ng/g Potato Chip 2 29.18 ng/mL 29.14 ng/mL 29.16 ng/mL 20.0 mL 1.995 292 ng/g
20000 18000 16000
Intentisy
Acrylamide 75 58 Area = 115877
RT: 2.94
14000 12000 10000 8000 6000 450000 400000 350000
RT: 2.91
Intentisy
300000 250000 200000 150000 100000 50000 0.0
Table 2: Results of acrylamide assay from food samples
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Time (min)
Figure 5: 1 ng/mL acrylamide standard analyzed directly after duplicate injections of the aqueous extract of the Potato Chip 2 sample
Conclusions
An LC-MS/MS method has been developed for the measurement of acrylamide on the TSQ Quantum Discovery. Using APCI for the analysis of acrylamide from 100% water, an LOQ of 0.25 ng/mL acrylamide or 2.5 pg on column was achieved. Incorporation of a columnswitching LC method prior to MS/MS detection of acrylamide eliminated the need to purify food sample extracts by SPE. The method was successfully demonstrated for the analysis of four brands of food samples using TSQ Quantum Discovery in conjunction with a columnswitching LC method.
References (1) Tareke, E.; Rydberg, P.; Karlsson, P.; Eriksson, S.; Tornqvist, M. J. Agric. Food Chem., 2002, 50, 4998-5006. (2) Rosen, J.; Hellenas, K. Analyst, 2002, 127, 880-882. (3) Musser, S. M. http://www.cfsan.fda.gov/~dms/acrylami.html (4) Becalski, A.; Lau, B.P.; Lewis, D., Seaman, S.W. J. Agric. Food Chem., 2003, 51, 802-808. (5) Brandl, F.; Demiani, S.; Ewender, J.; Franz, R.; Gmeiner, M.; Gruber, L.; Gruner, A.; Schlummer, M.; Smolic, S.; Stormer, A.; Wolz, G. Electron. J. Environ. Agric. Food Chem., 2002, 1(3), 1-8. (6) Castle, L.; Campos, M.J.; Gilbert, J. J. Sci. Food Agric. 1993, 54, 549-555. (7) Ikonomou, M.G.; Blades, A.T.; Kebarle, P. J. Am. Soc. Mass Spectrom., 1991, 2, 497-505. (8) McHale, K.J.; Winnik, W.; Paul, G. Proceedings of the 51st ASMS Conference on Mass Spectrometry and Allied Topics, Montreal, 2003.
In addition to these offices, Thermo Fisher Scientific maintains a network of representative organizations throughout the world.
Africa +43 1 333 5034 127 Australia +61 2 8844 9500 Austria +43 1 333 50340 Belgium +32 2 482 30 30 Canada +1 800 530 8447 China +86 10 5850 3588 Denmark +45 70 23 62 60 Europe-Other +43 1 333 5034 127 France +33 1 60 92 48 00 Germany +49 6103 408 1014 India +91 22 6742 9434 Italy +39 02 950 591 Japan +81 45 453 9100 Latin America +1 608 276 5659 Middle East +43 1 333 5034 127 Netherlands +31 76 587 98 88 South Africa +27 11 570 1840 Spain +34 914 845 965 Sweden / Norway / Finland +46 8 556 468 00 Switzerland +41 61 48784 00 UK +44 1442 233555 USA +1 800 532 4752
www.thermo.com
Legal Notices 2008 Thermo Fisher Scientic Inc. All rights reserved. All other trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientic Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
Thermo Fisher Scientic, San Jose, CA USA is ISO Certied.
AN62606_E 01/08S
View additional Thermo Scientic LC/MS application notes at: www.thermo.com/appnotes
Contaminants Melamine & Cyanuric Acid

t
424: Analysis of Melamine and Cyanuric Acid in Food Matrices by LC-MS/MS
Analysis of Melamine and Cyanuric Acid in Food Matrices by LC-MS/MS

PeterVarelis, National Center for Food Safety and Technology, Illinois Institute of Technology. Jonathan Beck, Kefei Wang, and Dipankar Ghosh; Thermo Fisher Scientic, San Jose, CA
Introduction
Key Words TSQ Quantum Ultra Cyanuric Acid Food Safety LC-MS/MS In March 2007, several North American manufacturers of pet food voluntarily issued nationwide recall notices for some of their products that were reportedly associated with renal failure in pets1. The raw material wheat gluten, used to manufacture the pet food, was imported from China and was identied as the source of contamination2. Although initial reports suggested that contamination was conned to pet food, further investigations revealed that melamine-tainted fodder may have been used to feed 3,4,5 animals intended for human consumption. In particular, it was discovered that melamine-contaminated ingredients had been used to prepare feed for chickens, swine, and 3,4 catsh. Consequently, the U.S. Food and Drug Administration (FDA)3 and the U.S. Department of Agriculture (USDA)4 have developed methods for the analysis of melamine residues in animal tissue. Both methods use tandem mass spectrometric detection and employ disposable strong cation exchange solid phase extraction (SPE) cartridges to prepare samples for liquid chromatographic analysis.
Sample Preparation Solid samples were homogenized using an Ultra-Turrax (IKA-Werke GmbH & Co. KG, Staufen, Germany) homogenizer. After extraction into aqueous 1:1 Water:MeOH, and addition of the internal standards, the samples were prepared by ofine ion exchange chromatography using SPE cartridges. Liquid Chromatography Aliquots (10-25 L) of the above extracts were chromatographed on a Thermo Scientic BioBasic AX analytical column (2.1 150 mm, 5 m), which was kept at 30C in an oven. The initial mobile phase was composed of acetonitrile-isopropanol-50 mM aqueous ammonium acetate in the ratio of 85:10:5, respectively, and was pumped through the column at a ow of 400 L per minute. After 5 min, the mobile phase composition and ow were immediately changed to 9:1 water-acetonitrile, and 500 L per minute, respectively. These conditions were maintained for 5 min before returning the mobile phase to the initial composition. After 5 min of equilibration, the ow through the column was returned to 400 L per minute. The column efuent was diverted to waste for the rst 1.5 minutes and then switched to the detector for the remaining run time.
MS Conditions Melamine
Experimental
Chemicals and reagents Unless stated otherwise, all organic solvents used in this work were HPLC grade quality and were purchased from Thermo Fisher Scientic (Fair Lawn, NJ, USA). Melamine, cyanuric acid, and 30% (w/w) aqueous ammonia were purchased from Sigma (St. Louis, MO, USA). The internal standards 13C3-melamine and -cyanuric acid were prepared using 13C3-cyanuric chloride, which was also obtained from Sigma. 18 M water was obtained from a Milli-Q (Millipore Corporation, Billerica, MA, US) purication system. Calibration Standards Individual solutions (1000 g/mL) of cyanuric acid and melamine were prepared by dissolving the crystalline compounds in 50% (v/v) aqueous methanol. Aliquots (1 mL) of these solutions were combined and then diluted with 1:3 water-acetonitrile, respectively, to obtain a 10 g/mL stock solution, from which eight working standards in the range of 1-1000 ng/mL were prepared by serial dilutions with acetonitrile. Calibration standards were prepared by adding 50 L of the stock solution of the internal standards to 1 mL of each of the eight working standards.
MS: Thermo Scientic TSQ Quantum Ultra Source: Heated Electrospray (H-ESI) Ionization: Positive ESI Sheath Gas: 65 units Auxiliary Gas: 35 units at 250C Ion Transfer Tube Temp: 350C Scan Time: 200 ms/transition Q1/Q3 Resolution: 0.7 FWHM SRM Transitions: Melamine: m/z 12768 @ 32 eV m/z 12785 @ 18 eV Melamine 13C3 (Internal Standard): m/z 13070 @ 32 eV m/z 13087 @ 18 eV
QED-MS/MS Conditions: Collision Energy: 30 eV Reverse Energy Ramp (RER): 50 eV
MS Conditions Cyanuric Acid
Results
A chromatogram showing a standard mixture of both melamine and cyanuric acid, along with their associated internal standards, is shown in Figure 1. Calibration curves ranging from 1-1000 ppb are shown in Figure 2 and Figure 3 for melamine and cyanuric acid, respectively. The calibrations are linear over the entire range, and a close-up of the lower portion of the calibration curve (1-100 ppb) is shown in the same gure. Melamine and cyanuric acid were spiked into a matrix of catsh and processed as described in the method section above. A chromatogram of this sample, spiked at 10 ppb for melamine and 50 ppb for cyanuric acid, is shown in Figure 4. Very low noise is observed, emphasizing the effectiveness of the cleanup procedure for a complicated matrix.
MS: TSQ Quantum Ultra Source: Heated Electrospray (H-ESI) Ionization: Negative ESI Sheath Gas: 75 units Auxiliary Gas: 10 units at 250C Ion Transfer Tube Temp: 350C Scan Time: 200 ms/transition Q1/Q3 Resolution: 0.7 FWHM SRM Transitions: Cyanuric Acid: m/z 12842 @ 17 eV m/z 12885 @ 11 eV Cyanuric Acid 13C3 (Internal Standard): m/z 13143 @ 17 eV m/z 13187 @ 11 eV
100 50
4.30
Cyanuric Acid
3.34 3.54 3.74 4.29 4.75
0 100 50
Cyanuric Acid 13C3

3.35 2.22 4.08 4.55
Relative Abundance
0 100 50
Melamine
0.30 0.83 1.20 1.57 1.69 2.22 2.37 2.57
0 100 50
Melamine 13C3
0.22 0.55 1.09 1 1.61 1.84 2
2.86 3 Time (min)
2.92 4
0 0
Figure 1. Melamine, cyanuric acid, and their internal standards at a concentration of 1 ppb. From top to bottom, cyanuric acid, cyanuric acid 13C3, melamine, and melamine 13C3.
Melamine Y = 0.0118128*X R^2 = 0.9998 W: Equal 12 10 8 6 4 2 0 0 200 400 600 ng/mL 800 1000
1.6 1.4 1.2
Melamine Y = 0.0118128*X R^2 = 0.9998 W: Equal
Height Ratio
Height Ratio
1.0 0.8 0.6 0.4 0.2 0.0 20 40 60 ng/mL 80 100
Figure 2. Calibration curve for melamine from 1-1000 ng/mL. The left gure shows the entire calibration range, while the right gure shows the expanded range from 10-100 ng/mL. Page 2 of 6
Additionally, full spectra data was collected using the standard Quantitation-Enhanced Data-Dependent MS/MS (QED-MS/MS) scan function. QED-MS/MS works by monitoring SRM data, and when the response of a particular SRM reaches a threshold level, the full scan MS/MS is activated. The resulting full scan spectra for melamine at 100 ppb and its internal standard are shown in Figure 5. The full scan data allows for further conrmation of results by eliminating false positives and also provides the opportunity to perform a library search. When a full scan QED-MS/MS spectra collected from a catsh sample
spiked at 10 ppb was searched against the library, the library search returned melamine as the most likely hit. The results of the library search are shown in Figure 6. The spectrum of the sample and the spectrum that is stored in the library are visible in the lower left quadrant of the gure. The top spectrum is the catsh sample, while the lower spectrum is the reference spectrum. There is good agreement between the two spectra, even though the reference spectrum was generated using standards without matrix.
Cyanuric_acid Y = 0.00790388*X R^2 = 1.0000 W: Equal 8 7 6

Area Ratio
Area Ratio
Cyanuric_acid Y = 0.00790388*X R^2 = 1.0000 W: Equal 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
5 4 3 2 1 0 0 200 400 600 ng/mL 800 1000
20
40 ng/mL
60
80
100
Figure 3: Calibration curve for cyanuric acid from 1-1000 ng/mL. The left gure shows the entire calibration range, while the right gure shows the expanded range from 1-100 ng/mL.
100 50
4.32
Cyanuric Acid
50 ppb spiked in Catfish
3.15 3.47 4.31 4.77
Relative Abundance
0 100 50 0 100 50 0 100 50 0 0
Cyanuric Acid Standard
3.42 2.30
3.73
4.13
4.57
Melamine
10 ppb spiked in Catfish
1.73 2.31
0.18
0.58
1.16
2.67
2.93
Melamine Standard
0.14
1.01 1
1.49
1.66
2.03 2
2.69 3 Time (min) 4
Figure 4: Chromatogram of cyanuric acid and melamine spiked into catsh matrix, at a level of 50 ppb for cyanuric acid, and 10 ppb for melamine
Page 3 of 6
100 95 90 85 80 75 70 65 60
44.17
Melamine 13C3
70.12
Relative Abundance
100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5
43.16
Melamine
68.08 127.05
Relative Abundance
55 50 45 40 35 30 25 20 15 10 5 0 30 40 50 60 70 80 m/z 90 100 110 120 130
130.06
87.06
85.07
30
40
50
60
70
80 m/z
90
100
110
120
130
Figure 5: QED-MS/MS spectra for melamine 13C3 (left) and melamine (right). Unique, rich, library-searchable spectra are collected in the same chromatographic run, allowing both quantitative and conrmatory full scan data in the same le.
Catfish Sample
Library Spectrum
Figure 6: Library search results for melamine spiked at 10 ppb into a catsh matrix. Melamine is the top hit in the search list.
Page 4 of 6
Conclusion
A simple, sensitive, and specic method for the detection and quantitation of melamine and cyanuric acid in food matrices has been demonstrated. The method is robust and allows for the analysis of a large number of samples, without degradation in column performance. Additionally, full scan spectra for Q3 are collected in the same chromatographic run using the QED-MS/MS scan function, permitting a library search of the results to eliminate any false positives.
References:
1
Weis, E. Nestl Purina, Hills join pet food recall. USA Today. Available at http://www.usatoday.com/news/nation/2007-03-31-pet-food-recall_N.htm. Accessed 10 December 2007. Aarthi Sivaraman. Melamine in pet food, wheat gluten from China: FDA. Reuters. Available at http://www.reuters.com/article/domesticNews/idUSWEN594320070330. Accessed 10 December 2007. Weise, E. and Schmit, J. Melamine in pet food may not be accidental. USA Today. Available at www.usatoday.com/money/industries/2007-04-24-fda-pet-food-probe_N.htm. Fish on U.S. sh farms fed melamine-contaminated feed; FDA discovers contaminated food products from China mislabeled. American Veterinary Medical Association. Available at www.avma.org/press/releases/070508_petfoodrecall.asp. Accessed 10 December 2007. Melamine contaminant found in chicken feed. Available at www.sciencedaily.com/releases/2007/05/070502072434.htm. Accessed 10 December 2007.
Page 5 of 6
Laboratory Solutions Backed by Worldwide Service and Support

Tap our expertise throughout the life of your instrument. Thermo Scientic Services extends its support throughout our worldwide network of highly trained and certied engineers who are experts in laboratory technologies and applications. Put our team of experts to work for you in a range of disciplines from system installation, training and technical support, to complete asset management and regulatory compliance consulting. Improve your productivity and lower the cost of instrument ownership through our product support services. Maximize uptime while eliminating the uncontrollable cost of unplanned maintenance and repairs. When its time to enhance your system, we also offer certied parts and a range of accessories and consumables suited to your application. To learn more about our products and comprehensive service offerings, visit us at www.thermo.com.
Legal Notices 2008 Thermo Fisher Scientic Inc. All rights reserved. Milli-Q is a registered trademark of Millipore Corporation. Ultra-Turrax is a registered trademark of IKA-Werke GmbH & Co. KG. All other trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientic Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
www.thermo.com

AN62732_E 05/08S
Contaminants Mycotoxins
377: Analysis of Mycotoxins in Various Cattle Forages and Food Matrices with the Thermo ScientificTSQ Quantum Discovery MAX
Analysis of Mycotoxins in Various Cattle Forages and Food Matrices with the TSQ Quantum Discovery MAX
Robert Huls1, Richard Zuiderent1, and Dipankar Ghosh2
1
Thermo Fisher Scientic BV, Breda, The Netherlands; 2Thermo Fisher Scientic, San Jose, CA, USA
Introduction
Key Words TSQ Quantum Discovery MAX Food and Environmental H-SRM Quantitation Mycotoxins are toxic metabolites produced by certain species of fungi that can infect and colonize on various agricultural crops in the eld and during storage. Environmental factors such as temperature and humidity inuence the occurrence of these toxins on grains, nuts and other commodities susceptible to mold infestation. In addition, any crop that is stored for more than a few days is a target for mold growth and mycotoxin formation. Most mycotoxins are relatively stable compounds that are not destroyed by food processing or cooking. Although the generating organisms might not survive processing, the toxin can still be present. Mycotoxins pose a potential threat to human and animal health through the ingestion of contaminated food products. Mycotoxins can have both chronic and acute effects on human and animal health. They can be teratogenic, mutagenic, or carcinogenic in susceptible animal species. They are linked to various diseases in domestic animals, livestock, and humans in many parts of the world. Most mycotoxins are toxic in very low concentrations and therefore require sensitive and reliable methods for their detection. This application note describes an LC-MS/MS method for the determination of mycotoxins in various cattle forages. Using this method it is possible to simultaneously measure the following 12 mycotoxins within 12 minutes: Nivalenol (NIV), Deoxynivalenol (DON), Aatoxin G1, Aatoxin G2, Aatoxin B1, Aatoxin B2, Fumonisin B1, Fumonisin B2, Diacetoxyscripenol (DAS), T2-Toxine, Ochratoxin A, and Zearalenon (ZEN). See Figure 1. The Thermo Scientic TSQ Quantum Discovery MAX triple quadrupole system has been evaluated for roundthe-clock analysis of different mycotoxins. Multiple samples with different matrices (cattle forages, food matrices) have been analyzed.
Goal
To demonstrate that the TSQ Quantum Discovery MAX, with its H-SRM capabilities and H-ESI source, is ideally suited for the rigorous demands of high-throughput analyses of mycotoxins in various matrices.
Experimental Conditions
Sample Preparation The samples analyzed were various extracts of cattle forages and food products. The following sample extraction procedure, adapted from TLR International Laboratories, was used. To begin, 25 g of grounded sample was dissolved in 100 mL of acetonitrile:water (80:20 v/v). The extract was then mixed for two hours. Afterwards, the extracts were ltered and diluted four times with water. The resulting solution was acetonitrile:water 20:80 v/v. HPLC HPLC analysis was performed using the Thermo Scientic Surveyor HPLC System. Each 20 L sample was injected directly onto a Thermo Scientic Hypersil GOLD 100 2.1 mm, 5 m analytical column. A gradient LC method used mobile phases A (0.1% formic acid in acetonitrile) and B (0.1% formic acid in water) at a ow rate of 0.3 mL/min. The gradient is described in Figure 2. Mass Spectrometry MS analysis was carried out on a TSQ Quantum Discovery MAX triple stage quadrupole mass spectrometer with a heated electrospray ionization (H-ESI) probe. The MS conditions were as follows: Ion source polarity: Positive ion mode Spray voltage: 4000 V Vaporizer temperature: 300C Sheath gas pressure (N2): 30 units Auxiliary gas pressure (N2): 30 units Ion transfer tube temperature: 350C Scan Type: SRM
The MS conditions and the H-SRM transitions were obtained by automatic optimization with the auto-tune software. Figures 3 and 4 show two examples of the collision energy optimization. Figure 5 summarizes all of the H-SRM transitions that were used.
Two product ions were measured for all compounds; one was used as the quantier ion and the other was used as the qualier ion. In this way, the ion ratio conrmation was done as an identity conrmation. See Figure 5 for further details.
Deoxynivalenol
Nivalenol
Diacetoxyscripenol
T2-Toxine
Zearalenon
Ochratoxin A
Fumonisin B1
Fumonisin B2
Aflatoxin B2
Aflatoxin B1
Aflatoxin G1
Aflatoxin G2
Figure 1: Structures of 12 mycotoxins
Column: Hypersil GOLD 100 x 2.1 mm, 5 m particles Flow: 0.3 mL/min Injection volume: 20 L Column temperature: 30 C Total time: 12 min
Figure 2: LC/MS conditions
Page 2 of 6
Figure 3: Optimization of collision energies of Aatoxin A1/B1/G2 Figure 4: Optimization of collision energies of Fumonsin B1/B2
Compound
Precursor
Product Ion (Quan)
Product Ion (Qual)
RT
Conc Range (ppb)
Aatoxin B1 Aatoxin B2 Aatoxin G1 Aatoxin G2 Fumonisin B1 Fumonisin B2 Ochratoxin Zearalenon Deoxynivanlenol Diacetoxyscripenol
Figure 5: H-SRM transitions
313 315 329 331 722 706 404 319 297 367
241 259 243 245 334 336 239 187 249 307
285 287 283 275 352 318 221 185 231 289
4.8 4.5 5.0 4.8 4.4 4.8 5.6 5.7 1.2 4.4
0.1100 0.1100 0.1100 0.1100 0.11000 0.11000 0.11000 0.1100 0.1100 0.1100
Nivalenol in corn, 0.5 ppm

100 95 90 85 80 75 70 65 60 Relative Abundance Relative Abundance 55 50 45 40 35 30 25 20 15 10 5 0 0 1 2 3 4 5 Time (min) 1.58 4.45 3.81 4.84 RT: 1.16 MA: 107932 100 95 90 85 80 75 RT: 1.19 MA: 101932
Diacetoxyscripenol in flour, 10 ppb

100 95 90 85 80 75 4.94 100 95 90 85
SRM
70 65 60
H-SRM
Relative Abundance
70 65 60 55 50
?
SRM
5.48 5.88 Relative Abundance RT: 4.40 MA: 6233 4.36
80 75 70 65 60 55 50 45 40 35 RT: 4.42 4.92 MA: 4475
H-SRM
55 50 45 40 35 30 25 20 15 10 5 0 0 1 2 3 4 5 Time (min)
45 4.72 40 35 30 25 20 15 10 5 0 3 4 Time (min) 5 6 4.01 3.80
30 25 20 15 10 5 0 3 4 Time (min) 5 6
Figure 6: Comparison of SRM and H-SRM data for two samples
Page 3 of 6

The TSQ Quantum Discovery MAX offers the unique capability of highly selective reaction monitoring (H-SRM). Setting the resolution of Q1 at 0.1 FWHM helps to decrease the background noise and eliminate isobaric interferences. This improves the signal-to-noise ratio and results into a lower limit of quantication. Figure 6 compares SRM and H-SRM data for two samples. The calibration curves were generated by dilutions in acetonitrile:water 20:80 v/v. Figure 7 presents the linear t calibration curves for ve mycotoxins using H-SRM.
The calibration curves have R2 values that are greater than 0.998, which indicate excellent linear ts over the dynamic range. The mycotoxin levels found in the various matrices were in the expected range. For example, in a QC-sample used as an internal control, Aatoxin B1 was expected on a level of 5 ppb (in extract). The detected amounts (ppb in solution) are presented in Table 1. This level for Aatoxin B1, is subjected to EU legislation as the low limit of quantication.
Fuminosine_B1 Y = -6629.54+96887.2*X-3.66559*X^2 R^2 = 0.9998 W: 1/X 100000000 95000000 90000000 85000000 80000000 75000000 70000000 65000000 60000000
Area
Area
Fuminosine_B2 Y = 565.239+135163*X-0.586979*X^2 R^2 = 0.9998 W: 1/X 140000000 130000000 120000000 110000000 100000000 90000000 80000000 70000000 60000000 50000000 40000000 30000000 20000000 10000000 0
55000000 50000000 45000000 40000000 35000000 30000000 25000000 20000000 15000000 10000000 5000000 0 0 100 200 300 400 500 ng/ml 600 700 800 900 1000 1100
Fumonisin B1 Range 0.11000 ppb R2=0.999 Weighting=1/x
Fumonisin B2 Range 0.11000 ppb R2=0.999 Weighting=1/x

0 100 200 300 400 500 ng/ml 600 700 800 900 1000 1100
Deoxynivanlenol_(DON) Y = -12942.9+4471.77*X R^2 = 0.9991 W: 1/X 4400000 4200000 4000000 3800000 3600000 3400000 3200000 3000000 2800000 2600000
Area
2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 0 100 200 300 400 500 ng/ml 600 700 800 900 1000 Ochratoxine_A Y = -11201.9+225947*X-28.6401*X^2 R^2 = 0.9996 W: 1/X 230000000 220000000 210000000 200000000 190000000 180000000 170000000 160000000 150000000 140000000 130000000
Area
Deoxynivalenol Range 0.11000 ppb R2=0.999 Weighting=1/x
Diacetoxyscripenol_(DAS) Y = -3583.47+11136.8*X R^2 = 0.9977 W: 1/X 48000000 46000000 44000000 42000000 40000000 38000000 36000000 34000000 32000000 30000000 28000000
Area
1 000000 120000000 110000000 100000000
26000000 24000000 22000000 20000000 18000000 16000000 14000000 12000000 10000000 8000000 6000000 4000000 2000000 0 0 500 1000 1500 2000 ng/ml 2500 3000 3500 4000
Diacetoxyscripenol Range 0.11000 ppb R2=0.999 Weighting=1/x
90000000 80000000 70000000 60000000 50000000 40000000 30000000 20000000 10000000 0 0 200 400
Ochratoxin A Range 0.11000 ppb R2=0.999 Weighting=1/x

600 ng/ml 800 1000 1200
Figure 7: Calibration curves for ve mycotoxins
Sample
Detected Amount (ppb)
Sample-01 Sample-02 Sample-03 Sample-04 Sample-05 Sample-06 Average RSD RSD% Average in Extract
1.19 1.28 1.43 1.25 1.15 1.37 1.28 0.1 8.3% 5.11
Table 1. Detected Amounts of Aatoxin B1 in Solution
Page 4 of 6
For the analysis of mycotoxins in various matrices, the heated electrospray ionization (H-ESI) probe provides signicant advantages. The dual desolvation zone design increases the ionization efciency and helps to get rid of the clustering solvents. (See Figure 8.) This leads to higher signals with better %RSDs. The H-ESI probe also handles
higher LC ows (up to 1 mL/min) without losing ionization efciency. This helps to speed up the method without the need to split the LC ow. Figures 9 and 10 describe the increased sensitivity of the H-ESI probe with two samples of mycotoxins.
Figure 8: H-ESI Heated Electrospray Ionization probe
100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 3.0 3.5 4.0 4.5 Time (min) 5.0 5.5 6.0 RT: 4.47 MA: 125169 RT: 4.78 MA: 159292
100
RT: 4.85 MA: 521821
ESI
90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 3.0
H-ESI
Aflatoxin B1 0.5 ppb
RT: 4.55 MA: 533364
Aflatoxin B2 0.5 ppb
3.5
4.0
4.5 Time (min)
5.0
5.5
6.0
H-ESI is 3 times more sensitive than ESI
Figure 9: Increased sensitivity with the H-ESI probe in Aatoxin data
Page 5 of 6
In addition to these offices, Thermo Fisher

100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 3.0 3.5 4.0 4.5 Time (min) 5.0 5.5 6.0 RT: 5.01 MA: 23282 RT: 4.64 MA: 14939 100 90 80 RT: 4.67 MA: 53001
Scientific maintains
ESI
70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 3.0
H-ESI
Fumonisin B1 0.5 ppb
a network of representative organizations throughout the world.
RT: 5.04 MA: 71964
Fumonisin B2 0.5 ppb
3.5
4.0
4.5 5.0 Time (min)
5.5
6.0
H-ESI is 3 times more sensitive than ESI
Figure 10: Increased sensitivity with the H-ESI probe in Fumonisin data
Conclusion
LC-MS/MS is a major technique for all kinds of environmental safety and food control laboratories. The TSQ Quantum Discovery MAX is the workhorse of the TSQ Quantum series for round the clock productivity. Matrix effects are always an issue with LC-MS/MS methods. However, this application note shows that the TSQ Quantum Discovery MAX can help overcome these effects with its unique features of H-SRM and H-ESI. The results presented here were obtained without extensive preparation. A wide range of matrices were analyzed and excellent results were obtained.
Acknowledgements
Drs. Ing. Harm Janssens and BSc Gerard Franken of TLR International Laboratories, www.tlr.nl, are acknowledged for supplying the basis for this method, which they developed for feed.
Australia +61 2 8844 9500 Austria +43 1 333 50340 Belgium +32 2 482 30 30 Canada +1 800 532 4752 China +86 10 5850 3588 Denmark +45 70 23 62 60 France +33 1 60 92 48 00 Germany +49 6103 408 1014 India +91 22 6742 9434 Italy +39 02 950 591 Japan +81 45 453 9100 Latin America +1 608 276 5659 Netherlands +31 76 587 98 88 South Africa +27 11 570 1840 Spain +34 91 657 4930 Sweden / Norway / Finland +46 8 556 468 00 Switzerland +41 61 48784 00 UK +44 1442 233555 USA +1 800 532 4752
www.thermo.com
Thermo Finnigan LLC, San Jose, CA USA is ISO Certied.
2007 Thermo Fisher Scientic Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
AN62247_E 04/09S
Contaminants Phenolic Pollutants

411: Analyzing Phenolic Pollutants in Water Using U-HPLC
Analyzing Phenolic Pollutants in Water Using U-HPLC

N. Jones, L. Pereira, D. Milton, Thermo Fisher Scientic, Runcorn, UK
Overview
Key Words Hypersil GOLD Columns Accela High Speed U-HPLC Surveyor Plus HPLC Phenols US EPA and EU Standards Water Pollutants This study demonstrates analysis optimization by variation of column chemistry, and the viability of reducing stationary phase particle size to signicantly increase analysis speed, while maintaining separation parameters and increasing sensitivity.
Materials and Methods

HPLC Columns The effect of particle/column size variation on analysis speed and separation efciency was studied using the following Thermo Scientic Hypersil GOLD columns and experimental conditions: 150 2.1 mm (5 m particle size) 100 2.1 mm (3 m particle size) 100 2.1 mm (1.9 m particle size). Mobile Phase: A) 0.1% Acetic Acid in Water B) 0.1% Acetic Acid in Methanol. Temperature: 60C Detection: UV Diode array (270-320 nm), Gradients, ow rates and injection volumes are listed in Table 1. Phenols were prepared at a concentration of 5 ppm in Water:Methanol (95:5). Stationary phase chemistry The effect of stationary phase chemistry on the separation of ve phenols (2-Chlorophenol, 4-Chlorophenol, 2Nitrophenol, 4-Nitrophenol and 2,4-Dinitrophenol), using 1.9 m particles, was studied using three column types (all 100 2.1 mm): Hypersil GOLD Thermo Scientic Hypersil GOLD aQ (polar endcapped C18) Thermo Scientic Hypersil GOLD PFP (peruorinated phenyl). Analysis conditions were equivalent to those described within U-HPLC Method 1 (Table 1). Instrumentation A Thermo Scientc Surveyor Plus HPLC system was used for 5 and 3 m particle analyses, and a Thermo Scientic Accela U-HPLC system was used for 1.9 m analyses.
Introduction
Phenolic compounds are of particular environmental importance due to their relatively high toxicity at low levels and their presence in environmental waters and organic matter, following degradation of a range of industrial products such as pesticides and herbicides, as well as naturally occurring humic substances and tannins. Previous studies(1,2) have shown that reversed-phase liquid chromatography (RP-LC) coupled to atmospheric pressure chemical ionization mass spectrometry (APCI-MS) can effectively separate and detect a range of phenolic compounds at low ppb levels, following various extraction methods. Such methods provide a realistic alternative to traditional analysis approaches using gas chromatography (GC), which involve lengthy sample preparation/analysis times and difculty in derivatization of certain phenols. In this study, the effect on the separation and analysis speed of a number of priority phenols cited within the U.S. Environmental Protection Agency (EPA) and European Union (EU) lists of priority pollutants(3) have been assessed by changing the chemistry and reducing the particle size of the stationary phase.
Results
Effect of particle/column size on analysis speed and quality The analysis times of eleven priority phenolic pollutants were signicantly improved by reducing column dimensions from 150 to 100 mm and particle size from 5 m to 3 m. Further improvements were achieved by changing to 1.9 m particles, using the Accela High Speed LC System.
Typical chromatograms demonstrating improvements in analysis speed are provided in Figures 1 to 3. Analysis time was further reduced by increasing the ow rate of the U-HPLC analysis to 1000 L/min, without any adverse effects on resolution (Figure 3). This is illustrated in the Table inset in Figure 3, which indicates peak width and resolution values for all separations.
Stationary phase chemistry The Hypersil GOLD 1.9 m phase produced the optimal overall separation of the chloro- and nitrophenols under the standard conditions used.
Method A (Column 150x2.1 mm, 5 m). Flow = 600 L/min. Injection Volume = 5 L.
Time (min) 0.0 1.5 19.5 21 21.01 22.5 Eluent B (%) 5 5 95 95 5 5
The Hypersil GOLD PFP (peruorinated phenyl) phase showed superior selectivity between chlorophenol components, likely due to the unique selectivity enabled by the presence of uorine in the stationary phase. However, the separation performance between the chloro- and nitrophenols was slightly reduced. Chromatograms illustrating the effect on the separation of using different stationary phases are given in Figure 4, along with resolution values between 4- and 2-chlorophenol (RS 6,7) and between 2-Nitro and 4-Chlorophenol (RS 4,6).
Method B (Column 100x2.1 mm, 3 m). Flow = 600 L/min. Injection Volume = 1 L.
Time (min) 0.0 1.0 13.0 14.0 14.01 15.0 Eluent B (%) 5 5 95 95 5 5
UHPLC Method 1 (Column 100x2.1 mm, 1.9 m). Flow = 600 L/min. Injection Volume = 1 L.
Time (min) 0.0 1.0 13.0 14.0 14.01 15.0 Eluent B (%) 5 5 95 95 5 5
UHPLC Method 2 (Column 100x2.1 mm, 1.9 m). Flow = 1000 L/min. Injection Volume = 1 L.
Time (min) 0.0 0.6 7.8 8.4 8.41 9.0 Eluent B (%) 5 5 95 95 5 5
Table 1: HPLC and U-HPLC gradients, ow rates, and injection volumes.
5 m Particles
No. Compound
1 2 3 4 5 6 7 8 9 10 11
Phenol 4Nitrophenol 2,4Dinitrophenol 2Nitrophenol 4Methylphenol 4Chlorophenol 2Chlorophenol 2,4Dimethylphenol 2,4Dichlorophenol 2,4,6Trichlorophenol Pentachloropenol
3 4
Rs (4,5) = 1.7
5,6 7 8 9 10 11
8 Time (min)
10
12
14
16
Figure 1: Separation of priority phenolic pollutants. using a 5 m particle packed column.
3 m Particles Rs (4,5) = 1.5
10
11
12
Time (min)
1.9 m Particles Rs (4,5) = 1.5
10
11
12
Time (min) Figure 2: Chromatographic effect of variation in column dimensions (3 and 1.9 m, 100 x 2.1 mm).
Column Dimensions 5 m. 150 x 2.1 mm 3 m. 100 x 2.1 mm 1.9 m. 100 x 2.1 mm 1.9 m. 100 x 2.1 mm
Method A B U-HPLC 1 U-HPLC 2
Average Peak Width (mins) 0.14 0.13 0.09 0.06
Resolution (peaks 4,5) 1.7 1.5 1.5 1.3
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Time (min)
Figure 3: Increased throughput using U-HPLC and 1.9 m particles. Comparison of peak width (at 10% height) and resolution.
In addition to these
Hypersil GOLD 1.9 m

4 Rs (6,7) = 8.8 Rs (4,6) = 4.2
Hypersil GOLD PFP 1.9 m

4 Rs (6,7) = 15.5 Rs (4,6) = 0.9
Hypersil GOLD aQ 1.9 m

4 Rs (6,7) = 7.9 Rs (4,6) = 1.1 3
offices, Thermo Fisher Scientific maintains a network of representative organizations throughout the world.
3 2 6 6 2 3 2
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
3.0
3.5
4.0
4.5 Time (min)
5.0
5.5
6.0
Time (min)
Time (min)
Figure 4: Comparison of 1.9 m stationary phase chemistries for the separation of chloro- and nitrophenols.
Conclusions
A number of priority phenols can be successfully separated in shorter analysis times by transferring to U-HPLC methods using Hypersil GOLD 1.9 m particle columns, without losing any signicant resolution. The increased peak efciency observed for 1.9 m particle packed columns indicates that low level phenol analyses in environmental matrices described in previous studies,(1,2) would be further enhanced with increased sensitivity. Different column chemistries create important differences in selectivity for method development purposes, which may aid studies involving, for example, the separation of halophenols using a Hypersil GOLD PFP phase.
References
1
M.C. Alonso, D. Puig, I. Silonger, M. Grasserbauer, D. Barcelo, J Chromatogr. A, 823 (1998) 231-239. J. Martinez Vidal, A. Belmonte Vega, A. Garrido Frenich. Analytical & Bioanalytical Chem. 1. 379 (2004) 125-130. EPA Method 625, Phenols, Environmental Protection Agency, Part VIII, 40 CFR Part 136, Washington DC, 1984.
Additional Information
For additional information, please browse our Chromatography Resource Center which can be accessed from: www.thermo.com/columns
www.thermo.com
Legal Notices 2008 Thermo Fisher Scientic Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientic Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
AN62531_E 01/08S
Contaminants Toxins
379: Analysis of Microcystins from Blue-green Algae Using the Thermo ScientificTSQ Quantum Ultra LC-MS/MS System
Analysis of Microcystins from Blue-green Algae Using the TSQ Quantum Ultra LC-MS/MS System
Mihoko Yamaguchi, Thermo Fisher Scientic, Yokohama, Japan
Introduction
Key Words TSQ Quantum Ultra Blue-green algae LC-MS/MS Microcystin SRM Overgrowth of algae is a common problem in many wetlands with advanced stages of eutrophication (the enrichment of chemical nutrients containing nitrogen or phosphorus in an ecosystem). This often results in a thick, colored layer on the water's surface, known as an algal bloom. Some of the algae that grow in these bodies of water, known as Cyanobacteria or blue-green algae, produce toxic compounds known as microcystins. Microcystins have a ring peptide structure consisting of seven amino acids, and more than 80 homologs are known. One of the most widely studied of the microcystins is known as Microcystin-LR, and is shown in Figure 1. Many of the microcystins are particularly toxic to the liver. (See References.) Among them are Microcystin-LR, YR and RR, which have been detected in wetlands in Japan. This application note reports on the analysis of these microcystins by using LC-MS/MS.
HOOC HN OCH3 H3 C O NH CH3 CH3 HN H2 N N H O H N CH3 N O O CH 2 NH O CH 3 CH 3
MS: Thermo Scientic TSQ Quantum Ultra Ionization: Positive ESI Spray voltage: 5000 V Sheath gas: 45 arbitrary units Auxiliary gas: 15 arbitrary units Sweep gas: 2 arbitrary units Capillary T: 350C Source CID: Off Collision gas: Ar, 1.2 mTorr Scan Time: 0.15 sec SRM setting: 519.9 135.0 @ 32 V (RR) 995.7 135.0 @ 65 V (LR) 1045.8 135.0 @ 70 V (YR)
SRM Chromatogram (STD 1.0 ppb)

The SRM chromatograms for 1.0 ppb standards are shown in Figure 2. The linear calibration curves of the standards (0.1 ppb1.0 ppm) are shown in Figure 3.
R T : 3 .00 - 6.0 0 10 9 8 7 Relative Abundance 6 5 4 3
H3C CH3 H HN N O O COO H
4.35 Microcystin-RR 4.72 Microcystin-YR 4.78 Microcystin-LR
Figure 1: Microcystin-LR
Method
HPLC: HTC PAL Autosampler and Thermo Scientic Surveyor MS pump Column: Thermo Scientic HyPURITY C18 2.1 50 mm, 5 Mobile Phase A: Water with 0.1% Formic Acid Mobile Phase B: Acetonitrile Gradient: 30%B (0.5 min) 80%B (in 3 min) 80%B (2 min hold) 30%B (7 min hold) Injection Volume: 20 L Flow: 0.2 mL/min Column temperature: Room temperature
2 1 0 3.0
3.5
4.0
4.5
Time
5.0
5.5
6.0
Figure 2: SRM Chromatogram (RT 4.35: Microcystin-RR, RT 4.72: Microcystin-YR, RT 4.78: Microcystin-LR)
Conclusion
Microcystin-LR, YR and RR can be quantitatively analyzed over four orders of dynamic range (0.1 ppb 1.0 ppm) by using the TSQ Quantum Ultra triple quadrupole LC-MS/MS system.
References
T Ohta; R Nishiwaki; M Suganuma; J Yatsunami; A Komori; S Okabe; M Tatematsu; H Fujiki. 1993. Signicance of the Cyanobacterial Cyclic Peptide Toxins, the Microcystins and Nodularin, in Liver-Cancer. Mutation Research, 292:286-287. JG Pace; NA Robinson; GA Miura; CF Matson; TW Geisbert; JD White. 1991. Toxicity and Kinetics of [H-3] Microcystin-Lr in Isolated Perfused Rat Livers. Toxicology and Applied Pharmacology, 107:391-401. R Nishiwaki; T Ohta; E Sueoka; M Suganuma; K Harada; MF Watanabe; H Fujiki. 1994. Two signicant aspects of microcystin-LR: Specic binding and liver specicity. Cancer Lett, 83:283-289. I Falconer; A Jackson; J Langley; M Runnegar. 1980. Liver Pathology of a Toxin from the Bloom-Forming Blue-Green Alga Microcystis Aeruginosa. Proceedings of the Australian Biochemical Society, 13:41-41.
Figure 3: Calibration Curves 0.1 ppb ~1.0 ppm
www.thermo.com
AN62272_E 01/07S
Drug Residues Antibiotics

t
465: Analysis of (Fluoro)quinolones in Honey with Online Sample Extraction and LC-MS/MS 435: Analysis of Sulfonamides in River Water using Thermo Scientific EQuan, an Online Concentration Analysis System 397: Determination of Sulfonamide Antibiotics in Wastewater by Liquid ChromatographyTandem Mass Spectrometry 361: Determination of Trace Level Nitrofuran Metabolites in Crawfish Meat by Electrospray LC-MS/MS on the Thermo Scientific TSQ Quantum Discovery MAX 358: Highly Selective Detection and Identification of Nitrofurans Metabolites in Honey using LC-MS/MS
t t t t
442: LC-MS/MS Analysis of Malachite Green, Leucomalachite Green, Ciprofloxacin, and Tetracycline in Food Samples using a TurboFlow Method 464: Multi-class Antibiotic Screening of Honey Using Online Extraction with LC-MS/MS 354: On-line Enrichment HTLC/MS/MS Assay for Multiple Classes of Antibiotics in Environmental Water Sources 407: Simple and Rapid Analysis of Chloramphenicol in Milk by LC-MS/MS
Analysis of (Fluoro)quinolones in Honey with Online Sample Extraction and LC-MS/MS

Yves-Alexis Hammel, Nestle Research Center, Lausanne, Switzerland; Frans Schoutsen, Thermo Fisher Scientific, Breda, The Netherlands; Cludia P. B. Martins, Thermo Fisher Scientific, Barcelona, Spain
Introduction
Key Words TurboFlow Technology Aria TLX-1 TSQ Quantum Ultra Food Safety The global food market has become more competitive and equally cost responsive. The need for analytical procedures that permit high sample throughput as well as higher sensitivity allied to good reproducibility is growing by the day.1,2,3 A method using automated online extraction with tandem mass spectrometry is presented as an alternative to the commonly used, time-consuming solid-phase extraction (SPE) method. Quinolones, including fluoroquinolones, are a group of synthetic antibacterial compounds used in the treatment of several diseases. There has been a significant and progressive increase in the use of quinolones in animal production, which has led to their residual presence in food. In the European Union, the maximum residue limits (MRLs) for several of these compounds are defined for different food matrices of animal origin, but not for honey.4 Furthermore, the presence of these compounds is an indication of unsafe practices of food production and deficient methods in the production of honey. The complexity of the matrix plays a fundamental role on the adoption of the method of analysis. Thermo Scientific TurboFlow technology enables the reduction of sample preparation as well as the elimination of interferences from complex matrices such as honey.
Experimental
Sample Preparation
To a sample of 1 g of honey, 1 mL of water was added and the mixture was homogenized. The sample was then filtered directly to the HPLC vial using a 0.22 m polyethersulfone membrane syringe filter. Different concentration levels were achieved by spiking the sample with different concentration levels of standard stock solution. The total sample preparation time was 40 minutes for 12 samples.
TurboFlow Method Conditions:
System: Online Extraction: Mobile Phase A: Mobile Phase B: Mobile Phase C: Mobile Phase D: Injection Volume:
HPLC conditions:
Thermo Scientific Aria TLX-1 controlled by Aria software (Figure 1) TurboFlow Cyclone 50 x 0.5 mm 0.1 % formic acid in water 0.1 % formic acid in acetonitrile 10 mM ammonium formate in water acetonitrile/isopropanol/acetone (4:3:3 v/v/v) 90 L
Goal
To develop a sensitive and reproducible liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitation of 12 fluoroquinolones and 4 quinolones in honey using automated extraction by TurboFlow technology.
Analytical Column: Thermo Scientific Hypersil GOLD 2.1 x 50 mm, 3 m column at 40 C Solvent A: 0.5 % formic acid in water Solvent B: 0.5 % formic acid in methanol/acetonitrile (1:1 v/v)
Figure 1: Aria software with LC Method Editor
MS Conditions
MS analysis was carried out on a Thermo Scientific TSQ Quantum Ultra AM triple stage quadrupole mass spectrometer. The MS conditions were as follows:
Ion Source Polarity: Spray Voltage: Vaporizer Temperature: Sheath Gas Pressure (N2): Auxiliary Gas Pressure (N2): Capillary Temperature: Collision Gas (Ar): Q1/Q3 Peak Resolution: Scan Time: Scan Width: Data Acquisition Mode: Positive Ion Mode 3000 V 350 C 40 units 35 units 325 C 1.5 mTorr 0.7 u (unit mass resolution) 0.025 s 0.010 m/z SRM
Table 1: Selected ion transitions (m/z), collision energy (CE) and tube lens voltages (TL) for studied compounds
Precursor Ion (m/z) Product Ion (m/z) CE (V) TL (V)
Analyte
1. Nalidixic Acid
233.064
2. Oxolinic Acid 3. Flumequine 4. Cinoxacin
262.032 262.050 263.029
5. Pipemidic Acid
304.062
The optimization of Selective Reaction Monitoring (SRM) parameters was performed by direct infusion of standards in the positive electrospray ionization mode. Collision induced dissociation (CID) mass spectra were recorded for each analyte and the optimum collision energies were obtained for the selected ion transitions. Table 1 summarizes these parameters and Figure 2 displays the MS method controlled by Thermo Scientific Xcalibur software.
6. Norfloxacin 7. Enoxacin 8. Ciprofloxacin 9. Lomefloxacin 10. Danofloxacin
320.096 321.083 323.100 352.104 358.120
11. Enrofloxacin 12. Ofloxacin 13. Marbofloxacin
360.128 362.107 363.066
14. Fleroxacin 15. Sarafloxacin
370.094 386.095
16. Difloxacin
400.107
104.143 215.020 187.025 130.106 244.012 199.998 243.962 105.202 189.014 217.049 245.011 189.000 217.029 286.075 276.058 302.055 206.012 302.981 231.024 314.018 265.010 308.067 82.215 314.097 340.089 245.025 315.958 261.041 318.055 70.067 72.073 276.064 320.022 269.023 326.061 298.979 342.078 367.878 299.009 356.017
40 15 25 33 19 34 19 37 29 22 16 29 19 20 17 21 29 21 36 22 23 17 39 18 24 26 19 27 19 34 22 14 14 27 19 28 18 22 29 20
78 78 78 82 82 61 61 59 59 59 59 82 82 82 70 70 65 65 74 74 78 78 75 75 75 72 72 109 109 66 66 66 66 112 112 105 105 105 75 75
Figure 2: MS method showing the SRM transitions and other conditions

The analysis of food samples normally requires long preparation times due to the complexity of the matrices. The Thermo Scientific Aria TLX-1 system powered by TurboFlow technology enables reduction of the sample preparation time. It took only 40 minutes to prepare the batch of samples for LC-MS/MS analysis, instead of an average time of 6 hours when using Solid Phase Extraction (SPE). Even when dealing with complex matrices, such as honey, the use of the TLX-1 system enables the elimination of possible interferences and creates less noisy chromatograms (Figure 3).
The results of a high-throughput, rapid, sensitive and linear method for the determination of 16 quinolones, including 12 fluoroquinolones, by LC-MS/MS using TurboFlow technology are presented (Table 2). The Limit of Detection (LOD) was calculated by using the statistical definition LOD = YB + 3SB, where YB is the blank signal and SB is the standard deviation of the blank.
Figure 3: Representative SRM chromatogram (20 g/kg) showing the selected ion transitions and retention times for the studied analyte
Table 2: Linearity, sensitivity and precision of the method

Range (g/kg) LOD (g/kg) RSD (%) R2 (1/X)
References and Acknowledgements

1. Mottier, P.; Hammel, Y.-A.; Gremaud, E.; Guy, P. A., Quantitative HighThroughput Analysis of 16 (Fluoro)Quinolones in Honey Using Automated Extraction by Turbulent Flow Chromatography Coupled to Liquid Chromatography Tandem Mass Spectrometry. Journal of Agricultural and Food Chemistry 2008, 56, 35-43. 2. Gunes, N.; Cibik, R.; Gunes, M. E.; Aydin, L., Erythromycin residue in honey from the Southern Marmara region of Turkey. Food Additives and Contaminants 2008, 25, 1313-1317. 3. http://www.cfsan.fda.gov/~comm/fluoroqu.html. U.S. Food and Drug Administration. Preparation and LC/MS/MS analysis of honey for fluoroquinolones residues 2006. 4. EU Comission Regulation No. 2377/90. Laying down a community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off. J. Eur. Communities 1990, L224, 1-8.
Analyte
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1-50 1-50 1-100 2-100 1-100 1-100 1-100 1-100 1-100 1-100 1-100 2-100 1-100 1-50 1-100 1-100
0.8 1.4 0.9 2.0 0.9 2.3 1.9 1.4 0.5 1.1 0.8 1.3 2.6 1.5 1.1 0.8
1.3 - 4.7 0.3 - 10.6 1.7 - 8.9 4.3 - 7.7 1.5 - 10.1 2.7 - 11.5 2.1 - 11.7 2.4 - 11.6 0.2 - 13.7 2.3 - 13.6 1.5 - 16.9 2.1 - 11.5 2.4 - 13.9 6.0 - 16.8 1.1 - 11.2 1.9 - 10.4
0.9943 0.9909 0.9902 0.9918 0.9964 0.9925 0.9928 0.9967 0.9954 0.9961 0.9907 0.9945 0.9939 0.9903 0.9966 0.9947
The method proved to be linear in the range studied. Three replicates were used for each point of the calibration levels, which, in addition to the relative standard deviation values, demonstrate the precision of the method.
Conclusion
A rapid, sensitive and reliable method for the quantitation of 16 quinolones, including 12 fluoroquinolones, was developed using a TurboFlow method in combination with a TSQ Quantum Ultra mass spectrometer. The use of TurboFlow technology enables a significant reduction of the sample preparation time. For 12 samples the preparation time was reduced from 5 hours to 40 minutes. Preliminary trials indicate this online extraction coupled with a TSQ Quantum Ultra is an excellent total solution for the quantification of a large number of compounds in food samples.
Africa-Other +27 11 570 1840 Australia +61 2 8844 9500 Austria +43 1 333 50 34 0 Belgium +32 2 482 30 30 Canada +1 800 530 8447 China +86 10 8419 3588 Denmark +45 70 23 62 60 Europe-Other +43 1 333 50 34 0 Finland / Norway / Sweden +46 8 556 468 00 France +33 1 60 92 48 00 Germany +49 6103 408 1014 India +91 22 6742 9434 Italy +39 02 950 591 Japan +81 45 453 9100 Latin America +1 608 276 5659 Middle East +43 1 333 50 34 0 Netherlands +31 76 579 55 55 South Africa +27 11 570 1840 Spain +34 914 845 965 Switzerland +41 61 716 77 00 UK +44 1442 233555 USA +1 800 532 4752
www.thermo.com
Legal Notices 2009 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

AN63070_E 06/09S
Analysis of Sulfonamides in River Water using EQuan, an Online Concentration Analysis System
Yoko Yamagishi, Thermo Fisher Scientic, Yokohama, Japan
Introduction
Key Words Antibiotics EQuan LC-MS/MS Online Concentration Sulfonamides Triple stage quadrupole LC-MS systems are often used for highly sensitive quantitative analyses of environmental pollutants. The performance of an LC-MS system is one important factor that determines if it will efciently and accurately detect pollutants present at low concentrations in environmental samples. Another factor is the process by which the system pre-treats the samples. In its sample pretreatment process, the Thermo Scientic EQuan system is able to quickly perform the time-consuming concentration process online, dramatically reducing the time required for analysis. Consequently, EQuan is capable of measuring multiple samples more efciently than systems using off-line sample preparation, while reaching lower detection limits than are achievable with a conventional LC-MS/MS analysis.
Calibration standards were prepared using a mixed standard solution of the nine LC target sulfonamides (Kanto Chemical Co., Ltd.). For the test samples, river water collected in Kanagawa Prefecture was passed through a 0.4 m lter prior to analysis.
HPLC
Analytical Column: Concentration Column: Mobile Phase A: Mobile Phase B: Gradient (for analysis): Injection Volume: Flow: Column Temperature: Thermo Scientic Hypersil GOLD C18 2.1 x 150 mm, 5 m Hypersil GOLD C18 2.1 x 20 mm, 12 m 1 mM ammonium formate, 0.05% formic acid in water 1 mM ammonium formate, 0.05% formic acid-methanol 5% B (1.5 min) to 90% B (in 10 min) to 90% B (5 min hold) 0.5 mL 0.2 mL/min 40 C
MS: Thermo Scientic TSQ Quantum

Ionization Mode: Spray Voltage: Sheath Gas: AUX Gas: Sweep Gas: Capillary Temperature: Skimmer Offset: Scan Time: Collision Gas Pressure: Mass Resolution (FWHM): Positive ESI 4500 V 50 20 0 360 C 7V 0.1 sec/SRM transition Argon, 1.2 mTorr Q1 & Q3 0.7 Da
Figure 1: EQuan system schematic
SRM Conditions
m/z 251.07 156.0 at 17 eV (sulfadiazine) m/z 254.07 156.0 at 16 eV (sulfamethoxazole) m/z 265.08 156.0 at 18 eV (sulfamerazine) m/z 268.08 156.0 at 14 eV (sulsoxazole) m/z 279.10 186.0 at 19 eV (sulfadimidine) m/z 281.08 156.0 at 18 eV (sulfamethoxypyridazine) m/z 281.08 156.0 at 18 eV (sulfamonomethoxine) m/z 301.08 156.0 at 17 eV (sulfaquinoxaline) m/z 311.09 156.0 at 19 eV (sulfadimethoxine)
The following application note presents a quantitative analysis of sulfonamide antibiotics using EQuan. These compounds are used widely as anti-inammatory medications for humans and livestock, and have recently become compounds of interest to regulatory agencies worldwide. Figure 2 shows the library spectra and chemical structures for the nine sulfonamide antibiotics used in this experiment.

Standard Sample Results
It was possible to detect all of the target compounds at a concentration of 1.0 ppt using the EQuan system (Figure 3). Furthermore, linearity was obtained over the range of 0.5 to 100 ppt (Figure 4).
Figure 2: Product ion spectra (from MS/MS library)
Figure 3: SRM chromatogram of 1.0 ppt standard samples. Signal-to-noise (S/N) value shown above each peak. range for calculating S/N.
on each chromatogram shows noise
Figure 4: Calibration curves
River Sample Assay Results

In the measurement of the river water, sulfamethoxazole was detected at a concentration of 12.3 ppt and four other components were detected at concentrations of about 1 ppt or less (Figure 5).
The mixed standard sample was spiked in the river water samples at the concentration of 1.0 ppt (except for Sulfamethoxazole, which was spiked at 10 ppt) and the samples were analyzed. A good recovery rate of 70% to 98% was obtained for each of the compounds. Furthermore, reproducibility for all replicates was 11% or less for the spiked samples (see Table 1).
Conclusion
With the EQuan online concentration analysis system, it was possible to measure the sulfonamide antibiotics that were present in the river water samples at low concentrations quickly and accurately.
Figure 5: SRM chromatogram of river samples. Signal-to-noise (S/N) value shown above each peak. chromatogram shows noise range for calculating S/N.
on each
1.0 ppt spiked samples (n = 4)
Compound
Concentration in river water sample (ppt)
Concentration in spiked samples (ppt)
Recovery rate (%)
CV (%)
Sulfadiazine Sulfamerazine Sulfadimidine Sulfamethoxypyridazine Sulfamethoxazole Sulfamonomethoxine Sulsoxazole Sulfadimethoxine Sulfaquinoxaline
0.35 0.39 NF NF 12.35 1.11 NF 1.42 NF
1.19 1.13 0.98 0.87 19.35* 1.85 0.95 2.19 0.85
84 73 98 87 70 74 95 77 85
10.4 5.1 11.3 7.1 2.8 3.3 8.5 1.4 3.3
* The spiking concentration was set at 10 ppt because sulfamethoxazole was detected in the river water samples at concentrations higher than 10 ppt.
Table 1: River water and spiked sample assay results and reproducibility
www.thermo.com
AN62734_E 09/08M
Determination of Sulfonamide Antibiotics in Wastewater by Liquid Chromatography Tandem Mass Spectrometry

Eleni Botitsi, Charalampia Frosyni, and Despina Tsipi; General Chemical State Laboratory, Athens, Greece
Introduction
Key Words TSQ Quantum Ultra Surveyor HPLC System Environmental Application Sensitivity Solid Phase Extraction (SPE)
Goal
Antibiotics are widely used in human and veterinary To develop methods for the determination of sulfonamide medicine for the prevention and treatment of bacterial antibiotics at trace levels in efuent wastewaters. infectious diseases. An important but often disregarded aspect of antibiotic use is the fate of antibiotic residues Experimental Conditions entering the environment.1 Pharmaceutical industry Sample Preparation wastewater, improperly-disposed of unused antibiotics, Samples of secondary efuent were collected from sewage and non-metabolized antibiotics excreted by humans can treatment plants in Greece and then vacuumed ltered. all enter the sewer system in low concentrations. Because Each 50 mL sample was diluted with 200 mL deionized sewage treatment plants are rarely equipped to lter these water. After acidication to pH 4, 5 ng of the internal drugs from wastewater, antibiotics are released into the standard d4-sulfamethoxazole (d4-SMX) was added water system. Veterinary antibiotics used in livestock before enrichment to assess possible losses during the operations are another major source of antibiotics in analytical procedure. The efuent samples were enriched the environment. Agricultural waste such as manure by solid phase extraction (SPE). The diluted wastewater and water runoff can carry these antibiotics into the samples were percolated through the cartridges at a ow soil and groundwater. rate of 5 mL/min. The cartridges were then washed with The effects of antibiotics in the environment are still 5 mL deionized water. Wastewater organics were eluted poorly understood. One major concern is the development with 2 4 mL methanol. The solvents were evaporated of antibiotic resistant strains of bacteria that could critically under a stream of nitrogen gas and then the extracts disturb the natural bacteria ecosystems and lead to a were redissolved in 0.5 mL mobile phase A (0.1% serious threat to human health. There are also concerns formic acid in water). that, exposure to environmental antibiotic residues might lead to carcinogenic or allergic reactions in humans and HPLC create hazards to aquatic and soil organisms.2,3 HPLC analysis was performed using the Thermo Scientic Sulfonamides (Figure 1) are a common class of Surveyor HPLC System. Each 20 L sample was injected synthetic antimicrobials that are widely used in human directly onto a 150 2.1 mm, 3.5 m, C18 HPLC column. and in veterinary medicine and as feed additives to proA gradient LC method used mobile phases A (0.1% mote growth in concentrated animal feeding operations. formic acid in water) and B (0.1% formic acid in They are regarded as emerging contaminants that are acetonitrile) at a ow rate of 0.2 mL/min. introduced into the environment predominantly in the USA and Europe. There is no regulation of the levels of these compounds in environmental matrices (water, sediment, soil). This is likely because Sulfapyridine (Mr = 249) Sulfathiazole (Mr = 255) of the limited knowledge of the input, O O fate, and effects of most pharmaceuN S S N NH2 NH S N H H ticals in the environment. Therefore, N O O sensitive and reliable analytical methods for detection of low Sulfamethoxazole (Mr = 253) concentrations (ng/L) of these O CH3 NH2 S N compounds are needed. H N O
2
Sulfamerazine (Mr = 264)

O NH2 S O N H CH3 N
NH2
Sulfamethazine (Mr = 278)

O S O N H CH3 N N CH3
Figure 1: Chemical structures of some sulfonamide compounds
MS MS analysis was carried out on a Thermo Scientic TSQ Quantum Ultra triple stage quadrupole mass spectrometer with an electrospray ionization source. The MS conditions were as follows: Ion source polarity: Positive ion mode Sheath gas pressure (N2): 40 units Ion transfer tube temperature: 350C Collision gas pressure (Ar): 1.0 mTorr Q1 resolution: 0.2 FWHM, Q3 resolution: 0.7 FWHM Dwell time: 0.2 s Scan Type: SRM
Table 1 summarizes the SRM transitions that were monitored. MS detection of the target compounds was divided into three time segments on the basis of their retention times during chromatography. The protonated molecular ion of the compound [M+H]+ was selected as the precursor ion. Detection was performed in the multiple reaction monitoring mode using, usually, the two most intense and characteristic precursor/product-ion transitions obtained from the MS/MS optimization procedure. Identication criteria for the target compounds were based on the LC retention time (tR) and on the ratio of the two monitored transitions for each compound. Method accuracy and precision were evaluated by recovery studies, using deionized water spiked with appropriate amounts of the sulfonamides at three concentrations (2 ng/L, 20 ng/L, and 200 ng/L). Calibration plots were obtained by analysis of standard solutions at eight concentrations in the range 0.1 g/L100 g/L (2 pg2000 pg injected).

The method validation data are summarized in Table 2. Linearity of the method was assumed because the r2 values were greater than 0.99 for the linear regression equations (1/x weighted) and the residuals were less than 20% for each calibration point in the concentration range 0.1 g/L100 g/L. Quantication was performed on the basis of external calibration plots using the peak area of the most intense transition of the analyte. For SMX, quantication was performed using the ratios of the peak areas of the most abundant monitored ion of the analyte to that of the respective ion of the surrogate standard d4-SMX. The accuracy of the method was determined by recovery studies conducted on spiked deionized water samples at three concentration levels: 2 ng/L, 20 ng/L, and 200 ng/L. The precision of the method was determined by repeated intra-day (n=3) and inter-day analysis (n=6) of samples spiked at the three concentrations. High rates of recovery were achieved, usually greater than 72%, and relative standard deviations for inter-day analysis (n=6) ranged between 3.1% and 19.0%. The SRM chromatograms obtained from deionized water spiked at a concentration of 2 ng/L are shown in Figure 2. The method was applied to the wastewater samples to investigate the occurrence of sulfonamide antibiotics. Sulfamethoxazole was detected in all of the samples. The median concentration was 150 ng/L. The SRM chromatograms of SMX in the wastewater efuent extract are shown in Figure 3.
Compound
Retention Precursor ion time (min) [M+H]+ m/z
Product ions m/z
CE (V)
Sulfapyridine, SPY Sulfamethoxazole, SMX Sulfathiazole, STZ Sulfamerazine, SMR Sulfamethazine, SMZ D4-Sulfamethoxazole, D4-SMX D4-Sulfathiazole, D4-STZ
10.1 26.2 10.3 12.0 18.6 26.0 10.0
250 254 256 265 279 258 260
108, 156 108, 156 108, 156 108, 156 186, 204 112, 160 112, 160
20 25 17 17 24 25 20
Table 1: Diagnostic ions of sulfonamide antibiotics
R2* concentration range LOD Table 1: Diagnostic ions of sulfonamideg/L Compound 0.1100 antibiotics(g/L)
Mean Recovery ( %RSD) 2 ng/L H2O (n=6) 20 ng/L H2O (n=6) 200 ng/L H2O (n=6)
Sulfapyridine, SPY Sulfamethoxazole, SMX Sulfathiazole, STZ Sulfamerazine, SMR Sulfamethazine, SMZ
0.997 0.999 0.999 0.998 0.999
0.053 0.055 0.054 0.110 0.110
77 (7.8) 102 (7.5) 79 (14.0) 80 (15.3) 72 (19.0)
83 (5.1) 99 (5.5) 83 (17.0) 87 (3.1) 77 (14.0)
120 (8.4) 110 (7.2) 106 (4.2) 120 (7.2) 116 (8.4)
*linear t calibration curves with 1/x weighting, (n=5 replicates)
Table 2: Validation data (linearity, accuracy, precision)
100
250 108, 156

50 0 100
SPY
254 108, 156
50 0 100
SMX
256 108, 156
Relative Abundance
50 0 100
STZ
258 112, 160
50
D4-SMX (I.S.)
0 100
260 112, 160

50 0 100
D4-STZ
265 108, 156
50 0 100
SMR
279 186, 204
50 0 0
SMZ
5 10 15 20 25 30 35
Time (min)
Figure 2: LC-ESI(+)-MS/MS SRM chromatograms of a spiked (2 ng/L) deionized water extract
100 80 60 40 20 0 100 80 60 40 20 0 100 80 60
TIC Sample
254 108, 156
SRM 254 156
Relative Abundance
40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 0
SRM 254 108

254 108, 156
TIC Standard (5 g/L)
SRM 254 156
+ H2N
SO S 2
m/z 156
+ H2N
SRM 254 108

5 10
m/z 108
15 20 25 30 35 40
Time (min)
Figure 3: LC-ESI(+)-MS/MS chromatograms of sulfamethoxazole in wastewater sample extract and of a standard solution of sulfamethoxazole
Conclusion
LC-ESI-MS/MS is a powerful analytical method for the sensitive determination of sulfonamide antibiotics in municipal wastewater at low ppt levels (ng/L). The solid phase extraction scheme for trace enrichment and separation of sulfonamide compounds from wastewater samples yielded high recovery rates and enabled their accurate quantication. Sulfamethoxazole (median concentration 150 ng/L) was detected in the wastewater efuent samples, which indicated that it was not completely eliminated in the sewage treatment plants. The described method proved to be a valuable tool for the detection of pharmaceuticals in wastewater efuents before they reached the aquatic environment.
References
1
Petrovic, M.; Hernando, M. D.; Diaz-Cruz, M. S.; Barcel, D. Liquid chromatography-tandem mass spectrometry for the analysis of pharmaceutical residues in environmental samples: a review; J, Chrom. A 2005, 1067(1-2), 1-14. Gbel, A.; McArdell, C. S.; Suter, M. J.-F.; Giger, W. Trace determination of macrolide and sulfonamide antimicrobials, a human sulfonamide metabolite, and trimethoprim in wastewater using liquid chromatography coupled to electrospray tandem mass spectrometry; Anal. Chem. 2004, 76(16), 4756-4764. Yang, S.; Cha, K.; Carlson, K.Quantitative determination of trace concentrations of tetracycline and sulfonamide antibiotics in surface water using solid-phase extraction and liquid chromatography/ion trap tandem mass spectrometry; Rapid Commun. Mass Spectrom. 2004, 18(18), 2131-2145.
www.thermo.com

AN62480_E 10/07S
Determination of Trace Level Nitrofuran Metabolites in Crawsh Meat by Electrospray LC-MS/MS on the TSQ Quantum Discovery MAX
Tao Ding,1 Jingzhong Xu,1 Chongyu Shen 1 and Kefei Wang2
1 2
Food Laboratory, APFIC, Jiangsu Entry-Exit Inspection and Quarantine Bureau of Peoples Republic of China, Nanjing, China Thermo Fisher Scientic, San Jose, CA, USA
Introduction
Key Words TSQ Quantum Discovery MAX Surveyor HPLC Food Residue Analysis SRM Veterinary Drugs Nitrofurans (furazolidione, furaltadone, nitrofurazone and nitrofurantoin) are a group of veterinary antibiotics banned in many countries because of human health concerns. The ban has stimulated signicant interest in development of analytical methods for detecting trace levels of these drug residues in animal products. Due to the rapid in vivo metabolism of the parent drugs, detection of nitrofurans in meat products relies on determination of their corresponding tissue-bound metabolites: 3-amino-2-oxazolidinone (AOZ), 3-amino5-morpholinomethyl-2-oxazolidinone (AMOZ), semicarbazide (SEM) and 1-aminohydantoin (AHD). These metabolites were removed from tissues by hydrolysis in acidic solution and derivatized to nitrobenzyl- (NB-)
NO 2 O N N O H2N N O N O N O
derivatives with 2-nitrobenzyladehyde (2-NBA). Figure 1 illustrates the transformation. LC-MS/MS utilizing selected reaction monitoring (SRM) of the corresponding four metabolite derivatives has become the method of choice. In this note we describe a sensitive and selective LC-MS/MS method for detecting trace level nitrofuran metabolites in crawsh using a Thermo Scientic TSQ Quantum Discovery MAX triple quadrupole mass spectrometer coupled to a Thermo Scientic Surveyor HPLC module. The limit of quantitation (LOQ) as low as <0.05 g/kg has been clearly demonstrated in fortied crawsh meat for all four nitrofuran metabolites. This LOQ represents twenty-fold better than the Minimum Required Performance Limit (MRPL) established by European Union (EU) in 2003.
O2N
Standards and Reagents
The following are a list of chemicals used in this work, and unless specied all chemicals are of at least reagent grade. AOZ and SEMHCl (Sigma-Aldrich, St. Louis, MO, USA) 2-NBA (Sigma-Aldrich) DMSO (Sigma-Aldrich) d4-AMOZ and d5-AMOZ (Cambridge Isotope Laboratory, MA, USA) 1-Amino-imidazolidin-2,4-dione-[2,4,5-13C] (WITEGA Laboratorien Berlin-Adlershof GmbH, Berlin, Germany) Semicarbazide hydrochloride-13C, 15N2 (WITEGA) Ammonium Acetate (NH4Ac), K2HPO4, and NaOH (Sigma-Aldrich) Methanol (HPLC grade, Thermo Fisher Scientic, Pittsburgh, PA, USA) Water (in-house distilled water, ltered with a 0.45 m lter)
Furazolidione
O2N O
AOZ
O O N N H2N NO 2 N
NBAOZ
N N
N N O O
O O
Furaltadone
O
AMOZ
O NO 2 NH
NBAMOZ
O
O O2N
N N O H2 N N
NH
N N O
NH
Nitrofuranzone
O2N O
AHD
NO 2 H N O
NBAHD
H N N
O NH 2
NH O H2N
H2N NH 2
Nitrofurantoin
SEM
NBSEM
Nitrofurans
Metabolites
NB-Derivatives
Figure 1: Nitrofurans, their metabolites, and 2-NBA derivatives
Analytical Equipment
HPLC: Surveyor HPLC module consisting of an AS Autosampler and MS Pump Mass spectrometer: TSQ Quantum Discovery MAX
Analytical Standard Preparation

The primary analytical standard solutions of 1 mg/mL were prepared by dissolving the corresponding solid standards into methanol. The working standard solutions were prepared by serial dilution of the primary standard solution with 95:5 water:methanol.
Gradient: Time (min) 0 8.5 9.5 10 15
%A 80 50 50 80 80
%B 20 50 50 20 20
Flow rate: 250 L/min Column temperature: Ambient (18-22C) Injection volume: 20 L (with loop)
Mass Spectrometry Conditions Sample Preparation

Note: Nitrofuran metabolite derivatives are sensitive to light; avoid prolonged exposure of sample to direct light sources during sample preparation. The extraction and derivatization of the nitrofurans from the crawsh were performed at Food Laboratory of Jiangsu Entry-Exit Inspectional and Quarantine Bureau at Nanjing, China, following the published procedure with some modication: To 2 g of homogenized crawsh meat inside a 50-mL glass tube, add 4 mL water, 0.5 mL of 0.5 M HCl solution, and 200 L of freshly prepared 50 mM 2-NBA in DMSO. Vortex for one minute and store the sample in the dark at 37C overnight (14-16 hours) After cooling the sample to room temperature, add 5 mL 0.1 M K2HPO4, adjust the pH of the mixture to 7.0-7.5 with 0.4 M NaOH solution. Centrifuge the mixture and collect the supernatant Extract the supernatant twice each time with 7 mL ethyl acetate. Combine the ethyl acetate extracts and evaporate to dryness under N2 at 40C Reconstitute the residues in 1.0 mL of water:methanol (95:5). Centrifuge the samples and lter the supernatant with 0.2 m syringe lter prior to injection to LC-MS system Note that the sample preparation results in a two-fold concentration that will be factored into the calculation of nitrofuran metabolite concentrations in meat samples. For fortied samples, the nitrofuran metabolites and their internal standards were added into the homogenized meat sample prior to the hydrolysis and derivatization. For calibration, the same procedures were followed except that 2 mL of working standard solutions was used instead of the meat samples. The mass spectrometer was calibrated routinely with 1,3,6-polytryosine, according to the standard operating procedures at the Nanjing laboratory. For method development, a standard solution containing 1 g/mL of derivatized nitrofuran metabolites including the internal standards was infused at 10 L/min with 250 L/min 50:50 (A:B) mobile phase into the ESI source. First, the spray voltage, sheath gas, auxiliary gas and tube lens were optimized with the automated tune of Thermo Scientic Xcalibur software. Second, the most abundant fragment ions and their optimized collision energy (CE) values were found in MS/MS optimization. For known SRM transitions, parent and product ions can be input directly to obtain the optimized CE value for each SRM transition. Finally, the Source CID (skimmer offset voltage), collision gas pressure, and ion transfer capillary temperature were adjusted manually for best signal sensitivity. The nal operation parameters are summarized as follows: Ion source (polarity): ESI (+) Spray voltage: 5000 V Sheath gas pressure: 30 units Auxiliary gas pressure: 8 units Ion transfer capillary temperature: 300C Source CID: 10 V Scan type: SRM Q1 and Q3 peak width (FWHM): 0.7 Da Collision gas and pressure: Ar at 1.3 mTorr For each parent ion, two SRM transitions were used, one for quantitation and one for conrmation, which would give 4 IP (identication points) to meet the EUs criteria for residue analysis in food. Based on the elution order of the nitrofuran metabolite derivatives, the chromatography run was divided into three segments for data acquisition. Table 1 lists SRM transitions and their parameters in each segment.
Chromatography Conditions
Analytical column: Thermo Scientic Hypersil GOLD, 5 m, 100 2.1 mm Eluent: A: 0.5 mM Ammonium Acetate in Water; B: Methanol

Figure 2 shows representative chromatograms of a 0.050 g/kg fortied crawsh sample. As shown, all four nitrofuran metabolite derivatives were detected with excellent signal quality as measured by signal-to-noise (S/N) ratio.
The LOQ values reported in literature, ranging from 0.02 to 0.1 g/kg for different nitrofuran metabolites in meat samples, have mostly been obtained from extrapolation based on S/N =10 of the signals of analytes at higher concentrations in standards or fortied samples. In reality, however, these extrapolated LOQs often cannot be achieved, because the S/N ratios of the signals deteriorate more than as predicted by the dilution factor. The current data in Figure 2 demonstrates that all four nitrofuran metabolites can be detected in fortied crawsh samples at 0.05 g/kg level, far lower than the MRPL value of 1 g/kg required by EU. Figure 3 shows seven-point calibration curves constructed from data from measuring standard solutions at concentration levels of 0.025, 0.2, 0.5, 1, 2.5, 5.0 and
Segment
Time (min)
Analyte
Parent Ion (m/z)
Product Ions (m/z)
CE (V)
07.4
NBAOZ d4-NBAOZ (IS) NBAHD

13
7.48.5
C3-NBAHD (IS) NBSEM
8.514.5
(13C, 15N2)-NBESEM (IS) NBAMOZ d5-NBAMOZ (IS)
236.045 236.045 240.037 249.040 249.040 252.037 209.000 209.000 212.048 335.092 335.092 340.134
104 134* 134 104 134* 134 166* 192 168 291* 262 296
19 22 14 22 14 14 11 13 11 12 19 12
Table 1: Segments of chromatography separation and SRM transitions

Note: * SRM transition for quantitation; IS : internal standard; CE: Collision Energy. For each segment, Scan Time (s) = 0.1 and Scan Width (m/z) = 0.002.
NBAHD
100
TIC
0
RT: 6.99 AA: 9031 SN : 352
NL: 1.70E3
NBSEM
100
TIC
0
RT: 7.82 AA: 31604 SN : 95
NL: 4.71E3
100
249>134
0
RT: 6.98 AA: 6490 SN : 502
NL: 1.21E3
100
209>166
0
RT: 7.88 AA: 14778 SN : 104
NL: 2.20E3
100
249>104
0
RT: 7.00 AA: 2542 SN : 187
NL: 5.62E2
100
209>192
0
RT: 7.82 AA: 17214 SN : 69
NL: 2.77E3
100
252>134
0 5 6
RT: 6.99 AA: 5915 SN : 69
NL: 1.07E3
100
212>168
0
RT: 7.88 AA: 13803 SN : 95
NL: 2.48E3
7 Time (min)
10
7 Time (min)
10
NBAOZ
100
TIC
0
RT: 7.13 AA: 38204 SN : 289
NL: 5.74E3
NBAMOZ
100
RT: 9.23 AA: 290684 SN : 284
NL: 4.59E4
TIC
0 RT: 9.23 AA: 55037 SN : 1014
NL: 7.88E3
100
236>134
0
RT: 7.13 AA: 26470 SN : 239
NL: 3.94E3
100
335>291
0
100
236>104
0
RT: 7.13 AA: 11734 SN : 344
NL: 1.80E3
100
335>262
0
RT: 9.22 AA: 234181 SN : 227
NL: 3.81E4
100
240>134
0 5 6
RT: 7.05 AA: 39116 SN : 869
NL: 5.81E3
100
340>296
0
RT: 9.13 AA: 114339 SN : 213
NL: 1.87E4
7 Time (min)
10
7 Time (min)
10
Figure 2: Chromatograms of shrimp meat sample containing 0.050 g/kg fortied nitrofurans and internal standard in the shrimp meat. For each panel from the top: TIC (total ion current), SRM for quantitation (bold and red) and conrmation (green), and internal standard (italic and blue). RT: retention time, AA: peak area counts, SN: signal-to-noise ratio.
NBAHD
Y = 0 .0 8 5 0 2 6 8 + 1 .0 6 8 5 7 *X R ^2 = 0 .9 9 5 6 W: 1 /X 7 10 6 8 5 4 3 2 2 0 0 2 4 pp b 6 8 10 1 0 0 2 4 6 8 10
NBAOZ
Y = 0 .0 5 1 7 6 0 3 + 0 .6 9 2 1 1 8 *X R ^2 = 0 .9 9 8 2 W : 1 /X
Area Ratio
6 4
Area Ratio
NBSEM
Y = 0 .0 5 6 2 9 1 2 + 1 .0 4 3 2 1 *X R ^2 = 0 .9 9 7 2 W : 1 /X 20 10 8 15
NBAMOZ
Y = 0 .1 9 2 4 3 5 + 1 .9 2 7 5 7 *X R ^2 = 0 .9 9 8 2 W : 1 /X
6 4 2 0 0 2 4 6 8 10
10
0 0 2 4 6 8 10
Figure 3: Seven-point calibration curves of nitrofuran metabolite, from 0.025 ng/mL (equivalent to 0.05 ng/mL after sample preparation) to 10 ng/mL
10.0 ng/mL. Excellent linearity was obtained for all four nitrofuran metabolites within the calibration range, with the correlation coefcient R2 > 0.995 (weight factor = 1/X). The method accuracy and precision were evaluated by performing triplicate preparation and analysis of one batch of homogenized crawsh meat samples fortied with nitrofuran metabolites at three different concentration levels of 0.05, 0.5 and 2.5 g/kg. The results are given in Table 2. As shown, recovery values in the range of 79%110% were obtained with standard deviations from 3 to 22%. It should be noted that standard deviations include the errors of both the sample preparation (major contributor) and analytical instrument.
Fortication Level (g/kg) 0.05 0.5 2.5 AHD 82 13% 88 4% 109 3% AOZ 110 22% 100 11% 86 18% SEM 89 15% 100 11% 79 19% AMOZ 98 14% 95 6% 100 3%
Conclusions
With use of the TSQ Quantum Discovery MAX, a sensitive and reliable LC-MS/MS method using SRM has been developed for detecting trace level nitrofuran metabolites with a quantitation limit of less than 0.050 g/kg in crawsh. The sample preparation procedure is relatively straightforward and setup of the instrument method is easy and fast.
References
1. US FDA: Detection of Nitrofuran Metabolites in Shrimp, http://www.cfsan.fda.gov/~comm/methnf.html 2. A. Leitner et al., Journal of Chromatography A, 939 (2001) pp 49-58. 3. Seu-Ping Khong et al., J. Agric. Food Chem. 2004, 52 pp 5309-5315.
Table 2: Mean recovery values (n=3) of crawsh samples fortied at three levels
Note: values given after are standard deviations.
Area Ratio
Area Ratio
www.thermo.com
AN62492_E 11/07S
Highly Selective Detection and Identication of Nitrofuran Metabolites in Honey Using LC-MS/MS
Eduardo Matus,1 Jean-Jacques Dunyach,2 Alejandro Albornoz3;
1
Food Science Laboratories, Buenos Aires, Argentina; 2Thermo Fisher Scientic, San Jose, CA; 3Eidomet, Buenos Aires, Argentina
Introduction
Key Words TSQ Quantum Discovery LC-MS/MS Quantitative Analysis Nitrofurans are broad-spectrum antibiotics used to treat bees and other animals with bacterial infections. As a result of dosing bees with these antibiotics their metabolites are sometimes found in honey. Female rats given nitrofurans in both low and high doses have exhibited increased incidences of ovarian granulose cell tumors. In the same study, newborn mice showed an increased incidence of pulmonary papillary adenomas.2 As a result, nitrofurans have been banned from use in food-producing animals in Australia (1993), the European Union (EU) (1995), The Philippines (2001), the United States (2002), Brazil (2002), Thailand (2002), and other countries. Several studies have shown that animals rapidly metabolize nitrofurans within a few hours so detection has focused on the metabolites rather than the native drug.3 The metabolites accumulate in tissue where they are stable and can be analyzed long after the nitrofurans have been administered. The EU has established a harmonized minimum required performance limit (MRPL) for the detection of residues of nitrofurans at one part per billion (ppb). Some European laboratories have been working to a detection limit of 0.3 ppb for several of the nitrofuran metabolites.4 The EU recently tightened its inspection policy for food imports after nitrofuran residues were found in shrimp, sh, and poultry imports. This signicantly reduced the volume of those imports. As a result, food exporting countries are required to detect nitrofuran metabolites at very low levels. There are several challenges that must be overcome. The rst is that honey, as well as other food products, provides a complex matrix which increases the difculty of sample preparation. Second, efcient chromatography is critical in order to provide good separation of the various metabolites from each other and any contaminants that might be present. The third and most important requirement is a very high level of sensitivity and linearity in the mass spectrometer in order to achieve the required high levels of accuracy in quantifying the metabolites. This note describes LC-MS methods developed on the Thermo Scientic TSQ Quantum Discovery by the Food Science Laboratories and Eidomet in Argentina in cooperation with Thermo Fisher Scientic. The method exceeds all current detection limits as set by the EU.
Goal
To demonstrate the ability to accurately quantitate nitrofuran metabolites at levels as low as 0.3 ppb in a matrix consisting of honey using the TSQ Quantum Discovery.
In this study, 2 grams of honey samples were treated with four nitrofuran metabolites, AOZ, AMOZ, SEM, and AHD.5 An aliquot of honey was dissolved in 125 mM HCl and derivatized with 2-nitrobenzaldehyde and the mixture was shaken for 3 minutes. The slurry was then incubated at 37C in a water bath for 17 hours. The mixture was then cooled to room temperature and neutralized by adding potassium phosphate to adjust the pH to approximately 7.0. Ethyl acetate was added to the slurry and it was hand shaken for 2 minutes and centrifuged for 15 minutes. The organic phase was collected into a tube, water added, and the mixture centrifuged. The supernatant was evaporated to dryness under a stream of nitrogen. The dry residue was reconstituted with water and injected into a lter cartridge. The residue was then washed with water and eluted with hexane, then analyzed by LC-MS/MS. HPLC was performed on a Thermo Scientic Surveyor MS Pump with a Thermo Scientic Surveyor Autosampler. A 100 2.1 mm, 3 m HPLC column was used. The mobile phase consisted of A (water containing 0.05% acetic acid) and B (methanol containing 0.05% acetic acid). The gradient program was as follows: 0-3.0 min. 90% A 10% B; 3.0-5.0 min. 85% A 15% B; 5.0 to 10.0 min. 75% A 25% B; 10.0-15.0 min. 70% A 30% B; 15.017 min. 65% A 35% B; 15-17 min. 65% A 35% B; 17.021.0 min. 60% A 40% B; and 21.0-25.0 min. 90% A 10% B. Sample analysis was performed on a TSQ Quantum Discovery mass spectrometer. The 0-13.4 min segment eluted AMOZ and d5-AMOZ while the 13.4 to 25 min segment eluted AOZ, d4-AOZ, SEM, and AHD. Samples were analyzed using positive electrospray ionization (ESI) in SRM mode. The scan width was 0.002 m/z and the scan time was 0.1 second. A peak width of 0.7 FWHM was used in both Q1 and Q3. Argon was used as the collision gas at a pressure of 1.5 mTorr.
Analyte
Precursor Ion
Product Ion
Collision Energy
AMOZ d5-AMOZ AOZ d4-AOZ SEM AHD
335 335 340 236 236 240 209 209 249 249
262 291 296 78 134 134 166 192 104 134
15 10 10 15 8 8 6 5 16 7
the average of results from a series of injections at a single concentration. The method is generally considered to be validated if the RSD is less than 15%. The recovery ratio was also calculated by injecting a known amount of sample and comparing it with the calculated amount delivered by the detector.

Tables 2 through 5 report the results obtained with the standard and QC samples in honey for each metabolite. Note that for clarity purposes, all areas reported in the tables are divided by 1000. The concentrations of the QC samples were calculated by comparing the area to the standards. Then the relative standard deviation for each set of QC samples was calculated. The RSD for AOZ ranged from 6.7% at 0.3 ppb to 2.7% at 4 ppb. The RSD for AMOZ ranged from 3.60% at 0.3 ppb to 2.50% at 4 ppb. The RSD for AHD ranged from 9.0% at 0.3 ppb to 2.9% at 4 ppb. The RSD for SEM ranged from 8.3% at 0.3 ppb to 4.2% at 4 ppb.
Table 1: Transition reactions for MS/MS
The nitrofuran metabolites were quantied with ve calibration standards at nominal concentrations of 0.63 ppb, 1.04 ppb, 2.09 ppb, 4.17 ppb, and 8.34 ppb. The area ratio of the analyte versus the quality control (QC) samples was plotted against the standard concentration ratio. The linearity of the MS response was determined by calculating the relative standard deviation (RSD) of
Equation Y = 0.5424 X + 0.0973
R2 = 0.9998
IDENT. LEVEL Std 0,6 ppb Std 1 ppb Std 2 ppb Std 4 ppb Std 8 ppb
NOMINAL CONC. (ppb) 0.63 1.04 2.09 4.17 8.34 CALCULATED CONC. (ppb) 4.204 4.222 4.253 4.155 4.451 2.111 2.252 1.938 2.166 2.150 1.068 1.038 1.017 1.138 1.112 0.556 0.567 0.565 0.477 0.513 0.335 0.280 0.304 0.293 0.300
AREA AOZ 217.3 344.1 671.2 1286.3 2601.2 Diff %
AREA ISTD (d4-AOZ) 559 524.3 532.3 567.2 580.6 RSD %
AREA RATIO 0.39 0.66 1.26 2.27 4.48
IDENT. LEVEL
AREA AOZ
AREA ISTD (d4-AOZ) 356.5 381.1 392.6 378.7 389.7 372.9 370.1 358.8 377.7 375.5 356.7 390.9 359.3 358.6 346.4 376.1 388.1 366.4 372.2 380.6 357.3 369.5 341.0 412.6 369.4
AREA RATI
SPECIFIED CONC. (ppb) 4.172 4.172 4.172 4.172 4.172 2.086 2.086 2.086 2.086 2.086 1.043 1.043 1.043 1.043 1.043 0.521 0.521 0.521 0.521 0.521 0.313 0.313 0.313 0.313 0.313
RECOVERY %
QC-4ppb QC-4ppb QC-4ppb QC-4ppb QC-4ppb QC-2ppb QC-2ppb QC-2ppb QC-2ppb QC-2ppb QC-1ppb QC-1ppb QC-1ppb QC-1ppb QC-1ppb QC-0.5ppb QC-0.5ppb QC-0.5ppb QC-0.5ppb QC-0.5ppb QC-0.3ppb QC-0.3ppb QC-0.3ppb QC-0.3ppb QC-0.3ppb
1609.6 1728.1 1849.5 1743.8 1919.4 864.7 912.9 789.1 924.2 912.2 436.6 466.2 431.3 477.7 451.5 258.3 271.1 260.2 228.8 249.0 162.6 146.9 145.5 171.5 156.0
4.52 4.53 4.71 4.60 4.93 2.32 2.47 2.20 2.45 2.43 1.22 1.19 1.20 1.33 1.30 0.69 0.70 0.71 0.61 0.65 0.46 0.40 0.43 0.42 0.42
0.77% 1.20% 1.94% -0.41% 6.69% 1.20% 7.96% -7.09% 3.84% 3.07% 2.40% -0.48% -2.49% 9.11% 6.62% 6.72% 8.83% 8.45% -8.45% -1.54% 7.03% -10.54% -2.88% -6.39% -4.15%
2.7%
5.5%
4.7%
7.4%
6.7%
100.8 101.2 101.9 99.6 106.7 101.2 108.0 92.9 103.8 103.1 102.4 99.5 97.5 109.1 106.6 106.7 108.8 108.4 91.6 98.5 107.0 89.5 97.1 93.6 95.8
Table 2: AOZ data
Page 2 of 6
Equation Y = 0.6123 X + 0.0413
R2 = 1.0000
NOMINAL CONC. (ppb) 0.6 1.0 2.0 4.0 8.0 CALCULATED CONC. (ppb) 4.200 4.040 3.938 4.017 3.974 2.000 2.100 1.914 1.991 1.959 1.010 1.020 0.967 0.952 0.981 0.517 0.482 0.489 0.455 0.477 0.314 0.314 0.298 0.295 0.291
AREA AMOZ 760.3 1320.8 2811.8 5582.3 11265.8 Diff %
AREA ISTD (d5-AMOZ) 1799.0 1936.8 2170.0 2166.6 2231.2 RSD %
AREA RATIO 0.40 0.69 1.37 2.68 5.32
IDENT. LEVEL
AREA AMOZ
AREA ISTD (d5-AMOZ) 1700.1 1376.6 1894.0 1464.6 1439.7 1246.4 1511.5 1477.9 1263.0 1662.7 1357.0 1687.2 1476.0 1853.7 1781.8 1710.4 1345.0 1602.7 1331.6 1640.8 1289.1 1139.9 1309.3 1719.1 1264.3
AREA RATIO
RECOVERY %
8814.0 6866.9 9431.2 7438.6 7235.0 3117.9 3951.3 3617.4 3214.0 4164.8 1742.6 2178.9 1864.6 2307.9 2281.8 1153.4 850.2 1067.5 830.8 1067.2 548.5 485.0 559.5 727.5 529.3
5.18 4.99 4.98 5.08 5.03 2.50 2.61 2.45 2.54 2.50 1.28 1.29 1.26 1.25 1.28 0.67 0.63 0.67 0.62 0.65 0.43 0.43 0.43 0.42 0.42
5.00% 1.00% -1.55% 0.43% -0.65% 0.00% 5.00% -4.30% -0.45% -2.05% 1.00% 2.00% -3.30% -4.80% -1.90% 3.40% -3.60% -2.20% -9.00% -4.60% 4.67% 4.67% -0.67% -1.67% -3.00%
2.50%
3.45%
2.90%
4.63%
3.60%
105.0 101.0 98.5 100.4 99.4 100.0 105.0 95.7 99.6 98.0 101.0 102.0 96.7 95.2 98.1 103.4 96.4 97.8 91.0 95.4 104.7 104.7 99.3 98.3 97.0
Table 3: AMOZ data
Equation Y = 0.1396 X + 0.0174
R2 = 0.9955
NOMINAL CONC. ( ppb ) 0.61 1.02 2.03 4.06 8.13 CALCULATED CONC. (ppb) 3.585 3.543 3.559 3.799 3.638 1.683 1.711 1.726 1.785 1.672 0.919 0.857 0.941 0.843 0.954 0.509 0.425 0.502 0.427 0.433 0.251 0.249 0.309 0.281 0.278
AREA AHD 70.9 112.2 200.3 416.4 797.2 Diff %
AREA ISTD (d4-AOZ) 559.0 524.3 532.3 567.2 580.6 RSD %
AREA RATIO 0.127 0.210 0.380 0.730 1.370
IDENT. LEVEL
AREA AHD
AREA ISTD (d4-AOZ) 356.5 381.1 392.6 378.7 389.7 372.9 370.1 358.8 377.7 375.5 356.7 390.9 359.3 358.6 346.4 376.1 388.1 366.4 372.2 380.6 357.3 369.5 341.0 412.6 369.4
AREA RATIO
RECOVERY %
436.6 461.5 397.0 408.3 402.6 222.6 224.4 179.1 194.8 181.8 123.0 126.8 100.6 90.6 98.3 78.7 70.6 57.7 50.8 52.6 44.5 45.8 35.4 39.6 35.1
1.22 1.21 1.01 1.08 1.03 0.60 0.61 0.50 0.52 0.48 0.34 0.32 0.28 0.25 0.28 0.21 0.18 0.16 0.14 0.14 0.12 0.12 0.10 0.10 0.10
-11.76% -12.80% -12.40% -6.50% -10.46% -17.26% -15.88% -15.14% -12.24% -17.80% -9.64% -15.73% -7.47% -17.11% -6.19% 0.20% -16.34% -1.18% -15.94% -14.76% -17.70% -18.36% 1.31% -7.87% -8.85%
2.9%
2.6%
5.5%
9.2%
9.0%
88.2 87.2 87.6 93.5 89.5 82.7 84.1 84.9 87.8 82.2 90.4 84.3 92.5 82.9 93.8 100.2 83.7 98.8 84.1 85.2 82.3 81.6 101.3 92.1 91.1
Table 4: AHD data
Page 3 of 6
Equation Y = 0.1396 X + 0.0174
R2 = 0.9955
NOMINAL CONC. ( ppb ) 0.61 1.02 2.03 4.06 8.13 CALCULATED CONC. (ppb) 3.585 3.543 3.559 3.799 3.638 1.683 1.711 1.726 1.785 1.672 0.919 0.857 0.941 0.843 0.954 0.509 0.425 0.502 0.427 0.433 0.251 0.249 0.309 0.281 0.278
AREA AHD 70.9 112.2 200.3 416.4 797.2 Diff %
AREA ISTD (d4-AOZ) 559.0 524.3 532.3 567.2 580.6 RSD %
AREA RATIO 0.127 0.210 0.380 0.730 1.370
IDENT. LEVEL
AREA AHD
AREA ISTD (d4-AOZ) 356.5 381.1 392.6 378.7 389.7 372.9 370.1 358.8 377.7 375.5 356.7 390.9 359.3 358.6 346.4 376.1 388.1 366.4 372.2 380.6 357.3 369.5 341.0 412.6 369.4
AREA RATIO
RECOVERY %
436.6 461.5 397.0 408.3 402.6 222.6 224.4 179.1 194.8 181.8 123.0 126.8 100.6 90.6 98.3 78.7 70.6 57.7 50.8 52.6 44.5 45.8 35.4 39.6 35.1
1.22 1.21 1.01 1.08 1.03 0.60 0.61 0.50 0.52 0.48 0.34 0.32 0.28 0.25 0.28 0.21 0.18 0.16 0.14 0.14 0.12 0.12 0.10 0.10 0.10
-11.76% -12.80% -12.40% -6.50% -10.46% -17.26% -15.88% -15.14% -12.24% -17.80% -9.64% -15.73% -7.47% -17.11% -6.19% 0.20% -16.34% -1.18% -15.94% -14.76% -17.70% -18.36% 1.31% -7.87% -8.85%
2.9%
2.6%
5.5%
9.2%
9.0%
88.2 87.2 87.6 93.5 89.5 82.7 84.1 84.9 87.8 82.2 90.4 84.3 92.5 82.9 93.8 100.2 83.7 98.8 84.1 85.2 82.3 81.6 101.3 92.1 91.1
Table 5: SEM data
Figure 1 shows the chromatograms of a negative and a positive unknown sample set in which the AOZ metabolite is clearly identied at the 0.6 ppb level. Table 6 summarizes the average method results for the four metabolites over multiple sample sets. The limits of detection (LODs) and
limits of quantication (LOQs) are reported and the data shows good accuracy at the LOQ levels for all the metabolites. The LODs and LOQs achieved on the four nitrofuran metabolites are all at sub ppb levels.
Time (min)
Time (min)
Figure 1: Identication of AOZ metabolite in honey at 0.6 ppb level
Page 4 of 6
ANALYTE AMOZ AHD AOZ SEM
MATRIX Honey Honey Honey Honey
LOD
(ppb)
LOQ
(ppb)
ANALYTICAL RANGE
(ppb)
% RECOVERY 99.3 88.5 101.2 94.3
RANGE % REC 88,8-109.8 71.2-105.8 84.2-118.5 70.6-117.8
CV % 3.5 6.5 5.6 8.3
INCERTITUDE % 7.1 13.0 11.3 16.6
0.04 0.06 0.06 0.08
0.09 0.16 0.14 0.18
0,04-4,0 0,06-4,063 0,06-4,172 0,08-4,064
Table 6: Summary of Method Results
Conclusion
An LC-MS/MS assay to detect and identify nitrofuran metabolites was developed using the TSQ Discovery. The extraction method appears to be extremely robust and reliable with good recovery efciency (better than 80%), allowing unambiguous routine identication and quantication of all nitrofuran metabolites in honey. The LCMS/MS-based method described here provides high speed, excellent sensitivity, and specicity of detection. The assay demonstrated the ability to easily meet the 0.3 ppb limit of quantitation that is required by the most stringent current requirements of food monitoring applications operating under FDA and EC regulations.
References
1
Proceedings of the National Academy of Sciences of the United States of America. Vol 73 No 10. pp 3386-3390. Nitrofurans, a Group of Synthetic Antibiotics, with a New Mode of Action: Discrimination of Specic Messenger RNA Classes. Peter Herrlich, Manfred Schweiger NTP (1988). Toxicology and carcinogenesis studies of nitrofurazone (CAS No. 59-87-0) in F344/N rats and B6C3F1 mice. Technical Report Series No. 337. US Department of Health and Human Services, Public Health Service/National Institutes of Health National Toxicology Program. Journal of Chromatography. 1997 Vol 691 pp 87-94. Determination of the furazolidone metabolite, 3-amino-2-oxazolidinone in porcine tissues using liquid chromatography-thermospray mass spectrometry and the occurrence of residues in pigs produced in Northern Ireland. R.J. McCracken, D.G. Kennedy. Food Standards Agency. June 30, 2003. Reporting limits for nitrofuran and chloramphenicol residues harmonized. Determination of Nitrofurans Residues in Honey by LC-MS/MS, Analysis Method, Food Science Laboratories, 2005.
Page 5 of 6

Tap our expertise throughout the life of your instrument. As an industry leader in analytical instruments, Thermo extends its support throughout our worldwide network of Thermo-trained and certied engineers who are experts in laboratory technologies and applications. Put our team of experts to work for you in a range of disciplines, from system installation, training and technical support, to complete asset management and regulatory compliance consulting. Improve your productivity and lower the cost of instrument ownership through our product support services. Maximize uptime while eliminating the uncontrollable cost of unplanned maintenance and repairs. When its time to enhance your systems,Thermo also offers certied parts and a range of accessories and consumables suited to your application. To learn more about our products and comprehensive service offerings, visit our Web site at www.thermo.com.
www.thermo.com

AN62490_E 10/07S
LC-MS/MS Analysis of Malachite Green, Leucomalachite Green, Ciprooxacin, and Tetracycline in Food Samples using a TurboFlow Method
Charles Yang and Dipankar Ghosh, Thermo Fisher Scientic, San Jose, CA, USA
Introduction
Key Words Aria TLX System Food Residue Analysis TSQ Quantum Access TurboFlow Technology Veterinary Drugs Accurate monitoring of chemical residue levels in food and agriculture products is essential to assure the safety of the food supply and manage global health risks. The analysis of chemical residues requires techniques sensitive enough to detect and quantify contaminants at or below the maximum residue limit (MRL) of the compound in a given sample matrix. In addition, because of increased food safety regulations and the growing numbers of samples to be analyzed, it is critical that the analytical techniques provide high sample throughput. With the continuing rapid growth of the aquaculture industry, there is increasing concern about the use of unapproved drugs and unsafe chemicals in aquafarming operations. Malachite green (MG), a triphenylmethane dye, is an effective and inexpensive fungicide used in aquaculture, particularly in Asian countries (Figure 1).
Figure 1: Structures of malachite green and leucomalachite green
During metabolism, malachite green reduces to leucomalachite green (LMG), which has been shown to accumulate in fatty sh tissues and can be found long after MG may no longer be detected.1 Both MG and LMG have demonstrated putative carcinogenic activity, and thus MG has been banned for use as an aquaculture veterinary drug in many countries including the United States and Canada, as well as the European Union (EU). For substances that are banned from use in food producing animals, EU legislation denes minimum required performance limits. For malachite green, an analytical test method must be able to determine the sum of MG and LMG residues in sh muscle at the minimum required performance limit of 2 g/kg (ppb).2 Ciprooxacin is a broad-spectrum antibiotic belonging to the uoroquinolone group (Figure 2). Fluoroquinolones have been shown to be very effective in combating various diseases in animal husbandry and aquaculture and are used extensively worldwide. However, because of concerns that uoroquinolone residues in food products may lead
to the development of antibacterial resistance to these drugs in humans, the FDA has prohibited extra-label use of uoroquinolones in food Figure 2: Structure of Ciprooxacin animals.3 According to the EU legislation on veterinary drug residues, the maximum residue limits for the sum of enrooxacin and its metabolite ciprooxacin are 100 g/kg (ppb) in muscle for all food producing species and 200 g/kg (ppb) in pork liver.4 Tetracycline is a polyketide antibiotic that is highly effective against a number of gram-positive and gramnegative bacteria (Figure 3). As with other veterinary antibiotics, when tetracycline is used in food animals, it has the potential to generate drug residues in the animals and animal products which can lead to increases in microbial resistance. The MRLs for tetracycline are 100 g/kg (ppb) in muscle and 300 g/kg (ppb) in liver for all food producing Figure 3: Structure of Tetracycline species.5 Here we show a solution that combines the Thermo Scientic Aria TLX system utilizing TurboFlow technology with a Thermo Scientic TSQ Quantum Access mass spectrometer. Compared to traditional ofine extraction methods, this solution provides fast and reliable sample analysis of chemical residues in food by online sample extraction followed by LC-MS/MS. The Aria TLX system uses TurboFlow technology to retain small molecules and lter out proteins and larger materials by diffusion, size exclusion, and column chemistry. This enables users to directly inject samples into the LC-MS system for analysis, greatly simplifying sample preparation and increasing throughput.
Goal
To demonstrate a reduction in overall analytical time compared to traditional methods, such as liquid-liquid or solid phase extraction, while also minimizing ion suppression and matrix interference in food samples.
Sample Preparation
TurboFlow Method Conditions
Samples were prepared for analysis using a simple extraction procedure with acetonitrile. Individual samples, weighing 3 to 4 g, of shrimp (with head attached), tilapia, and pig liver were homogenized with approximately 30 mL of acetonitrile and then left at room temperature for 10 minutes. The liquid phase was then aspirated from the sample into a 0.22 m pore centrifuge tube and spun at 10,000 rpm for approximately 10 minutes. The supernatant was aspirated into a 50 mL scintillation vial. This portion of the sample preparation took 25 minutes. A stock mix solution of malachite green, leucomalachite green, ciprooxacin, and tetracycline was prepared at a concentration of 1 mg/L. All four analytes were mixed in one vial at 0.1 mg/mL (1000 g/mL) in methanol. Calibration solutions in the concentration range 10 g/kg to 5 ng/kg were prepared by serial dilution of the stock solution into the three sample matrices. The total sample preparation time was approximately 30 to 40 minutes.
The samples were processed on the Thermo Scientic Aria TLX-1 System. The Multiple Column Module (MCM), which allows 6 loading columns and 6 analytical columns to be tested at once, was used to facilitate method development. First, the loading column that gave the best recovery of each analyte was selected. Because all of the analytes were in one stock solution, the run time was minimized. Then the analytical column that gave the best performance was selected. The nal TurboFlow method conditions were as follows:
Loading Column: Analytical Column: Thermo Scientic TurboFlow XL C18 column Thermo Scientic Hypersil GOLD 50 x 3 mm 5 m column Mobile Phase A: 0.1% formic acid in water Mobile Phase B: 0.1% formic acid in acetonitrile Mobile Phase D: 20% acetone/40% methanol/40% acetonitrile Autosampler Injection Size: 10 L Sample Extraction Solution: 50:50 (A/B)
The Aria TLX TurboFlow method is shown in Figure 4. The analytical run was completed in less than 6 minutes.
Figure 4: Aria TLX method
MS Conditions

As the international food trade continues to grow, so does the need for careful monitoring of the food supply to ensure that levels of drug residues and other chemical contaminants are below established standards. In this study, ciprooxacin, tetracycline, and malachite green/leucomalachite green were examined in several food matrices. LC-MS/MS assays of chemical residues in food matrices typically require extensive sample preparation prior to analysis, which can be time consuming and expensive. The Aria TLX system with TurboFlow technology eliminates the need for lengthy sample preparation steps. In this study, each sample was centrifuged once to clean out any oating particles and then immediately injected onto the column. Most of the time-consuming steps in the sample preparation process were removed, which increased sample throughput and helped to minimize errors and variability. The Aria TLX system software allows both HPLC and TurboFlow methods to be run on a single system, injection to injection. To evaluate the differences in performance between a standard HPLC
MS analysis was carried out on a Thermo Scientic TSQ Quantum Access triple stage quadrupole mass spectrometer. The MS conditions were as follows:
Ion Source Polarity: Spray Voltage: Vaporizer Temperature: Sheath Gas Pressure (N2): Auxiliary Gas Pressure (N2): Ion Transfer Tube Temperature: Skimmer Offset: Collision Gas (Ar): Q1/Q3 Peak Resolution: Scan Mode: Positive ion mode 3500 V 472 C 40 arb 50 arb 270 C 5V 1.5 mTorr 0.7 u (unit mass resolution) Selected Reaction Monitoring
The MS method is shown in Figure 5.
Figure 5: MS method showing the SRM transitions that were monitored
method and a TurboFlow method, each sample was analyzed by both methods. In the standard HPLC method, only the analytical HPLC column was used. In the TurboFlow method, the TurboFlow column and the analytical column were used. Figure 6 compares representative standard HPLC and TurboFlow method chromatograms of 500 ng/kg (parts per trillion) tetracycline in the sh matrix. The
TurboFlow method chromatogram shows that interferences present in the standard HPLC chromatogram have been removed. The turbulent ow properties successfully remove matrix interferences that cause ion suppression. Similar results were observed in the analysis of drugs in other food matrices. Figure 7 compares representative standard HPLC and TurboFlow method chromatograms
Figure 6: Chromatogram comparison of tetracycline at 500 ng/kg in sh (tilapia) matrix in standard HPLC and TurboFlow method
Figure 7: Chromatogram comparison of LMG at 500 ng/kg and 50 ng/kg in shrimp matrix in standard HPLC and TurboFlow method
for leucomalachite green at both 500 ng/kg (ppt) and 50 ng/kg (ppt) in the shrimp matrix. The TurboFlow method chromatograms show improved signal-to-noise ratios and signicant reduction in ion suppression. The most dramatic results are shown in the analysis of tetracycline at 500 ng/kg (ppt) in pig liver (Figure 8). Peak areas were used for quantitation and the resultant linearity of responses is plotted in Figure 9 for both the standard HPLC and the TurboFlow methods for ciprooxacin in pig liver. Excellent R2 values were observed for the TurboFlow method. A data summary showing the improvement in the TLX system results over those of
the standard HPLC results is shown in Table 1. The R2 values for the TLX system results were all greater than 0.99 for the linear regression equations (1/x weighted) in the concentration ranges tested. Table 1 shows the results of the assay for ciprooxacin, MG, LMG, and tetracycline in sh, shrimp, and pork liver extracts. The limits of quantitation (LOQs) achieved for all four analytes using online extraction followed by LC-MS/MS were signicantly better compared to that achieved by standard HPLC. This indicates the removal of endogenous tissue by the Aria TLX system, thus reducing ion suppression effects and increasing detection limits.
Figure 8: Chromatogram comparison of tetracycline at 500 ng/kg in pig liver matrix in standard HPLC and TurboFlow method Figure 9: Ciprooxacin calibration 1/x on standard HPLC vs. TurboFlow method in pig liver matrix
Fish
LOD (g/kg) 0.5 0.1 0.5 5.0 0.1 0.1 0.5 0.5 %RSD n=3 32.0 23.0 7.4 10.5 21.4 20.5 38.8 14.8 Standard HPLC LOQ %RSD (g/kg) n=3 1.0 8.0 0.5 1.4 0.5 7.4 5.0 0.5 0.5 1.0 1.0 10.5 7.2 11.0 10.4 10.5 LOD (g/kg) 0.1 0.1 0.1 0.5 0.05 0.05 0.1 0.1 TurboFlow Method %RSD LOQ %RSD n=3 (g/kg) n=3 15.9 0.5 7.1 6.8 0.1 8.2 12.0 0.1 12.0 16.0 7.9 13.5 29.0 11.5 1.0 0.05 0.1 0.5 0.5 2.0 7.9 11.2 8.6 11.3
R2 0.9968 0.9984 0.9983 0.9906 0.9990 0.9988 0.9969 0.9953
Ciprooxacin MG LMG Ciprooxacin MG LMG Ciprooxacin Tetracycline
0.9875 0.9988 0.9990 0.9580 0.9991 0.9975
Shrimp
Pig Liver
0.9707 0.9932
Table 1: Data summary showing the improvement in the TurboFlow method results over those of the standard HPLC results. Note that results are only shown for a compound in the matrix in which it would be found; for example, MG and LMG would be found in sh but not in pig liver.
Conclusion
A rapid, sensitive and reliable method for the quantitation of veterinary drugs in food matrices was developed using the Aria TLX-1 system with the TSQ Quantum Access mass spectrometer. Minimal sample preparation was required because the TurboFlow method allows direct injection of samples into the system. The overall processing time for analysis was signicantly shortened compared to methods using ofine sample preparation. In addition, the Aria TLX system reduced ion suppression and matrix effects compared to standard HPLC runs.
References
1. Plakas, S. M.; Doerge, D. R.; Turnipseed, S. B. Disposition and Metabolism of Malachite Green and Other Therapeutic Dyes in Fish. In Xenobiotics in Fish; Smith, D. J., Gingerich, W. H., Beconi-Barker, M. G., Eds.; Plenum Press: New York City, 1999; p. 149-166. 2. European Commission, Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results, as amended by Decision 2003/181/EC(4), (Ofcial Journal of the European Communities L 221, 17.08.2002, p. 8-36). 3. U.S. Food and Drug Administration. Federal Register: May 22, 1997 (Volume 62, Number 99). 4. European Parliament and Council, Regulation (EC) 1181/2002 of 1 July 2002, amending Annex I of Council Regulation (EEC) No 2377/90 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin food (Ofcial Journal of the European Communities L 172, 2.07.2002). 5. European Parliament and Council, Regulation (EC) 508/1999 of 4 March 1999, amending Annex I of Council Regulation (EEC) No 2377/90 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin food (Ofcial Journal of the European Communities L 60, 9.3.1999).
www.thermo.com
AN62883_E 10/08M
Multi-class Antibiotic Screening of Honey Using Online Extraction with LC-MS/MS

Catherine Lafontaine, Yang Shi, Francois A. Espourteille, Thermo Fisher Scientific, Franklin, MA, USA
Introduction
Key Words Aria TLX-1 System TSQ Quantum Ultra Food Testing TurboFlow Method Antibiotics are commonly used in bee hives to control bacterial disease in honey bees. Use of these antibiotics requires caution to prevent persistent residues from occurring in food-grade honey. If antibiotic residues are present in sufficient quantities, allergic reactions and bacterial resistance can develop. Many countries now monitor antibiotic residues in honey. LC-MS/MS is currently a common analytical approach for the quantification of antibiotic contamination in honey. Sample preparation for LC-MS/MS analysis can be time and labor intensive, often involving pH modification, hydrolysis, liquid-liquid extraction, solid phase extraction, solvent evaporation, and pre-concentration. A quick, comprehensive, online screening liquid chromatography (LC) method using a Thermo Scientific Aria TLX system powered by Thermo Scientific TurboFlow technology has been developed here to monitor several classes of antibiotics.
Sample Preparation
A McIlvaine/0.1 M EDTA buffer was used as a 1:1 w:v (gram weight honey: milliliter volume buffer) diluent for wildflower honey, the testing matrix in this study.1 A stock solution was prepared for sulfapyridine, sulfathiazole, tilmicosin, tylosin, oxytetracycline, and erythromycin in 3:1 methanol:water at 100 g/mL. Additionally, one was prepared for doxycycline, demeclocycline, streptomycin, and dihydrostreptomycin in water at 100 g/mL. These stocks were each spiked into 1:1 honey:buffer matrix and used as a spiking stock to make a set of calibration standards and quality controls (QCs). All blanks, standards, and QCs were prepared and analyzed in polypropylene vials. Injection volumes were 0.050 mL.
Aria TLX-1: TurboFlow Method Parameters
TurboFlow Cyclone MAX and TurboFlow Cyclone-P columns (0.5 50 mm), in-tandem BETASIL Phenyl/Hexyl column, 100 3 mm, 3 m Aria operating system 1.6.2 software
Goal
To develop a broad, generic, automated LC-MS/MS method for screening multi-class antibiotics in honey.
Loading Pump Mobile Phases

Mobile Phase A: 1.0% formic acid in water Mobile Phase B: 0.1% formic acid in acetonitrile Mobile Phase C: 10 mM ammonium acetate in water, pH 9 Mobile Phase D: 50 mM ammonium acetate in methanol with 0.1% formic acid
Experimental
Method Information
Residues representative of several classes of antibiotics (macrolides, sulfonamides, aminoglycosides, and tetracyclines) were extracted from wildflower honey using buffer containing ethylenediaminetetraacetic acid (EDTA). The extract cleanup was accomplished using a TurboFlow method involving two TurboFlow columns placed in tandem, a mixed mode anion exchange column and a polar polymer-based column. Simple sugars were un-retained and moved to waste during the loading step while the analytes of interest were retained on the extraction column set. This was followed by organic elution to an end-capped silica-based mixed mode reversed phase analytical column (Thermo Scientific BETASIL Phenyl/Hexyl) and gradient elution to a Thermo Scientific TSQ Quantum Ultra triple stage quadrupole mass spectrometer with a Heated Electrospray Ionization (H-ESI) source operating in positive selective reaction monitoring (SRM) mode. The total LC-MS/MS method run time was less than 18 minutes. Positive SRM transitions and other MS parameters for individual analytes are shown in Table 1.
Elution Pump Mobile Phases

Mobile Phase A: 1 mM NFPA*, 0.5% formic acid, 0.04% TFA** in water Mobile Phase B: 0.5% formic acid, 0.04% TFA in 1:1 methanol:acetonitrile
*NFPA is nonafluoropentanoic acid. **TFA is trifluoroacetic acid.
TSQ Quantum Ultra Mass Spectrometer (MS) Parameters

Ion Polarity: Ionization Source: Spray Voltage: Vaporizer Temperature: Capillary Temperature: Sheath Gas Pressure (N2): Auxiliary Gas Pressure (N2): Ion Sweep Gas Pressure (N2): Skimmer Offset: Collision Pressure: Chrom Filter Peak Width: Scan Type: Scan Time: Scan Width: Peak Width Q1 Da. (FWHM): Peak Width Q3 Da. (FWHM): Positive H-ESI 4000 V 400 C 370 C 30 arb units 60 arb units 0.0 arb units 5 V (for streptomycin), 0 V (for all others) 1.2 mTorr 8.0 s SRM 0.020 s 0.100 m/z 0.700 0.700

Results were packaged using Thermo Scientific LCQUAN 2.5.6 data quantitation software and included subtraction of background due to the presence of a few endogenous analytes in the store-bought honey. Figure 1 shows a representative chromatogram of the 10 analytes at 100 ng/mL in 1:1 honey/buffer. Matrix-matched calibration standards showed linear response of two orders of magnitude (r2 > 0.99) for all of the analytes investigated (Table 2). All %CVs (n=3) were less than 19% for the lower limit of quantifications (LLOQ) and less than 8% for all other points of the curves. Figure 2 shows an LCQUAN representative linear regression using oxytetracycline as an example. QC sample variability was determined by processing and analyzing three replicates of each of four QC samples (2, 50, 100, and 500 ng/mL). All % RSDs were lower than 7% (except for erythromycin which was below 15%). Data was not used for any QC level that fell below the analytes determined LLOQ.
Structural Class
Sulfonamides Tetracyclines
Analyte
Sulfapyridine Sulfathiazole Doxycycline Oxytetracycline Demeclocycline Streptomycin Dihydrostreptomycin Erythromycin Tilmicosin Tylosin
Precursor Ion
250.1 256.1 445.3 461.2 465.2 582.3 584.3 734.5 869.6 916.5
Product Ions
156.0 (Q), 108.1 (C), 92.1 (C) 156.1 (Q), 92.0 (C), 108.1 (C) 154.0 (Q), 428.5 (C) 426.4 448.4 (Q), 430.4 (C) 263.0 (Q), 246.0 (C), 203.9 (C), 221.0 (C) 262.9 (Q), 245.9 (C) 576.2 696.3 772.3
Aminoglycosides Macrolides
NOTE: (Q)=Quantification Ion; (C)=Confirmation Ion.
Table 1: The 10 analytes and their positive SRM transition ions
Figure 1: Example chromatogram of 100 ng/mL calibration standard in 1:1 honey/buffer
Figure 2: LCQUAN view of oxytetracycline calibration curve and LLOQ (left window) vs. ULOQ (right window) chromatograms
Analyte
Sulfapyridine Sulfathiazole Doxycycline Oxytetracycline Demeclocycline Streptomycin Dihydrostreptomycin Erythromycin Tilmicosin Tylosin
R2 (1/x weighting)
0.9980 0.9988 0.9990 0.9990 0.9996 0.9960 0.9980 0.9877 0.9917 0.9958
Dynamic Range* (ng/mL)**

50-500 50-500 10-500 5-500 10-500 50-500 50-500 50-500 2-50 10-100
Limit of Detection (ng/mL)

10.0 10.0 5.0 2.0 5.0 10.0 10.0 10.0 0.5 5.0
Percent Carryover (%)

8.95 5.46 10.80 11.70 18.70 11.60 6.47 1.16 16.80 13.70
*Based on analysis using 8 point standard curve (ng/mL): 0.500, 2.00, 5.00, 10.0, 50.0, 100, 200, & 500. **The level of carryover was included in the determination of dynamic range (kept to 20% or less).
Table 2: Calibration curve statistics of the 10 analytes
Conclusion
During the honey quality monitoring process, it is always an analytical challenge to deal with a large number of antibiotics belonging to different classes. This often requires multiple LC-MS methods. In this study, a novel application was introduced using dual online TurboFlow extraction columns with different chemistries. The results reveal that this design facilitates the separation and quantification of all of the representative compounds in the complex honey matrix. Sample preparation time was minimal, requiring only the addition of a buffer to reduce sample viscosity. These factors enabled a broad screening for antibiotic contaminants to be performed quickly for a given sample, thus increasing sample throughput. Additionally, multiplexing with an Aria TLX-4 system would further reduce total LC-MS/MS run time four-fold and enable screening of 12 samples per hour. Future work could involve screening a larger range of antibiotic and environmental contaminants and lowering detection limits for all analytes thus combining a screening method with accurate quantification.
References
1. Qualitative Identification of Tetracyclines in Tissues, CLG SOP No: CLGTET2.01, Rev 01, p. 6, United States Department of Agriculture, Food Safety and Inspection Service, Office of Public Health Science, 9/25/03.
www.thermo.com

AN63043_E 05/09M
On-line Enrichment HTLC/MS/MS Assay for Multiple Classes of Antibiotics in Environmental Water Sources
Kevin J. McHale,1 Chris Esposito,2 and Francois Espourteille2
1
Thermo Fisher Scientic, Somerset, NJ, USA; 2Thermo Fisher Scientic, Franklin, MA, USA
Introduction
Key Words TSQ Quantum Ultra Aria TLX-2 H-SRM Turbulent-ow Extraction There is a growing concern over the presence of antibiotics in environmental sources of water. This has caused environmental and government labs to develop LC/MS methods to monitor water supplies for the presence of antibiotics.1-4 However, the low-level concentration of antibiotics in environmental water sources often requires extensive sample preconcentration and cleanup. Preparation of water samples (100-1000 mL) prior to LC/MS analysis, even with an unlimited sample volume, is time consuming and reduces sample throughput. This report presents a method that signicantly decreases sample preparation time by applying on-line preconcentration and extraction in conjunction with detection using the Thermo Scientic TSQ Quantum Ultra in highly-selective reaction monitoring (H-SRM) mode for assaying antibiotics at low pg/mL concentrations.
Scientic Aria TLX-2 system, water samples in 1 mL volumes were injected onto a TurboFlow column without any further sample preparation. Targeted antibiotics were focused and concentrated on the turbulent-ow extraction column, then transferred to the analytical column. Analyte separation was accomplished using a reverse-phase gradient prior to detection with the TSQ Quantum Ultra in highly-selective reaction monitoring (H-SRM) mode.
Goal
Develop a method to screen for antibiotics in surface water by applying on-line preconcentration and analyte extraction with LC/MS/MS detection.
On-line TurboFlow Extraction Aria TLX-2 TurboFlow Column: 0.5 50 mm Thermo Scientic Cyclone MAX Autosampler: CTC PAL (Leap Technologies) Injection Volume: 1.0 mL Loading Pump Mobile Phase: (A) 10 mM ammonium bicarbonate, (B) 0.5% HAc + 0.04% TFA, (C) ACN + 0.1% HCOOH Flow Rate: 2.0 mL/min Liquid Chromatography Analytical Column: 4.6 100 mm, 3 m Thermo Scientic Hypersil GOLD Flow Rate: 1.2 mL/min Eluting Pump Mobile Phase: (A) 0.5% HAc + 0.04% TFA, (B) ACN + 0.5% HAc + 0.04% TFA Flow Split: post-column, 0.5 mL/min to ESI source Mass Spectrometry TSQ Quantum Ultra Ionization mode: Positive ion ESI Ion Transfer Tube Temperature: 375C Selective Reaction Monitoring (SRM) Parameters Q2 Pressure: 1.5 mTorr argon SRM Transitions: see Table 1 SRM Scan Time: 40 ms per transition Q1 Resolution: Unit (0.7 Da FWHM) and H-SRM (0.15 Da FWHM) Q3 Resolution: Unit (0.7 Da FWHM)
The antibiotics assayed in this method (Table 1) were purchased from Sigma (St. Louis, MO) and used without further purication. Stock solutions of the antibiotic standards were prepared at 1.0 mg/mL in methanol and stored in amber polypropylene vials at -20C until needed. Prior to High-Throughput HPLC (HTLC/MS/MS) analysis, water samples were prepared in 2 g/mL Na2EDTA (aq) to inhibit binding of the tetracycline antibiotics to glass surfaces and to metal ions in solution.1 Using the Thermo
Compound Sulfamethoxazole Sulfamerazine Sulfamethizole Sulfamethazine Lincomycin Tetracycline Doxycycline Chlortetracycline Dehydroerthromycin Erythromycin Roxithromycin Tylosin Precursor m/z 254 265 271 279 407 445 445 479 716 734 837 916 Product m/z 108, 156 156, 172 108, 156 156, 186 126, 359 154, 410 321, 428 444, 462 158, 558 158, 576 158, 679 174, 772 Resolution H-SRM H-SRM H-SRM H-SRM H-SRM H-SRM H-SRM H-SRM H-SRM Unit H-SRM Unit H-SRM Unit H-SRM
Table 1: List of antibiotics and SRM transitions for the HTLC/MS/MS assay

Details on HTLC/MS/MS Method
The 13 antibiotics studied (Table 1) were preconcentrated on a new mixed-mode TurboFlow extraction column, the Cyclone MAX, which has reverse-phase and anion exchange characteristics. After sample loading and ushing the TurboFlow column at 2.0 mL/min to remove matrix interferences (see Figure 1), valve 1 is switched to allow the extraction solvent plug (50% ACN + 0.1% HCOOH) to transfer the antibiotics to the analytical column. The organic content of the extraction solvent plug, which contains the antibiotic analytes, is diluted by the highly aqueous mobile phase of the eluting pumps at 1.2 mL/min. This dynamic mixing occurs before the analytical column so that the antibiotics can be effectively refocused prior to the reverse-phase separation step. During the LC/MS/MS data acquisition, the TurboFlow column is reconditioned for the next sample injection.
TurboFlow Column
matrix to waste Valve 1 analytes retained Valve 2
For unit SRM and H-SRM detection on the TSQ Quantum Ultra, data acquisition was sectioned into ve time segments, whereby eight SRM transitions per segment were employed. Two SRM transitions were monitored for each antibiotic compound (see Table 1) for conrmation purposes and to improve ion statistics.
Sensitivity and Calibration Data for HTLC/MS/MS Assay

Figure 2 presents the HTLC/MS/MS chromatograms of the 13 antibiotic standards and dehydroerythromycin at their limits of quantitation (LOQs), which ranged from 0.5-5 pg/mL, using 1 mL injections. Calibration data were generated from the LOQ to 100 pg/mL for the 13 antibiotic standards in deionized water. Linear t calibration curves with 1/x weighting were used for 11 of 13 antibiotics. Sulfamethoxazole and sulfamethizole provided the best results by employing quadratic t calibration curves. All sample standard regressions yielded R2 0.990 (n = 4 replicates).
Spike of Antibiotics into Surface Water Sample

Figure 3 shows the results for the HTLC/MS/MS assay for a surface water sample that was spiked at 25 pg/mL with the antibiotic standards. Prior to this experiment, the surface water sample was screened using the described method, and it was found to be devoid of the target antibiotics. Figure 4 presents a comparative HTLC/MS/MS assay for a neat standard solution at 25 pg/mL. The sulfonamide class showed a minor signal suppression in the surface water sample, while the response for the macrolides were slightly enhanced. The tetracyclines, however, showed a signicant difference in response in the surface water sample vis--vis the neat standard. This difference may be attributed to binding of these tetracycline antibiotics to residual metals in the water sample.
Waste Analytical Column
HTLC TurboFlow column
A
Transfer Loop
Autosampler
Loading Pumps
TSQ Quantum Ultra

Eluting Pumps
Figure 1: Schematic of the Aria TLX-2 system coupled to the TSQ Quantum Ultra
10000 5000
AA: 76974
30000
Sulfamerazine 0.5 pg/mL

AA: 45434
20000 10000
AA: 53467
Doxycycline 2.5 pg/mL
6000 4000 2000 20000 15000 10000 5000
10000 5000
AA: 34513
Lincomycin 1 pg/mL
Sulfamethoxazole 1 pg/mL
AA: 120532
30000 20000 10000 40000
AA: 115192
Sulfamethazine 0.5 pg/mL
Sulfadimethoxine 1 pg/mL
AA: 10458
Intensity
Intensity
AA: 75164 10000 5000
Sulfamethizole 0.5 pg/mL
30000 20000 10000 15000 10000 5000
Erythromycin 0.5 pg/mL

AA: 51026
8000 6000 4000 2000
AA: 42810
Oxytetracycline 5 pg/mL
Tylosin 2.5 pg/mL
8000 6000 4000 2000
AA: 32106
Tetracycline 2.5 pg/mL
1500 1000 500
AA: 5359
Dehydroerythomycin
AA: 3185
AA: 33060 10000 5000 15000
AA: 51325
Chlortetracycline 5 pg/mL
10000 5000
Roxithromycin 2.5 pg/mL

8 9 10 11 12
10
Time (min)
Time (min)
Figure 2: Chromatograms for antibiotics at their LOQs using HTLC/MS/MS
AA: 2259422 20000 10000
AA: 77977 20000 10000 AA: 75565
Sulfamerazine
Doxycycline
60000 40000 20000
AA: 399786
100000 50000
AA: 408175
Lincomycin
Sulfamethoxazole
AA: 3193035 40000 20000 400000
AA: 1748334
Sulfamethazine
Sulfadimethoxine
200000
Intensity
200000 150000 100000 50000 4000 3000 2000 1000 10000
AA: 1501694
Intensity
Sulfamethizole
20000 10000
AA: 816634
Erythromycin
AA: 18567
AA: 959990 200000
Oxytetracycline
Tylosin
100000
AA: 27617
Tetracycline
5000
40000 30000 20000 10000
AA: 166426 AA: 134268
Dehydroerythomycin
8000 6000 4000 2000
AA: 25406
600000 400000 200000
AA: 2440704
Chlortetracycline
Roxithromycin
10
10
11
12
Time (min)
Time (min)
Figure 3: 25 pg/mL antibiotics spiked into a surface water sample
400000 300000 200000 100000
AA: 2930945
AA: 597641 20000 10000
Sulfamerazine
Doxycycline
150000 100000 50000
AA: 1036150 100000
AA: 763241
Lincomycin
50000
Sulfamethoxazole
600000 400000 200000
AA: 4017893
AA: 2674376 400000 200000
Sulfamethazine
Sulfadimethoxine
Intensity
AA: 2886119 400000 200000
Intensity
AA: 541236 20000 10000
Sulfamethizole
Erythromycin
40000 30000 20000 10000
AA: 207609
Oxytetracycline
200000
AA: 663101
Tylosin
100000
AA: 325303 60000 40000 20000
40000 30000 20000 10000
Tetracycline
Dehydroerythomycin
AA: 73328 AA: 66932
40000 30000 20000 10000
AA: 145222
600000 400000 200000
AA: 1001733
Chlortetracycline
Roxithromycin
10
10
11
12
Time (min)
Time (min)
Figure 4: Neat 25 pg/mL antibiotics standard
HTLC/MS/MS Screening for Antibiotics in Environmental Water Samples

Surface water samples from multiple locations in California, Florida and Ontario were screened for antibiotics using the described HTLC/MS/MS method. Of the water samples screened only the Lake Ontario sample showed measurable levels of the targeted antibiotics (Figure 5). Insets in Figure 5 show chromatographic traces for the two monitored SRM transitions for the observed antibiotics, providing additional conrmation and higher condence in these results.
Conclusions
A method for assaying antibiotics in water samples at the low pg/mL level using on-line sample clean-up and preconcentration has been demonstrated. The capability of on-line turbulent-ow extraction of large sample volumes (1 mL) signicantly reduces sample analysis time from a matter of hours to a matter of minutes. Detection using highly-selective reaction monitoring (H-SRM) provides an additional level of selectivity and condence over unit resolution SRM. This HTLC/MS/MS method, when applied to screening surface water samples, was able to detect and quantitate the observed antibiotics at the low pg/mL level.
254
4000
8000
Intensity
20000 15000
AA: 78525
108
Intensity
10000 5000
Sulfamethoxazole Calc Conc = 2.3 pg/mL
14000
254
7000
156
9 .0
9 .2
9 .4
Time (min)
9 .6
9 .8
1 0 .0
Intensity
AA: 41832
4500
10000 6000 2000

7000
734
2000
158
Erythromycin Calc Conc = 1.9 pg/mL

Intensity
7000
734
3500
756
Intensity
9 .6
9 .8
1 0 .0
1 0 .2
1 0 .4
1 0 .6
Time (min)
10000 6000 2000
Intensity
916
3000
174
AA: 40234
Tylosin Calc Conc = 2.1 pg/mL
3500
916
1500
772
AA: 17829
9 .8
1 0 .0
1 0 .2
1 0 .4
1 0 .6
1 0 .8
Time (min)
3500
10000 6000 2000 8.0
Intensity
716
158
AA: 37424
1500
Dehydroerythromycin
716 558
8000
4000
AA: 13955
1 0 .3 1 0 .5
Time (min)
1 0 .7
1 0 .9
1 1 .1
AA: 5150
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
12.5
13.0
Time (min)
Figure 5: Chromatograms of the detected antibiotics in a Lake Ontario water sample References
1 Lindsey, M.E.; Meyer, M.; Thurman, E.M. Anal Chem., 2001, 73, 4640-4646. 2 Yang, S; Cha, J.; Carlson, K. Rapid Commun. Mass Spectrom., 2004, 18, 2131-2145. 3 Gobel, A.; McArdell, C.S.; Suter, M.J.-F.; Giger, W. Anal. Chem., 2004, 76, 4756-4764. 4 Shang, D.; Dyck, M.; Jia, X.; DiCicco, A.; Alleyne, C.; Nicolidakis, H.; Mori, B. Proc. 48th ASMS Conf. on Mass Spectrom. and Allied Topics, TPH #264.
Intensity
Intensity
www.thermo.com
AN62485_E 11/07S
Simple and Rapid Analysis of Chloramphenicol in Milk by LC-MS/MS

Ting Liu1, Peter Wang1, and Kefei Wang2 1 Thermo Fisher Scientic, Shanghai, China; 2Thermo Fisher Scientic, San Jose, CA, USA
Introduction
Key Words TSQ Quantum Access Accela High Speed LC System Antibiotic Food Residue Analysis SRM Chloramphenicol (CAP) is a broad-spectrum antibiotic with historical veterinary uses in all major food-producing animals (see Figure 1 for structure). It has serious side effects on humans that may cause aplastic anemia, and the suspected carcinogen effect is also thought to be dose independent. Consequently, chloramphenicol has been banned for use in all food-producing animals by the European Union (EU), USA and Canada. A minimum required performance limit (MRPL) for chloramphenicol determination was recently set by the EU at 0.3 g/kg (ppb) in all foods of animal origin, such as meat, seafood, egg, milk, honey, etc. However, residues of CAP at unacceptable levels continue to be found in food imports, as a result of illegal use in some countries to mask the poor hygiene conditions of animal-raising farm and to augment animal growth. The growing food safety concerns call for intensive surveillance of chloramphenicol in food products.
Goal
To develop a simple, rapid, and sensitive LC-MS/MS method for analyzing chloramphenicol in milk. The method should be suitable for both screening and conrmatory purposes.
Sample Preparation Standards and Regents: Chloramphenicol (98%) was purchased from Sigma-Aldrich (St. Louis, MO) and d5chloramphenicol (100 g/mL in acetonitrile) as internal standard from Cambridge Iosotope Lab (Andover, MA). Regent grade water, acetonitrile and methanol were from Thermo Fisher Scientic (Pittsburgh, PA).
Procedures:
0.5 g Milk + d5CAP (0.3 ppb) as IS
+ 0.75 mL CH3CN, vortex 1 min, Centrifuge @ 14000 rpm for 10 min
Take 0.7 mL Supernatant + 0.3 mL Water, store at 4C for 1 hr
Figure 1: Structure of chloramphenicol
Analysis of residual of chloramphenicol in foodstuff is challenging because of the complicated sample matrices and stringent requirements of both low quantitation limit (<0.3 ppb) and method validation. The technique of liquid chromatography separation followed by tandem mass spectrometry detection, LC-MS/MS, is the technology of choice because of its sensitivity and specicity. A sample cleanup process is generally required to remove the sample matrix prior to the LC-MS/MS run. Typically, this involves the costly and labor-intensive solid phase extraction (SPE) and/or liquid-liquid extraction (LLE) procedures. In this work, we report a simple sample preparation procedure involving only the acetonitrile protein precipitation and dilution to extract the CAP from milk, followed by a high-speed LC separation and detection by a triple quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode. The sample preparation is simple, fast, and inexpensive, and the method exceeds the sensitivity and specicity requirements for both screening and conrmatory assays. Validation according to the European Commission Decision 2002/657/EC has also been performed.
Pipette 0.8 mL upper solution for LC-MS/MS Analysis
Chromatography Conditions HPLC Module: Thermo Scientic Accela High Speed LC System Column: Thermo Scientic Hypersil GOLD 50 mm 2.1 mm and 1.9 m particle size Column Temperature: Ambient Mobile Phase: A: Methanol B: Water Gradient: Time (min) A% 0.0-0.6 5% 2.3 100% 2.35-3.0 5% Flow Rate: 500 L/min Injection Volume: 20 L (with loop)
Mass Spectrometer Conditions Mass Spectrometer: Thermo Scientic TSQ Quantum Access triple stage quadrupole mass spectrometer Source: ESI-, 3000 V Sheath Gas: 45 unit Auxiliary Gas: 10 unit Capillary Temperature: 300C Source CID: -7 V Q1 and Q3 Peak Width (FWHM): 0.7 Da Scan Time: 0.1 s Collision Gas: Ar (1.5 mTorr) SRM Transitions: 3 SRMs for CAP, 1 SRM for d5-CAP (see Table 1)
Product Ion (Collision Energy) 152 (17)* 257 (15) 194 (16) 157 (17)*
The results of relative ion abundance measured at various concentrations are given Table 2. Both relative ion abundance ratios of 257/152 and 194/152 meet the requirements set by Decision 2002/657/EC. Note that we found the 321>257 transition is more likely subjected to matrix interferences in many other cases of different matrices, thus if two SRM transitions need to be selected (4.0 IPs) for the method, 321>152 and 321>194 are preferred. Method Performance: Figure 2 shows representative SRM chromatograms for a blank and 0.05 g/kg spiked milk samples. As shown, with high-speed LC, each chromatographic run is only 3 min, allowing high throughput for screening assay. All three SRM traces for CAP at 0.05 g/kg spiked samples can be well quantied. Note that the 0.05 g/kg spiked in milk is equivalent to 0.46 pg injected on column by assuming a full recovery. It should also be noted that with the high-speed LC separation of only 3 min for each chromatographic run, the CAP peak width (at 10% above baseline) is as narrow as 6 s. Under current MS acquisition conditions, there are 13-14 points across each peak, enough for maintaining a well-dened peak shape for accurate integration. A representative calibration curve from standards prepared in milk is shown in Figure 3. Good linearity from 0.05 to 1.0 g/kg with correlation coefcient of R2= 0.9954 (Weighting factor W = 1/X) was obtained. Table 3 shows excellent recovery and within-laboratory reproducibility of the method (at four different days). Decision Limit (CC) and Detection Capability (CC): According to Decision 2002/657/EC, the Decision Limit CC is the minimum CAP concentration at which a sample is really non-compliant with an error probability of 1% ( =0.01), and the Detection Capability (CC) is the minimum amount of CAP that can be quantied and conrmed with an error probability of 5% ( =0.05). Two methods can be used for calculating the CC according to the Decision. One is to use the S/N ratio of 3:1 of blank samples, similar to those for estimation of limit of detection. The other is to use the intercept of calibration curve at low levels and the within-laboratory reproducibility. The former method does not work well for LC-MS/MS because the very low background (noise count ~0) of SRM chromatogram often yields unrealistically low values for CC. Thus we use the latter approach by using cali-
Precursor Ion
CAP (M -H -)
320.93
d 5 -CAP (M -H -)
* Product ion used for quantitation
326.93
Table 1: SRM transitions for CAP and d5-CAP (IS)

Sample Preparation: A major goal for the method development in this study is to avoid using the labor intensive and time-consuming SPE or LLE procedures as in literatures. In curret work, the proteins from milk were removed with acetonitrile precipitation at ratio of 1.5:1 (v/v Acetonitrile:Milk), followed by dilution with water, which is necessary for gradient chromatographic separation. At such ratio, protein removal was not complete, trace amount of precipitates of proteins appeared after the sample was stored at 4C for some time. Thus, the supernatant was taken for LC-MS/MS analysis after the sample was stored at 4C for 1 hr. Choice of Quantitation and Qualication Ions: Three product ions were chosen to give an Identication Points (IPs) of 5.5 to meet the requirement of 4.0 IPs by the Decision 2002/657/EC for conrmatory assay of the prohibited substances such as CAP. The m/z 152 was chosen as quantitation ion, the m/z 257 and 194 as conrmation ions, consisting with those reported in literatures.
CAP Spiked Level (g/kg) 0.05 0.15 0.30 0.50
Relative Ion Abundance of 257/152 Mean n= 6 96% 92% 93% 90% %RSD n= 6 16% 7.6% 15% 3.4% 20% Tolerance by Decision 2002/657/EC
Relative Ion Abundance of 194/152 Mean n= 6 26% 28% 31% 31% %RSD n= 6 21% 25% 15% 17% 25% Tolerance by Decision 2002/657/EC
Note: Relative ion abundance values were calculated by relative peak area ratios
Table 2: Relative ion abundances at various CAP concentrations in milk and the associated tolerances required by Decision 2002/657/EC
bration data of (0.05-0.15-0.30 g/kg) to obtain the Y-intercept and its standard deviation, SDY-intercept,
CC=Y-intercept + 2.33*SDY-intercept
with the within-laboratory reproducibility data of 0.15 g/kg spiking level, thus,
CC =CC + 1.64*SD0.15 g/kg
Similarly, the CC can be calculated from CC and the standard deviation of 20 measurement of samples spiked at CC level. Here the latter term is approximated
Where SD0.15 g/kg is the within-laboratory reproducibility (in standard deviation) of the 0.15 g/kg in Table 3. The calculated values of CC and CC are 0.087 g/kg and 0.12 g/kg, respectively.
CAP Spiking Level (g/kg) 0.05 0.15 0.30 0.50 Mean (%) 97% 101% 104% 94%
Within-laboratory Reproducibility (n= 20) SD (g/kg) 0.0065 0.020 0.037 0.042 %RSD 14% 13% 11% 8.0%
Table 3: Recovery and Reproducibility Data
Figure 2: SRM chromatograms for milk blank and 0.050 g/kg spiked milk samples
CAP Y = 0.148836 + 2.55752*X R^2 = 0.9954 W: 1/X 3.5 3.0 2.5

Area Ratio
2.0 1.5 1.0 0.5 0.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
g/kg
Figure 3: Calibration of CAP in milk
Conclusions
A simple, rapid and sensitive method for analysis of CAP in milk by LC-MS-MS has been developed and validated. The sample preparation by protein precipitation and dilution is very simple to perform and avoids the use of SPE or LLE. With the high-speed Accela LC coupled to a triple quadruple TSQ Quantum Access, each analytical run is as short as 3 min. The method can be used for the purposes of both high-throughput screening and rapid conrmatory assays. For screening assay, the method can detect < 0.050 g/kg CAP in milk. For conrmatory assay, the method validated according to Decision 2002/657/EC gives a CC =0.087 g/kg and CC = 0.12 g/kg, both below the MRPL of 0.3 g/kg.
References
1. Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/ECD concerning the performance of analytical methods and the interpretation of results, Ofcial Journal of the European Communities, L 221, 2002, 8-36. 2. Bogusz, M.J. et al. Rapid determination of chloramphenicol and its glucuronide in food products by liquid chromatographyelectrospray negative ionization tandem mass spectrometry; J, Chrom. B 2004, 807(2), 343-356. 3. Tao, D. et al. Effects of sample preparation and high resolution SRM on LC-MS-MS determination of chloramphenicol in various food products; Poster Presentation, 53rd ASMS Conference, San Antonio, TX, USA, June 5-9, 2005. 4. Gallo, P. et al. Development of a liquid chromatography/electrospray tandem mass spectrometry method for conrmation of chloramphenicol residues in milk after alfa-1-acid glycoprotein afnity chromatography; Rapid Commun. Mass Spectrom. 2005, 19(4), 574-579. 5. Vinci, F. et al. In-house validation of liquid chromatography electrospray tandem mass spectrometry method for conrmation of chloramphenicol residues in muscles according to Decision 2002/567/EC; Rapid Commun. Mass Spectrom. 2005, 19(22), 3349-3355.
www.thermo.com
AN62530_E 11/07S
Drug Residues Antibiotics & Antimicrobials

t
372: LC/MS/MS Analysis of Anti-Infectives In Raw and Treated Sewage
LC-MS/MS Analysis of Anti-Infectives In Raw and Treated Sewage

P.A. Segura1, A. Garcia Ac1, A. Lajeunesse2, D.Ghosh3, C. Gagnon2 and S. Sauv1
1 2
Dpartement de Chimie, Universit de Montral, C.P. 6128, succursale Centre-ville, Montral, QC, Canada H3C 3J7 Centre Saint-Laurent, Environnement Canada, 105, rue McGill, Montral, QC, Canada H2Y 2E7 3 Thermo Fisher Scientic, San Jose, CA, USA
Introduction
Key Words TSQ Quantum Ultra Surveyor HPLC Antibiotics SPE Anti-infectives is a general term that refers to several classes of biologically active compounds used to treat or prevent infections. Therapeutic agents such as antimicrobials (synthetic) and antibiotics (natural or semi-natural) are examples of anti-infectives. The widespread utilization of anti-infectives in urban centers as well as their resistance to biodegradation or elimination in wastewater treatment plants (WWTPs) has led to their appearance in efuents and surface waters[1-3]. In the last few years there has been a growing concern about the environmental fate and the possible effects of these agents on the aquatic environment[4,5]. The rst report on the occurrence of anti-infective traces in the aquatic environment was published as early as 1983[6]. A later study[7] acknowledged that pharmaceuticals would enter the water cycle mainly via a domestic route (i.e. by the excreta of individuals taking medication at homes, hospitals or clinics). It is therefore important to know the amounts of these substances released in the aquatic environment to be able to evaluate potential effects. A sensitive and robust method was developed for the determination of some of the most prescribed anti-infectives in trace amounts (lower nanogram-per-liter range) in raw and treated wastewaters.
Goals
Quantify several anti-infectives at the lower nanogramper-liter level in raw and treated wastewaters. Apply two specic single reaction monitoring mode (SRM) transitions and their peak ratio to avoid the presence of false positives.
Method
Raw sewage (north and south inuent) was collected and treated (efuent) 24-h composite samples at the municipal wastewater treatment plant of the City of Montral (Qubec, Canada). This plant has physico-chemical treatments only and its efuent is one of the largest in North America. We analyzed six of the most prescribed compounds (sulfamethoxazole, trimethoprim, ciprooxacin, levooxacin, clarithromycin and azithromycin) (Figure 1), by using solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The compounds were selected based on drugstore sales.
a.
O N O HN S O O O O N N NH2 NH2
b.
c.
O O N N NH2 N H2
NH2
Sulfamethoxazole
O F N HN N O OH
Trimethoprim
O F N N O N O OH Cl N HN NH2 N NH2
Diaveridine (IS)
O F N F N O OH
Ciprofloxacin
O O OH O O O O O O OH N O OH
Levofloxacin
N HO O O O O O OH OH O OH N O OH
Pyrimethamine (IS)
Lomefloxacin (IS)
HO
HO
O OH O O O O N O O O OH O O
O O
Clarithromycin
Azithromycin
Josamycin (IS)
Figure 1: Molecular structures of the anti-infectives studied (a), the surrogate standard (b), and the internal standards (c).
Sample Preparation
Wastewater samples were ltered using 1.2 m pore-size ber glass lters and then 0.45 m pore-size mixed cellulose membranes. 50 mM of formic acid and 1 mL of a 5% Na2EDTA (w/v) solution were added to 250 mL of wastewater and the pH adjusted to 3 with NaOH 1.0 M. Pyrimethamine was used as a surrogate standard and spiked at a concentration of 500 ng L-1. Analytes were pre-concentrated and extracted using a 200 mg reversed phase polymeric SPE cartridge on top of a 200 mg mixed mode polymeric SPE cartridge. Retained analytes were eluted from the cartridges using 2 2.5 mL ACN: MeOH 1:1 (reversed phase) and 2 2.5 mL 5% NH3 in ACN: MeOH 1:1 (mixed mode). The eluates were recovered from both cartridges and were collected on the same conical-bottom centrifuge tube and then evaporated to dryness with N2(g). Extracted analytes were reconstituted to 250 L with 0.1% formic acid in 90% H2O/5% MeOH/5% ACN solution containing the internal standards (diaveridine, lomeoxacin and josamycin).
Table 1: Instrument Parameters
HPLC Column Column temperature Mobile phase A Mobile phase B Injection volume Flow rate Gradient MS Thermo Scientic BetaBasic C18 (50 2.1 mm, 3 m) 30C 0.1 % formic acid/H2O 0.1% formic acid/MeOH:ACN 1:1 20 L 200 Lmin-1 t=0 min, A=90%, B=10% t=2 min, A=80%, B=20% t=15 min, A=75%, B=25% t=17 min, A=50%, B=50% t=20 min, A=5%, B=95% t=25 min, A=5%, B=95% t=30 min, A=90%, B=10%
LC-MS/MS Conditions
HPLC separation was done with a Thermo Scientic Surveyor HPLC system. Detection and quantication of the analytes was performed with a Thermo Scientic TSQ Quantum Ultra using the single reaction monitoring mode (SRM) (Table 1). Two specic single reaction monitoring (SRM) transitions were used for each compound as well as their peak area ratios to reliably conrm the presence of the targeted anti-infectives. This reduced the possibility of false positives given that some interfering matrix components areco-extracted with the analytes and could have the same SRM transition.[8]

MS/MS in the SRM mode proved to be highly selective. Instrument response was linear (r2 0.99) in the dynamic range (251000 ng L-1) in spite of the presence of high concentrations of organic as well as inorganic interferences in the matrix. Limits of detection ranged from
Ionization mode Spray voltage Ion transfer capillary temperature Sheath gas pressure Auxiliary gas pressure Collision gas pressure Source CID
ESI+ 3500 V 350 C 21 mTorr 4 mTorr 1.5 mTorr 12 V
Table 2: SRM transitions used for detection and quantication (SRM #1) and conrmation (SRM #2)
Compound Pyrimethamine Sulfamethoxazole Diaveridine Trimethoprim Ciprooxacin Lomeoxacin Levooxacin Clarithromycin* Azithromycin* Josamycin
SRM #1 249.10 177.07 254.08 92.11 261.15 123.11 291.16 123.10 332.16 231.07 352.17 265.13 362.17 261.12 748.55 590.36 375.33 82.96 828.53 108.87
CE (V) 40 36 34 33 49 34 35 19 25 46
SRM #2 254.08 108.10 291.16 230.17 332.16 288.15 362.17 221.05 748.55 115.99 749.54 158.04 828.53 173.96
CE (V) 37 34 27 43 35 38 47
Tube Lens 70 91 82 92 96 74/112 126
Quantied using diaveridine as the internal standard, Quantied using lomeoxacin as the internal standard, *Quantied using josamycin as the internal standard
Table 3: Analytical method parameters

Compound Sulfamethoxazole Trimethroprim Ciprooxacin Levooxacin Clarithromycin Azithromycin
r 2 matrix* 0.9995 0.9998 0.9996 0.9996 0.9997 0.9900
Limit of Detection Standard SRM (ngL-1) ratioSD 22 1.53 0.03 7 4.2 0.1 21 5.5 0.8 4 3.65 0.07 0.3 1.67 0.04 12 1.2 0.1
Sample SRM ratioSD 1.6 0.2 4.39 0.07 6.59 0.05 3.83 0.06 1.59 0.09 0.44 0.1
SRM ratio difference^ -2.6 -3.3 -18.9 -5.0 4.3 6.4
*Determination coefcient of the calibration curve made using the WWTP efuent diluted by a factor of 10; **Calculated from the efuent data based on a S/N=3; Standards spiked WWTP efuent diluted by a factor of 10, n=4; WWTP efuent, n=3; ^Percentage difference between the standard and sample SRM ratio.
100 80 Recovery/% 60 40 20 0
cin ole pr ac cin my ox ho lox flo ro th et of ro my xa az cin im in
Reversed phase Reversed phase + mixed mode
Figure 2: Analytes mean percentage recovery (spiked in the efuent at 500 ng L-1, n=2)
100
9.21
50
Sulfamethoxazole 254.08 92.11
100
9.16
50
Sulfamethoxazole 254.08 108.10
100
* 6.96
Trimethoprim 291.16 123.70
50
100
6.99
Trimethoprim 291.16 230.17
50
0.3 to 22 ng L-1 (Table 3). As suggested by Hernandez[8], the use of two SRM transitions in the analytical method (Figure 3) as well as their peak ratios effectively and unambiguously conrmed the presence of the studied anti-infectives in all the samples. SRM peak ratios were reproducible (RSD <10%) and differences with SRM peak ratios of spiked standards were not higher than 20% except for AZI (64%). The tandem-SPE approach utilized to pre-concentrate and extract the analytes from untreated and treated sewage improved the recovery on all six analytes (Figure 2). The combination of reversed-phase and ion-exchange surface chemistry proved to be a suitable way to extract compounds having different chemical properties such as pKa and pKow. All targeted anti-infectives were found in the wastewater samples in concentrations ranging from 391 to 2767 ng L-1 (Figure 4). Anti-infective daily mass ows in the St. Lawrence River were estimated using the ow of the sampling day (35 m3 s-1) (Table 4). These results show that while anti-infective concentration in urban wastewaters are typically in the low nanogramper-liter range, their daily discharged inputs in surface waters can be substantial.
vo
ith
Tri m
me
pr
Le
ar
Ci
Su
lfa
100
Cl
* 9.61
Az
ith
Ciprooxacin 332.76 288.15
50
100
9.67
50
Ciprooxacin 332.16 231.07
10
12 14 Time (min)
16
18
20
22
24
26
100
8.77
50
Levooxacin 362.17 261.12
100
8.75
50
Levooxacin 362.17 221.05
Figures 3a-b: Chromatograms showing two SRM transitions of the studied compounds in treated wastewater. Peaks due to interferences are marked by asterisks(*).
100
20.23
50
Clarithromycin 748.55 590.36
100
20.23
50
Clarithromycin 748.55 115.99
100
20.31
50
Azithromycin 375.33 82.96
100
20.30
50
Azithromycin 749.54 158.04
10
12
14 Time (min)
16
18
20
22
24
26
North Influent
300
South Influent Effluent
Concentration (ng/L)
200
100
Figure 4: Occurrence of the studied anti-infectives in the dissolved phase of raw and treated sewage of the City of Montral (n=3)
Table 4: Removal efciency of the Montral wastewater treatement plant and average mass ow of the studied anti-infectives.
Compound Sulfamethoxazole Trimethroprim Ciprooxacin Levooxacin Clarithromycin Azithromycin Mean mass ow in the St. Lawrence River (g day-1) 340 30 310 20 320 10 118 2 830 60 310 20
Conclusions
The developed analytical method allowed the extraction, detection and quantication of six of the most used anti-infectives in untreated and treated sewage. Detection limits ranged from 0.3 to 22 ng L-1 and instrument response was linear (r2 0.99) in the dynamic range (251000 ng L-1). The use of two specic SRM transitions and their peak area ratios proved to be a reliable and effective way to reduce false positives and conrm the presence of targeted substances. All the studied antiinfectives were found in the wastewater samples in concentrations ranging from 39 to 276 ng L-1. More studies are necessary to elucidate the fate of these anti-infectives after they are discharged into the St. Lawrence River as well as their effects on aquatic biota and the environment.
References
1
Kolpin D.W., Furlong E.T., Meyer M.T., Thurman E.M., Zaugg S.D., Barber L.B., and Buxton H.T. (2002) Environmental Science & Technology 36:1202-1211. Hirsch R., Ternes T., Haberer K., and Kratz K.L. (1999) Science of the Total Environment 225:109-118. Metcalfe C.D., Koenig B.G., Bennie D.T., Servos M., Ternes T.A., and Hirsch R. (2003) Environmental Toxicology & Chemistry 22:2872-2880. Wilson B.A., Smith V.H., Denoyelles F., and Larive C.K. (2003) Environmental Science & Technology 37:1713-1719. Richards S.M., Wilson C.J., Johnson D.J., Castle D.M., Lam M., Mabury S.A., Sibley P.K., and Solomon K.R. (2004) Environmental Toxicology and Chemistry 23:1035-1042. Watts CD, Crathorne B, Fielding M, and Steel CP (1983) Analysis of Organic Micropollutants in Water. D. Reidel Publishing Company, Dordrecht. Richardson M.L. and Bowron J.M. (1985) Journal of Pharmacy & Pharmacology 37:1-12. Hernandez F., Ibanez M., Sancho J.V., and Pozo S.J. (2004) Analytical Chemistry 76:4349-4357.
cin
le
in
zo
ac
cin
im
pr
xa
my
xa
ho
fl o
ho
of
ro
et
vo
et
Ci pr
i th
Tri m
Su lf
Cl ar
am
Az it
Le
hr
om
lox
yc
in
www.thermo.com
AN62489_E 11/07S
Drug Residues Pharmaceuticals

t
30162: The Thermo Scientific Exactive Benchtop LC/MS Orbitrap Mass Spectrometer
The Thermo Scientic Exactive Benchtop LC/MS Orbitrap Mass Spectrometer

Andreas Wieghaus, Alexander Makarov, Ulf Froehlich, Markus Kellmann, Eduard Denisov, Oliver Lange, Thermo Fisher Scientic, Bremen, Germany
Overview
Key Words Exactive Accurate Mass High Resolution Polarity Switching Scan Speed Review of a new benchtop mass spectrometer based on a stand-alone Thermo Scientific Orbitrap mass analyzer. Key features of the instrument layout, analytical parameters and typical applications are described. Ions are transferred from the source through four stages of differential pumping using RF-only multipoles into a curved RF-only trapping quadrupole (the C-trap). In the C-trap ions are accumulated and their energy dampened using a bath gas (nitrogen). Ions are then injected through three further stages of differential pumping using a curved lens system into the Orbitrap analyzer where mass spectra are acquired via image current detection. The vacuum inside the Orbitrap mass analyzer is maintained below 1E-09 mBar.
Introduction
Over the past three years the combination of Orbitrap technology with a linear ion trap has become an established platform for high resolution, accurate mass LC/MSn analysis. The high resolving power, mass accuracy and dynamic range of the Orbitrap analyzer allow rigorous characterization of complex mixtures even in the absence of precursor ion mass selection. We now describe the development of a non-hybrid mass spectrometer comprising of an atmospheric-pressure ion source (API) and a standalone Orbitrap mass analyzer.
Methods
All experiments were performed on a prototype of the new Thermo Scientific Exactive mass spectrometer using an electrospray ionization (ESI) source.
Automatic Gain Control (AGC) Automatic control of the number of ions in the Orbitrap is performed by measuring the total ion charge using a pre-scan and by calculating the ion injection time for the analytical scan from this. For very high scan rates, the previous analytical scan is used as a prescan to optimize the scan cycle time without compromising automatic gain control. Ion gating is performed using a fast split lens setup that ensures the precise determination of the ion injection time. Higher Energy Collision Induced Dissociation (HCD) In a HCD experiment ions are passed through the C-trap into a multipole collision cell where they are fragmented. After that, the HCD cell voltages are ramped and ions are transferred back into the C-trap from where they are injected into the Orbitrap for detection.
Instrument Layout Overview Figure 1 shows the schematic layout of the instrument. Samples can be introduced introduced into the API source by a variety of methods includingdirect infusion or an U-HPLC system (Thermo Scientific Accela). The source is similar to the commercial source of the Thermo Scientific TSQ Quantum Ultra.
Figure 1: Schematic layout of the instrument.
Results
Automatic Gain Control (AGC) A requirement of any ion trap device is the ability to control the ion population within the trap. When the ion population is not accurately maintained it can result in large variations in the quality of data. The correct AGC functionality of the Exactive instrument is exemplified in Figure 2 by two mass spectra acquired in the middle and at the end of an eluting LC peak of Buspirone. Scan Speed The use of a single mass analyzer with very high transmission characteristics in combination with the use of fast digital and analog electronics allow high resolution mass spectra to be detected, processed and recorded at
In both cases the mass resolution, mass accuracy and signal-to-noise ratio are excellent. The AGC feature in combination with the precise determination of the ion injection time allows the instrument to be used for accurate quantitative analyses.
high scan rates of up to 10 Hz. This is compatible with the narrow peak widths observed in fast chromatography analyses (Figure 2).
Figure 2: LC peak and mass spectra of Buspirone acquired at a scan rate of 10 scans per second.
Mass Resolution At a scan rate of 10 Hz the resolving power of the instrument is > 10,000 at m/z 200. Increasing the transient detection time by a factor of 10 (corresponding to a scan rate of 1 Hz) the mass resolution can be increased beyond 100,000.
291.07571 R=94800 276.06339 R=97300 313.05768 R=89900 417.08408 R=77900
To demonstrate the resolving power of the instrument a pesticide mixture was measured showing well resolved isobaric peaks of Dimethon (m/z 231.0273) and Asulam (m/z 231.0434) within a full scan spectrum (Figure 3).
100 Relative Abundance 80 60
445.11548 R=75400 551.11932 R=68300
603.12616 R=64200
20 0 150 200 250 300 350 400 m/z 450
Relative Abundance
199.05530 R=114600 245.04321 169.00880 R=101600 40 R=124400
100 90 80 70 60 50 40 30 20 10 0
231.02757 R=104600 C6H16O3PS2 1.18536 ppm
231.04359 R=106400 C8H11O4N2S 0.82379 ppm
500
550
600
231.02 231.03 231.04 231.05 m/z
Figure 3: Full scan spectrum of a pesticide mixture demonstrating a resolving power of up to 100,000.
Mass Accuracy and Stability Using fully automated AGC and mass calibration procedures, mass spectra with high mass accuracy are recorded. The mass accuracy, precision and stability is equally as good as that obtained in ion trap based hybrid instruments, i.e. Thermo Scientific LTQ Orbitrap or LTQ FT Ultra.
m/z 195 RMS error: 0.45 ppm 3 deviation (ppm)
Figure 4 shows the mass accuracy and its stability over time for different molecular ions of an ESI calibration mixture. The full scan spectra were acquired at a resolution setting of 100,000 in an infusion experiment applying an external calibration, i.e. no lock masses were used.
m/z 1122 RMS error: 0.48 ppm 3 deviation (ppm) 2 1 0 -1 -2 -3
2 1 0 -1 -2 -3 0 30 60 90 time (min) 120 150
30
60
90 time (min)
120
150
m/z 524 RMS error: 0.45 ppm 3 deviation (ppm) deviation (ppm) 2 1 0 -1 -2 -3 0 30 60 3 2 1 0 -1 -2 -3
m/z 1722 RMS error: 0.63 ppm
90 120 150 0 30 60 90 time (min) time (min) Figure 4: Mass accuracy and stability of ions at different m/z values acquired in an infusion experiment without using lock masses.
120
150
Fast Polarity Switching Due to the use of a novel power supply design it is possible to perform fast polarity switching without sacrificing mass accuracy in any scans. Figure 5 demonstrates this feature by means of two experiments. In the first experiment the polarity was changed from scan to scan to check mass accuracy at fast alternating polarity
switching corresponding to a full cycle of 1 positive and 1 negative scan within 1 second. In the second experiment the polarity was switched every 5 minutes to check for potential drift effects. In both cases full scan spectra were acquired at a resolution setting of 30,000 in an infusion experiment using an ESI calibration solution applying an external calibration, i.e. no lock masses were used.
Figure 5: Mass deviations of m/z 524 (positive ions) and m/z 514 (negative ions) observed in polarity switching experiments.
Dynamic Range The dynamic range of the instrument varies by sample and with the instrument settings but it is typically about 3 to 4 orders of magnitude. Figure 6 shows that it is possible to acquire full scan spectra with an in-scan dynamic range of more than 13,000. The spectrum was acquired in an infusion experiment using a mix of Buspirone (m/z 386) and Caffeine (m/z 195).
The ratio of the Buspirone signal to the Caffeine signal is greater than 13,000. Both peaks show mass accuracies of less than 1 ppm. Thus this spectrum demonstrates not only the high in-scan dynamic range in terms of signal but also the high dynamic range in terms of mass accuracy of this instrument analogous to the performance of a hybrid LTQ Orbitrap mass spectrometer.
Figure 6: Spectrum of a mixture of Buspirone (m/z 386) and Caffeine (m/z 195) showing an in-scan dynamic range of > 13,000 and sub-ppm mass accuracies.
All Ion Fragmentation (HCD) The instrument design allows high efficiency All Ion Fragmentation experiments by means of Higher Energy Collision Induced Dissociation (HCD).
As an example, Figure 7 shows full scan spectra of Verapamil with and without HCD fragmentation and demonstrates the high fragmentation efficiency and the excellent mass accuracy of the HCD fragments.
100 80 60 40 20 0 100 80 165.0912 167.0126 239.1614
HCD off
455.2890
311.2189
O
1.3 ppm
N + N
1.2 ppm 303.2071
HCD on
O + O O
O + O N
1.4 ppm 260.1642
60 40 20 0 150
MH+ 2.1 ppm 455.2895
200
250
300 m/z
350
400
450
Figure 7: Full scan spectra of Verapamil with and without HCD fragmentation.
Applications As a result of the described performance characteristics of this new benchtop Orbitrap mass spectrometer several key applications are ideally suited to the use of the Exactive mass spectrometer. Some of these are: 1. Exact mass measurements of organic compounds 2. Early drug discovery metabolism and pharmacokinetics (DMPK) 3. General unknown screening 4. Multiple residue analysis (Pesticides, Mycotoxins, veterinary drugs) 5. Metabolomics
For all of these applications high resolution, accurate mass measurements together with high dynamic range is required for unequivocal results in full MS mode. Where it is needed, additional information, can be provided by use of by high resolution/high mass accuracy MS/MS experiments in an All Ion Fragmentation mode. Figure 8 shows an extracted ion chromatogram of 116 pesticides and mycotoxins at a level of 50 ppb in a very complex matrix of horse feed extract at a mass resolution of 50,000. This exemplifies the high selectivity and sensitivity of the instrument working in full scan mode, which is a prerequisite for a successful screening approach, since resolving matrix interferences from the target analytes is essential.
Conclusions
A new benchtop mass spectrometer has been developed based on an API ion source combined with a stand-alone Orbitrap mass analyzer. The key performance features are as follows: Mass resolutions of up to 100,000 Scan speeds of up to 10 Hz High in-scan dynamic range (4 orders of magnitude) Mass accuracies of better than 2 ppm in full scan and All Ion Fragmentation mode Fast polarity switching (full cycle of 1 positive and 1 negative scan within 1 second) High efficiency All Ion Fragmentation Higher Energy Collision Induced Dissociation (HCD) The instrument is very easy to operate and with its performance characteristics are ideally suited for discovery work, screening applications, quantitative analyses and elemental composition determinations.
Acknowledgements
We would like to thank the other Exactive project team members Frank Czemper, Florian Grosse-Coosmann, Thomas Heise, Oliver Hengelbrock, Sebastian Kanngiesser, Alexander Kholomeev, Sascha Moehring, Uwe Rickens, Ronald Seedorf and Stevan Horning.
Figure 8: Extracted ion chromatogram of 116 pesticides and mycotoxins at a level of 50 ppb in a complex matrix of horse feed extract.

www.thermo.com
Thermo Fisher Scientic (Bremen) GmbH is certied DIN EN ISO 9001:2000
AN30162_E 07/08C
Drug Residues Pharmaceuticals, Personal Care Products, and Pesticides

t
466: Detection of Pharmaceuticals, Personal Care Products, and Pesticides in Water Resources by APCI-LC-MS/MS
Detection of Pharmaceuticals, Personal Care Products, and Pesticides in Water Resources by APCI-LC-MS/MS
1
Liza Viglino1, Khadija Aboulfald1, Michle Prvost2, and Sbastien Sauv1 Department of Chemistry, Universit de Montral, Montral, QC, Canada; 2Dpartement of Civil, Geological, and Mining Engineering, cole Polytechnique de Montral, Montral, QC, Canada
Introduction
Key Words TSQ Quantum Ultra Water Analysis Solid Phase Extraction Pharmaceuticals (PhACs), personal care product compounds (PCPs), and endocrine disruptors (EDCs), such as pesticides, detected in surface and drinking waters are an issue of increasing international attention due to potential environmental impacts1,2. These compounds are distributed widely in surface waters from human and animal urine, as well as improper disposal, posing a potential health concern to humans via the consumption of drinking water. This presents a major challenge to water treatment facilities. Collectively referred to as organic wastewater contaminants (OWCs), the distribution of these emerging contaminants near sewage treatment plants (STP) is currently an area of investigation in Canada and elsewhere3,4. More specifically, some of these compounds have been detected in most effluent-receiving rivers of Ontario and Qubec5,6. However, it is not clear whether contamination is localized to areas a few meters from STP discharges or whether these compounds are distributed widely in surface waters, potentially contaminating sources of drinking water. A research project at the University of Montreals Chemistry Department and Civil, Geological, and Mining Engineering Department was undertaken to establish the occurrence and identify the major sources of these compounds in drinking water intakes in surface waters in the Montreal region. The identification and quantification of PhACs, PCPs, and EDCs is critical to determine the need for advanced processes such as ozonation and adsorption in treatment upgrades. The establishment of occurrence data is challenging because of: (1) the large number and chemical diversity of the compounds of interest; (2) the need to quantify low levels in an organic matrix; and (3) the complexity of sample concentration techniques. To address these issues, scientists traditionally use a solid phase extraction (SPE) method to concentrate the analytes and remove matrix components. After extraction, several different analytical techniques may perform the actual detection such as GC-MS/MS and more recently, LC-MS/MS7,8. Another analytical challenge resides in the different physicochemical characteristics and wide polarity range of organic compounds making simultaneous preconcentration, chromatography separation, and determination difficult. Analytical
methods capable of detecting multiple classes of emerging contaminants would be very useful to any environmental monitoring program. However, up to now, it has often been a necessity to employ a combination of multiple analytical techniques in order to cover a wide range of trace contaminants9. This can add significant costs to analyses, including equipment, labor, and time investments.
Goals
To develop a simple method for the simultaneous determination of trace levels of compounds from a diverse group of pharmaceuticals, pesticides, and personal care products using SPE and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Determine which selected substances are present in significant quantities in the water resources around the Montreal region.
Materials and Method

Analyte selection
Compounds were selected from a list of the mostfrequently encountered OWCs in Canada4-6 (Figure 1).
Sample collection
Raw water samples were taken from the Mille Iles, des Prairies, and St-Laurent rivers. Three samples were collected at the same time from each river in pre-cleaned, four-liter glass bottles and kept on ice while being transported to the laboratory. These water sources vary widely due to wastewater contamination and sewer overflow discharges. All samples were acidified with H2SO4 for sample preservation and stored in the dark at 4 C. Immediately before analysis, samples were filtered using 0.7 m poresize fiberglass filters followed by 0.45 m pore size mixedcellulose membranes (Millipore, MA, USA). Samples were extracted within 24 hours of collection.
Personal Care Products

Caffeine (CAF) MW: 194.19, pKa =10.4, Stimulant
Hormones
Estrone MW: 270.4 Estrogen
Triclocarban (TCC) MW: 315.19, Anti-bacterial agent Estriol MW: 288.4 Estrogen
Pharmaceuticals
Carbamazepine (CBZ) MW: 236.27, pKa=13.9, Anticonvulsant Clofibric acid MW: 214.65 Metabolite lipid regulator
Estradiol MW: 272.4 Estrogen
Progesterone MW: 314.15 Progestogen
H H H O
H3C O
Gemfibrozil (GEM) MW: 250.33, Anti-cholesterol
CH3 H3C CH3 OH O
H H O H
CH3 O
CH3
Salicylic acid MW: 138.12 Metabolite of acetylsalicylic acid (aspirin)
Pesticide
Atrazine (ATRA) MW: 215.68, pKa =1.7, Herbicide
Trimethoprim (TRI) MW: 290.30, pKa=7.12 Anti-infective Figure 1: Molecular structures of selected compounds
Concentration and Extraction Procedure

The solid phase extraction procedure is illustrated in Figure 2. Briefly, analytes were concentrated and extracted using a 200 mg C18-like analytical cartridge. Retained analytes were eluted from the cartridges using 3 mL MTBE:MeOH 90/10 and 3 mL MeOH. They were then collected on the conical-bottom centrifuge tube for evaporation to dryness with N2 (g). Extracted analytes were reconstituted to 200 L with 90% water/formic acid 0.1% and 5% MeOH solution containing the internal standards. Search | Contents | Index
Naproxen (NAPRO) MW: 230.26, pKa=4.15 Analgesic
OH
17--Ethinylestradiol MW: 296.4 Synthetic estrogen
LC-MS/MS conditions
HPLC separation was done with a Thermo Scientific Surveyor HPLC system. Separation conditions are given in Table 1. Detection and quantification of the analytes were performed with a Thermo Scientific TSQ Quantum Ultra triple stage quadrupole mass spectrometer using selective reaction monitoring (SRM) (Table 2). Preliminary experiments were performed with two atmospheric pressure ionization (API) sources ESI and APCI to detect all compounds. Although some compounds showed a slightly higher intensity with the ESI source (i.e. atrazine), APCI was selected because of the higher sensitivity provided for steroids. This endocrine disruption class is an important analytical challenge due to the low detection limits (1 ng/L) required for the determination of these compounds. These compounds are known to affect the living organisms at very low concentrations. Given that the aim was to develop a simple analytical method to detect as wide a range of compounds as possible, we selected the APCI source. The small loss in sensitivity for some easily measured molecules was more than compensated by the gain in sensitivity for other compounds that could not have been detected using ESI. Moreover, APCI ionization is known in some cases to be less susceptible to matrix interferences than ESI ionization10. Lastly, some authors demonstrated signal suppression for analysis of various organic waste compounds in water samples using ESI-LC-MS/MS11. The identification of analytes was confirmed by the LC retention time12,13. Instrument control and data acquisition were performed with Thermo Scientific Xcalibur software.
Solid Phase Extraction (200 mg, C18-like SPE cartridges)
1 Conditioning:
MTBE (3 mL) MeOH (3 mL) Reagent water (3 mL)

Surrogate [13C3]-caffeine
2 Load:
1 L filtered sample
3 Washing:
Reagent water 3 mL
4 Drying:
Nitrogen 40 min
5 Elution:
10/90 MeOH/MTBE (3 mL) MeOH (3 mL)

Internal standard [13C3]-atrazine and [13C3]-estradiol
6 Evaporate:
Nitrogen (Vf = 200 L)
7 Detection:
LC-APCI-MS/MS Analysis
Figure 2: SPE enrichment procedure
Table 1: Instrument Parameters
HP LC Column: Column temperature: Mobile phase A: Mobile phase B: Injection volume: Flow rate: Gradient:
Thermo Scientific Hypersil GOLD (50 x 2.1 mm, 3 m) 30 C 0.1% Formic acid/H2O MeOH 20 L 500 L/min T=0, A=90%, B=10% T=1, A=90%, B=10% T=15, A=1%, B=99% T=16.5, A=1%, B=99% T=17, A=90%, B=10% T=22, A=90%, B=10%
MS Ionization mode: Discharge current: Vaporizer temperature: Capillary temperature: Sheath gas pressure: Aux. gas pressure: Collision gas pressure: Source CID:
APCI+ 3 A 500 C 250 C 40 arb units 20 arb units 1.5 mTorr -10 V
APCI4 A 500 C 250 C 30 arb units 15 arb units 1.5 mTorr 15 V
Table 2: SRM transitions used for detection and quantification
Compound Trimethoprim Caffeine Estriol Carbamazepine Atrazine Naproxen 17--Ethinylestradiol Estradiol Estrone Progesterone TCC Gemfibrozil Salicylic acid* Clofibric acid* *APCI-
Precursor ion (m/z) 291.16 195.10 271.24 237.11 216.11 231.11 279.16 255.16 271.24 315.26 316.99 251.09 137.04 213.17
Product ion (m/z) 230.16 138.10 157.10 194.10 174.10 185.10 133.10 159.10 157.10 109.10 127.04 129.10 93.10 127.10
CE (eV) Tube lens (V) 22 90 18 77 18 80 20 80 34 97 13 101 31 86 17 79 18 80 38 118 32 99 20 118 31 72 32 102
Figure 3: Mean recoveries for the extraction of selected compounds using C18-like cartridges (spiked in Milli-Q water and Mille Iles River water at 50 ng/L, n=6)
The compounds of interest were investigated using Reproducibility (%RSD), ranging from 3% to 11% for all samples from various surface waters. Figure 4 shows representative LC-MS/MS chromatograms of selected analytes, was very good. Accuracy (recovery percentages), compounds in surface water. The concentrations are ranging between 72% to 94% for all compounds in illustrated in Figure 5. The selected compounds were spiked matrix, was satisfactory and indicated high performance of our method. Results are shown in Table 3. detected in all river samples at various concentrations depending on sampling locations (Figure 5 a and b). Matrix effects are very important when developing an The highest concentrations were found for caffeine LC-MS/MS method and can affect reproducibility and accuracy14. This phenomenon was evaluated by comparing (16-24 ng/L), atrazine (1.5-39 ng/L), salycilic acid (10-33 ng/L) and gemfibrozil (4-14 ng/L). The lowest recovery percentages in Milli-Q water and surface water concentrations were found for carbamazepine (3-5 ng/L), samples (Mille Iles River) spiked at 50 ng/L (n = 6). We clofibric acid, and two hormones (progesterone and can consider a very low matrix effect in surface waters estradiol). Trimethoprim, triclocarban and other selected since signal suppression varies from 1% to 13%, except hormones were detected at trace levels (Trace limit of for atrazine and TCC showing an enhancement signal of detection). 6% and 2%, respectively (Figure 3). Overall, concentrations of most of the compounds Good linearity in surface water samples was observed analysed were similar to those reported from other areas over a concentration range from <LOD to 100 ng/L with in Canada and Europe3,4. correlation coefficients greater than 0.99 for all compounds. Detection limits in surface water were in the range of 0.03 to 2 ng/L (Table 3). Search | Contents | Index
Table 3: Retention time, limit of detection (LOD), linearity, recoveries and RSD (%) data for each detected compounds in tap water.
Compound Trimethoprim Caffeine Estriol Carbamazepine Atrazine Naproxen 17--Ethinylestradiol Estradiol Estrone Progesterone TCC Gemfibrozil Salicylic acid Clofibric acid
Retention time (min) 5.46 5.79 10.14 10.76 11.41 12.62 12.85 12.88 12.94 14.44 15.10 15.17 8.82 12.00
LOD* (ng/L) 0.50 0.07 0.30 0.09 0.03 2.00 0.50 0.10 0.60 0.08 0.20 2.00 0.90 0.60
R2** 0.9998 0.9995 0.9981 0.9999 0.9995 0.9996 0.9931 0.9979 0.9989 0.9994 0.9970 0.9991 0.9993 0.9989
Recovery***(%) 91 87 84 86 86 85 73 72 79 94 81 84 77 83
RSD (%) 7 9 9 5 3 9 10 6 9 4 10 6 6 11
*LOD in surface water (Mille Iles River) **Value for calibration line in river water (0-100 ng/L) ***Recoveries over the total method (surface samples spiked at 50 ng/L, n = 6).
Caffeine 195.10 138.10 Salicylic acid 137.04 91.10
Carbamazepine 237.11 194.10 Atrazine 216.11 174.10 Clofibric acid 213.17 127.10 Naproxen 231.11 185.10 Estradiol 255.16 159.10
* *
Gemfibrozil 251.09 129.10
Figure 4: Representative SRM chromatograms of some selected compounds detected in water matrix (Mille Iles River). Peak due to interferences are marked by asterisks (*)
Conclusion
We developed and successfully applied an APCI-LC-MS/MS method for quantifying a wide range of compounds from a diverse group of pharmaceuticals, pesticides, and personal care products at concentration in the low ng/L range in surface waters with good precision and accuracy. Results confirmed the presence of pharmaceuticals, personal care products, and endocrine disruptors in all water resources around the region of Montreal. The concentrations of compounds fluctuated with sampling locations due to the variation of these sources, wastewater contamination and combined sewer overflow discharges.
References
1. T. A. Ternes and A. Joss, Eds., Human Pharmaceuticals, Hormones and Fragrances: The Challenge of Micropollutants in Urban Water Management, IWA, 2006, p 443. 2. A. Kortenkamp, Env. Health Perspective, 2007, pp 1-42. 3. C. Hao, L. Lissemore, B. Nguyen, S. Kleywegt, P. Yang, K. Solomon, Anal. Bioanal. Chem., 2006, 384, 505-513. 4. C. D. Metcalfe, X-S Mia, W. Hua, R. Letcher, M. Servos, Pharmaceuticals in the Canadian Environment. In Pharmaceuticals in the Environment: Sources, Fate, Effects and Risks, second edition; K. Kummerer, Ed.; SpringerVerlag, 2004, 67-87. 5. F. Gagn, C. Blaise, C. Andr, Ecotoxicology and Environmental Safety 2006, 64, 329-336. 6. P. A. Segura, A. Garcia-Ac, A. Lajeunesse, D. Ghosh, C. Gagnon and S. Sauv, J. Environ. Monitor., 2007, 9, 307-313. 7. R. A. Trenholm, B. J. Vanderford, J. C. Holady, D. J. Rexing and S. A. Snyder, Chemosphere, 2006, 65(11), 1990-1998. 8. C. Zwiener and F. H. Frimmel, Anal. Bioanal. Chem, 2004, 378, 862-874. 9. T. A. Ternes, Trends Anal. Chem., 2001, 20(8), 419-434. 10. S. Souverain, S. Rudaz, J-L. Venthey, J. Chrom. A, 2004, 1058, 61-66.
Figure 5: (a) The highest mean concentrations of selected compounds in water samples collected from Mille-Iles River, des Prairies River and St-Laurent River (n = 6). (b) The lowest mean concentrations of selected compounds in water samples collected from Mille Iles River, des Prairies River and St-Laurent River (n = 6).
11. B. J. Vanderford, R. A. Pearson, D. J. Rexing and S. A. Snyder, Anal. Chem., 2003, 62656274. 12. J. B. Schilling, S. P. Cepa, S. D. Menacherry, L. T. Barda, B. M. Heard and B. L. Stockwell, Anal. Chem., 1996, 68, 1905. 13. L. Y. T. Li, D. A. Campbell, P. K. Bennett and J. D. Henion, Anal. Chem., 1996, 68, 3397. 14. T. Reemtsa, Trends Anal. Chem., 2001, 20, 533-542.
www.thermo.com
Legal Notices 2009 Thermo Fisher Scientic Inc. All rights reserved. Milli-Q is a registered trademark of Millipore Corporation. All other trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientic Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

AN63041_E 06/09S
Natural Compounds Flavanoids

420: Metabolomic Analysis of Green and Black Tea Extracts Using the Thermo Scientific LTQ Orbitrap XL Hybrid Linear Ion Trap Mass Spectrometer
Metabolomic Analysis of Green and Black Tea Extracts Using an LTQ Orbitrap XL Hybrid Linear Ion Trap Mass Spectrometer
Donna L. Wilson and Charles Yang; Thermo Fisher Scientic, San Jose, CA, USA
Overview
Key Words Accela High Speed UHPLC SIEVE Software Higher Energy Collisional Dissociation (HCD) Mass Frontier Software Natural Product Analysis Purpose: To show a complete analytical metabolomic workow including (1) data acquisition using a high resolution accurate mass instrument that is equipped with a Higher Energy Collisional Dissociation (HCD) cell and coupled to a high pressure LC (Figure 1), (2) metabolite differential abundance analysis, and (3) structural elucidation of relevant metabolites using accurate mass and HCD fragmentation information to highlight the component differences between green and black tea. Methods: Green and black tea extracts were analyzed using the Thermo Scientic LTQ Orbitrap XL with an HCD cell. Chromatography was performed using the Thermo Scientic Accela High Speed LC equipped with a 2.1 mm ID Thermo Scientic Hypersil GOLD column packed with 1.9 m particles. Data Dependent analysis was performed using an LTQ Orbitrap XL with full scan data acquired at a resolving power of 30,000 and MSn data acquired at a resolving power of 7500 following HCD fragmentation. Results: The study included a comparative analysis of green and black tea using differential analysis software to identify compositional variations between the two tea samples. Using a UHPLC coupled with a small particle column afforded a fast analysis time while maintaining very high chromatographic resolution. The high mass accuracy data (better than 3 ppm with external calibration) was used to determine elemental composition and for tentative identication of compounds via database searching. HCD fragmentation facilitated structural identication and conrmation. This was demonstrated with the example of epigallocatechin gallate (EGCG).
Introduction
Metabolomics, the comprehensive and quantitative analysis of wide arrays of metabolites in biological samples, marks promising new research territory. The numerous analytes in these samples have diverse chemistries and polarities. In addition, metabolites occur at a range of concentrations within a particular sample. Consequently, comprehensive metabolomics investigations create many analytical challenges that can be addressed using LC-MS/MS. Tea contains a wide range of components including vitamins, amino acids, and polyphenols, many of which are structurally similar and may differ only in the type and location of a side chain. The use of high resolution chromatography is essential for the separation of such a complex mixture. Furthermore, acquisition of accurate mass data in both full scan and MSn modes enables complete structural characterization. Here, we highlight an untargeted metabolomic workow from data acquisition through metabolite ID. The study included differential and structural characterization of polyphenolic catechin (avan-3-ol) derivatives and theaavin components of green tea and black tea.
Methods
Samples Green tea and black tea aqueous extracts were examined without any pre-treatment. Each sample was analyzed in quadruplicate.
Figure 1: Schematic of the LTQ Orbitrap XL mass spectrometer with an HCD collision cell. The LTQ Orbitrap XL features an HCD collision cell to provide additional exibility for low level components in complex mixtures. Ions can be selected in the linear ion trap and fragmented either in the ion trap (CID) or in the HCD collision cell. For HCD, ions are passed through the C-trap into the gas-lled collision cell, providing high sensitivity and high signal-to-noise fragmentation.
Chromatography Conditions Chromatographic separation was performed using the Thermo Scientic Accela UHPLC system. The LC conditions were as follows: Column: Thermo Scientic Hypersil GOLD, 100 2.1 mm, 1.9 m particle size Mobile phase: (A) water with 0.1% formic acid; (B) acetonitrile with 0.1% formic acid Flow rate: 500 L/min Injection volume: 10 L Gradient: Linear gradient of 100%1% A over 20 minutes Mass Spectrometry Conditions MS analysis was carried out using the Thermo Scientic LTQ Orbitrap XL mass spectrometer. The MS conditions were as follows: Positive electrospray ion source voltage: 5.0 kV All methods: Full scan MS in the Orbitrap with a mass resolution of 30,000. Data Dependent MS/MS in the Orbitrap on the top three most intense ions from the full scan at a mass resolution of 7500.
Results
Considerable interest has developed in the potential health benets of teas, particularly in the antioxidant and other properties of some of the polyphenolic catechins and theaavins (Figure 2). The analysis described here focused on detection, relative quantitation, and identication of these low molecular weight components in green and black tea samples. The HPLC separation of tea samples shows excellent peak separation and low noise, with most components eluting in less than 10 min. High resolution full scan spectra were acquired at a mass accuracy of better than 3 ppm. After data acquisition, SIEVE software was used to determine statistically signicant differences in the metabolite proles of green and black tea samples (Figure 3). By comparing peak intensities between the two chromatographically aligned samples, metabolites present at different levels in the two teas were identied.
OH OH HO O
O OH
B
R1 O R2
OH
O R1 OH
A
OH
OH OH
O HO HO O OH
OH O
Epicatechin (EC) Structures

OH OH HO O R1 O R2 OH
Galloyl (gal) Unit
R1 = H or OH R2 = H or gal
OH
O R2
Theaflavin Structures
Theaflavin 3,3digallate gal gal 868
Catechin (C) Structures

EC or C R1 R2 MW (amu) H H 290 EGC or GC OH H 306 ECG or CG H gal 442 EGCG or GCG OH gal 458 R1 R2 MW (amu)
Theaflavin H H 564
Theaflavin 3monogallate H gal 716
Theaflavin 3monogallate gal H 716
OH
HO OH
A
H
OH
HO
R1
H+
HO
R1
H+
B
R1
O R2 OH
OH
O R2
OH
O R2
OH
m/z 139, A-ring ion
B-ring ion C,EC; R1=R2-H; m/z 152 GC,EGC; R1 =OH, R2=H; m/z 168 ECG; R1=H, R2=gal; m/z 304 EGCG; R1=OH, R2=gal; m/z 320
For theaflavin derivatives the proton is retained by the benzotropolone part (or Bring part) presenting the larger aromaticity than the A-ring part.
OH O OH O OH
R1 =
OH HO O OH O OH
A and B-ring ions formed by retro-Diels-Alder (RDA) mechanism in catechin polyphenols.
RDA fragmentation mechanism for theaflavin polyphenols.
Figure 2: Three types of tea are produced from Camellia sinensis leaves: green tea (nonfermented), oolong tea (semi-fermented), and black tea (fermented). Catechins are polyphenolic antioxidant plant metabolites of the class avanoids called avan-3-ols and are highly present in tea plants. Fermentation induces enzymatic oxidation of avan-3-ols and leads to the formation of two major pigments in black tea, theaavins (TFs) and thearubigns (TRs). Catechins are expected to be more abundant in green tea and theaavins more abundant in black tea. The proposed fragmentation pathway for these compounds proceeds via a Retro-Diels-Alder rearrangement as outlined here.
After differentially abundant metabolites of interest were detected, the accurate mass and the MSn data were used for structural identication. The elemental formula, as determined by the accurate mass data, and the accurate mass value itself were used for metabolite database searching. The EGCG metabolite was tentatively assigned using this combination of tools.
Further metabolite characterization was accomplished using MSn spectra and Mass Frontier software. Mass Frontier allowed condent metabolite identication using its comprehensive spectral library and predictive fragmentation algorithms to facilitate structural elucidation (Figure 4). The compounds in Figure 3 were identied using this workow.
A)
Green Tea Blue Black Tea Red
Catechins Eluting
Theaflavins Eluting
B)
EC or C Ratio = 1.97
EGC or GC Ratio = 4.53
ECG or CG Ratio = 0.94
EGCG or GCG Ratio = 2.91
Catechins
Theaflavin Ratio = 0.07 Theaflavin 3-monogallate Ratio = 0.03 Theaflavin 3,3-digallate Ratio = 0.01 Caffeine Ratio = 0.56
Theaflavins
Caffeine
Figure 3: Differential metabolite abundance analysis with SIEVE software. A) Chromatographic alignment of the various LC sample traces is the rst step in the SIEVE process. Differences between the green tea samples (Blue) and the black tea samples (Red) can be identied. The Accela UHPLC provided highly resolved chromatographic peaks and high signal-to-noise ratios. B) After alignment, the corresponding peak intensities are compared for green tea (Blue) and black tea (Red). The relative abundances of several compounds of interest are shown with their abundance ratios. These metabolites were identied using a combination of accurate mass database searching and MSn spectra interpretation via Mass Frontier software.
Figure 4: A metabolite of interest from Figure 3 was chosen for further characterization. This ion was present at levels ~3 times that of black tea and was identied as the potent antioxidant, EGCG. Accurate mass was used to identify the metabolite via database searching, and Mass Frontier software was used to conrm the EGCG identication by using the fragmentation spectra of the parent ion.
Conclusions
The analytical metabolomic workow described here encompasses data acquisition, discovery of differentially abundant metabolites, and metabolite identication. The LTQ Orbitrap XL coupled to an Accela U-HPLC system afforded fast analysis times while maintaining high chromatographic resolution. Accurate mass measurements increased the condence in elemental composition assignments and ultimately metabolite identication. Thermo Scientic SIEVE differential analysis software enabled large-scale evaluation of multiple complex LC-MS data and comparison of metabolite proles between green and black tea samples. The spectral database and fragmentation algorithms of Mass Frontier software facilitated structural assignments of metabolites of interest utilizing MSn fragmentation spectra.
References
Menet, MC., Sang, S. Yang, C.S., Ho, CT, and Rosen, R.T., Analysis of Theaavins and Thearubigins from Black Tea Extract by MALDI-TOF Mass Spectrometry. J. Agric. Food Chem. 2004, 52, 2455-2461.
www.thermo.com
Legal Notices 2008 Thermo Fisher Scientic Inc. All rights reserved. Mass Frontier is a trademark of HighChem, Ltd. All other trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientic Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
AN62740_E 04/08S
Natural Compounds Flavonoids

t
30173: Direct Analysis of Red Wine Using Ultra-Fast Chromatography and High Resolution Mass Spectrometry
Direct Analysis of Red Wine Using Ultra-Fast Chromatography and High Resolution Mass Spectrometry
Eugen Damoc, Michaela Scigelova, Anastassios E. Giannakopulos, Thomas Moehring, Thermo Fisher Scientic, Hanna-Kunath-Str. 11, 28199 Bremen, Germany Frantisek Pehal, NNH Hospital Na Homolce, Roentgenova 2, 150 30 Prague, Czech Republic Martin Hornshaw, Thermo Fisher Scientic, 1 Boundary Park, Hemel Hempstead HP2 7GE, UK
Overview
Key Words LTQ Orbitrap U-HPLC Flavonoids Red Wine Red wine is a very complex mixture and a rich source of beneficial anti-oxidants. Identification and quantitation of these natural products is challenging. Ultra High Pressure Liquid Chromatography (U-HPLC) coupled to the Thermo Scientific LTQ Orbitrap XL mass spectrometer was used for analysis of French red wine, which enabled simultaneous detection and relative quantitation of the wine's anti-oxidant constituents. The phenolic compounds (such as quercetin) responsible for most of the health benefits associated with the consumption of red wine were identified and their variable content across two different harvest years was observed. Direct wine analysis approach was then applied to monitor the progressive changes in red wine after its exposure to air. This work demonstrated the feasibility of analyzing complex mixtures without any prior sample preparation by making use of the high resolving power of both U-HPLC and the Orbitrap mass analyzer detector.
Introduction
Free radicals derived from molecular oxygen are considered major causative agents of tissue damage.1,2 Both recent and historical evidence suggests that regular drinking of wine in moderation has a positive impact on human health thanks to its high content of antioxidants.3,4 Red wine in particular contains a complex mixture of phenolic compounds which are important contributors to the organoleptic quality of wines as well as essential components in the evolution of wine. Quercetin is of special interest for its commercial use as an anti-oxidant food supplement with a proven record of promoting vascular relaxation, inhibiting human platelet aggregation in vitro, and modulating eicosanoid synthesis towards a pattern likely to be protective against coronary heart disease.5 Reversed-phase HPLC is well established for the analysis of flavonoids in red wine, including quantitative analysis.6,7,8 Coupling reversed-phase HPLC to a mass spectrometer adds considerable benefits such as the ability to: 1) analyze complex mixtures without much sample fractionation 2) monitor hundreds of compounds in a single analysis over a wide dynamic range of concentrations 3) provide an unambiguous identification and structural characterization of the compounds based on accurate mass measurement and informative fragmentation spectra.
Recent advances in both HPLC and mass spectrometry techniques are having a significant impact on the analyses of complex mixtures such as those represented by food and agricultural products. First, the use of small particles (< 2 m) in HPLC columns can provide remarkable increase in speed of analysis while maintaining or even improving the separation efficiency. Second, the new generation of powerful but easy-to-use hybrid mass spectrometers, like the LTQ Orbitrap XL, combines extremely high mass accuracy and resolution with the capability of multiple levels of fragmentation.9 The combination of these powerful techniques provides a robust and confident means of profiling complex mixtures as well as successful identification and advanced structural characterization of detected compounds. As a result, we are seeing rapidly growing interest in the area of metabolomic analysis being applied in nutrition and health research.10,11 We investigated the potential of a direct analysis of red wine using U-HPLC coupled to a linear ion trap Orbitrap hybrid mass spectrometer. Of particular interest was the ability of the designed workflow to pinpoint statistically significant differences between individual harvest years for wines of a specific origin (area, label). In addition to that, we used the developed methodology to monitor the trend in oxidative changes of red wine after exposure to air.
Methods
Two bottles of French red wine Les Charmes de Kirwan, Margaux (cuvee, Bordeaux region, France), years 2003 and 2005, were obtained from a specialized wine merchant. The wine was stored at room temperature in the dark until analyzed. Immediately after opening the bottle, a glass vial (20 mL) was filled with the wine to the very top, quickly closed to ensure minimum oxidation, and stored at 4C in the dark. This sample was collected just in case there was a need for repeated analysis of the profiling experiments or structural elucidation studies. A second 20 mL aliquot of wine was poured from the original bottle into a glass beaker. From this beaker a sample vial was immediately filled to the rim and placed in the chilled (4 C) Thermo Scientific Accela autosampler tray, awaiting analysis. For a wine oxidation trend analysis, further samples were taken from this open beaker 1, 5 and 24 hours after the bottle opening. Chromatography was performed using an Accela U-HPLC injecting 20 L sample from a cooled tray (4C) directly onto a Thermo Scientific Hypersil GOLD column (2.1 mm x 100 mm, 1.9 m particles, equilibrated in 95% solvent A (0.1% aqueous solution of formic acid), 5% solvent B (acetonitrile containing 0.1% formic acid). The compounds were eluted using flow rate 300 L/min by linearly increasing solvent B concentration from 5% to final 40% over 15 min, and from 40% to 95% over 1 min. The column was then washed with 95% solvent B (2 min) and re-equilibrated in 95% solvent A, 5% solvent B. The total run time, including column wash and equilibration, was 20 min. A Thermo Scientific LTQ Orbitrap XL mass spectrometer was operated in positive ion mode at 30,000 resolving power (defined as FWHM @ m/z 400) for full scan analysis (mass range 150 1500 u) followed by data dependent MS/MS on the most intense ion from the full scan at 7,500 resolving power (~0.3 sec per scan). The measurements were done in triplicate with external calibration. The settings for the higher energy collisional dissociation (HCD) fragmentation mode were 65% normalized collision energy, isolation width 3 u. Thermo Scientific SIEVE 1.2 software was used for comparative and trend analyses. The software allows for processing a large number of samples, presenting the statistically significant differences between populations and various time points. Data were normalized on total spectral ion current. Results were filtered using pValue < 0.001 and at the same time requiring a minimum 2-fold change in peak height. The results from SIEVE were further subjected to multivariate analysis with SIMCA P+, version 11 (Umetrics, Umea, Sweden). Mass Frontier (HighChem, Slovakia) software was used to confirm a suggested compound identity and structure based on observed fragmentation patterns.
Results
Due to the large number and the chemical complexity of phenolic compounds in wine matrix, analytical methods in the past involved sometimes difficult and complicated traditional chromatographic techniques. One of the major problems underlying separation of the phenolic compounds is their similarity in chemical characteristics. As many phenolics show similar UV spectra with maxima in a narrow range of 280-320 nm, extensive fractionation steps might be needed prior to HPLC analysis. Rather large initial volumes required and variable losses occuring due to incomplete extraction or oxidation can be an issue. The use of modern chromatographic techniques coupled to mass spectrometric detection can alleviate these problems. Our approach avoids entirely the sample fractionation step: red wine is injected directly on the reverse phase column. Moreover, the use of small particles (< 2 m) and relatively high flow rates (300 L/min) enable swift analysis with excellent chromatographic resolution. The observed peak width for individual compounds was, on average, 7 sec, back pressure not exceeding 350 bar. With 20 min total cycle time per injection, this setup allows for high throughput analysis while the total sample consumption remains negligible (20 L per injection). U-HPLC coupled to the LTQ Orbitrap XL proved to be very robust, allowing for an uninterrupted analysis of 24 untreated red wine samples which corresponds to an 8-hour continuous analysis without any requirement for a system cleanup or column change.
Figure 1: Overview of differences between harvest years 2003 and 2005. The result from differential analysis software (SIEVE 1.2) highlights the compounds having at least two-fold higher concentration in year 2005 compared to year 2003 ( blue shaded area) and compounds whose concentration in year 2005 was less than a half of that in year 2003 ( red shaded area). The purple horizontal line represents 1:1 ratio between concentrations in the year 2005 and 2003. The grayed area covers the features with less pronounced concentration difference and those with low statistical significance, i.e. pValue > 0.001.
Variables like wine varietals, soil composition, and harvest year will play an important role by providing the basic pool of compounds for these biotransformations. With accurate mass acting as a highly selective filter we could monitor hundreds of compounds across multiple samples, enabling advanced comparative studies and trend analyses. Initially, we were interested in comparing the wine of the same origin (area, label) but harvested in different years. Our differential analysis of the Les Charmes de Kirwan, Margaux, contrasted wine from production years 2003 and 2005 using SIEVE software. The features (peaks) were filtered for their statistical significance (pValue < 0.001) and significant change defined as a minimum 2-fold concentration difference between the two harvest years (Figure 1). We observed 75 individual compounds which showed at least 2-fold higher content in year 2005 compared to year 2003 (blue shaded area in Figure 1). Kaempferol and quercetin concentration increased 25- and 8-fold, respectively, in year 2005 compared to 2003 (Figure 2). On the other hand, there were 36 other compounds whose concentration in the 2005 sample was significantly less than in the 2003 sample (red shaded area in Figure 1). Some flavonoids (myricetin) showed no change in concentration between the two harvest years.
Total anti-oxidant status refers to overall antioxidant properties of wine, and can be largely ascribed to a group of compounds comprising vanillic acid, trans-polydatin, catechin, m-coumaric acid, epicatechin, quercetin, cis-polydatin and trans-resveratrol.12 In our analysis we detected vanillic acid, (epi)catechin, coumaric acid, and quercetin. When compared to wine produced in 2003, the wine produced in 2005 contained 50, 40 and 20% less coumaric acid, vanillic acid and (epi)catechin, respectively, while the amount of quercetin increased 8-fold (Table 1).
Calc m/z Formula MW Name Change 24h/0h Change 2003/2005
165.0546 169.0495 199.0601 391.1387 229.0859 291.0863 303.0499 319.0448 287.0550
C9H8O3 C8H8O4 C9H10O5 C20H22O8 C14H12O3 C15H14O6 C15H10O7 C15H10O8 C15H10O6
Coumaric acid Vanillic acid Syringic acid Polydatin Resveratrol (Epi)catechin Quercetin Myricetin Kaempferol
0.32 0.40 0.59 Not found Not found 0.66 0.78 0.69 1.00
0.51 0.61 0.92 Not found Not found 0.82 7.96 1.00 20.96
Table 1: Overview of some compounds of interest and the changes in their content between year 2003 and 2005 (column Change 2003/2005), and after 24 hours following exposure to air (column Change 24h/0h). The compounds highlighted are the major contributors to the total anti-oxidant status.12
The remarkable difference in the content of quercetin between the two harvest years is interesting. Quercetin is one of the most abundant natural flavonoids found in fruits, vegetables and wine.
Figure 2: Extracted ion chromatogram for kaempferol and quercetin (left and right pane, respectively; 3 injections each) shows remarkable difference in concentration of these compound in wine harvested in year 2005 (blue trace) and 2003 (red trace). The mass deviation did not exceed 0.7 ppm for kaempherol (calculated m/z = 287.0550) and 1.3 ppm for quercetin (calculated m/z = 303.0499). Note the reproducibility of the retention time (RT) values and peak height calculations (AH) for 3 replicate injections.
At present, labeling requirements for red wine are far from comprehensive, basically limited to listing the total alcohol content and the comment that it contains sulfites. Including more specific information about compounds with strong anti-oxidant properties would improve a general public awareness and be helpful in the current climate of debate on healthy balanced diet. A fast but highly informative analysis of wines as described herein can thus help maintain consistency and quality, and provide useful information about products nutritional value. Reliable accurate mass measurements over a broad dynamic range of concentration are helpful for unambiguous identification of compounds of interest. The mass deviation of our measurements did not exceed 2 ppm using external calibration. Such an accuracy supported by reliably measured isotope abundancies in the LTQ Orbitrap XL enabled a confident assignment of elemental composition to individual peaks. For confident identification of a compound, the elemental composition suggestions based on mass accuracy need to be complemented with the evidence from the fragmentation spectra. Our method was set up to collect higher energy collision dissociation (HCD) spectra. On average 700 such spectra were collected during each 20-minute LC-MS run. The MS/MS spectrum acquired in the multipole collision cell of the LTQ Orbitrap XL serves for confirming identity of a known compound or even determining identity of an unknown. Such an approach was demonstrated for the analysis of antioxidant compounds in olive oil.13 Rich fragmentation, accurate mass measurement of both parent and fragment ions, and spectrum interpretation provided by Mass Frontier software were all crucial for this challenging task (Figure 3).
The anti-oxidant properties of wine are clearly beneficial to a consumer. On the other hand, wines with higher polyphenolic concentration are more susceptible to oxidation. We were interested to observe a trend of changes in the wine samples over the period of 24 hours after opening the bottle. The groups of samples from time points 0, 1, 5, and 24 hours (triplicate injections) were processed with SIEVE and further subjected to principal component analysis. The progressive changes caused by exposure to air are well observable and statistically significant (Figure 4).
Figure 4: Wine sampled in triplicate at 0, 1, 5, and 24 hours after exposure to air. The sample groups are easily separated by the first two principal components.
Figure 3: Confirmation and structural characterization of quercetin. Assignment of fragments in HCD spectrum using the Mass Frontier software relying on its extensive database of fragmentation mechanisms.
At this point, a potential effect of evaporation of more volatile constituents of wine has to be considered. In general, the partial pressure for compounds with molecular weight 300 and higher is considered negligible such compounds should not be lost to evaporation at room temperature. Kaempferol (MW 286), (epi)catechin (MW 290), myricetin (MW 318) and quercetin (MW 302) would fall into such a category. Kaempferol showed no change over this period. Thus the decrease of 20% for quercetin and 30% for (epi)catechin and myricetin observed over the period of 24 hours following the bottle opening could be confidently ascribed to oxidation. For coumaric (MW 164), vanillic (MW 168) and syringic (MW 198) acids we observed a more pronounced drop in concentration (60, 70 and 40%, respectively) after 24 hours following the exposure to air (Figure 5). It might prove difficult, however, to distinguish between the effect of evaporation and oxidation under the employed experimental conditions.
Figure 5: Changes over a 24-hour period of air exposure. The amount of a given compound at time 0 h defined as 100%. Panel A shows decrease in content of higher molecular weight compounds such as (epi)catechin (MW 290), myricetin (MW 318) and quercetin (MW 302). Lower molecular weight components including coumaric (MW 164), vanillic (MW 168) and syringic (MW 198) acid display much higher rate of disappearance (panel B). Error bars show the standard deviation for three repetitive analyses of samples at each time point.
Conclusions
As consumers are becoming increasingly aware of the harmful as well as helpful content of what they eat and drink, modern powerful analytical tools will undoubtedly play a crucial role to supply that information more accurately and quickly. Albeit a very complex mixture, red wine is perfectly suitable for mass spectrometric supported by SIEVE differential expression software. Such fingerprinting analysis can be applied in quality control and process monitoring, and for highlighting relevant nutritional value to consumers. U-HPLC affords fast analysis times while maintaining very high chromatographic resolution (peak width 7 seconds at half height). The mass deviation of the LTQ Orbitrap XL measurements was always smaller than 2 ppm using external calibration up to one day old. Higher collision energy dissociation MS/MS spectra confirm the identity and structure of compounds in complex mixtures. Accurate mass measurements also significantly improve the precision of quantitation by eliminating nearly isobaric interferences. This is a particularly important aspect for complex mixture analyses, which red wine undoubtedly is. The methodology described here is extremely robust, allowing for an uninterrupted analysis of 24 untreated red wine samples (continued analysis over an 8-hour period).
References
1
Packer L., Interactions among antioxidants in health and disease: Vitamin E and its redox cycle. Proc. Soc. Exp. Biol. Med. 1992, 200, 271-276. Mehra M. R., Lavie C. J., Ventura H.O., Milani R. V., Prevention of atherosclerosis: the potential role of antioxidants. Postgrad. Med. 1995, 98, 175- 184. Maxwell S., Cruikshank A., Thorpe G., Red wine and antioxidant activity in serum. Lancet 1994, 344, 193-194. Whitehead T. P., Robinson D., Allaway S., Syms J., Hale A., Effect of red wine ingestion on the antioxidant capacity of serum. Clin. Chem. 1995, 41, 32-35. Tomera J. F., Current knowledge of the health benefits and disadvantages of wine consumption. Trends Food Sci. Technol., 1999, 10, 129-138. Hyoung S. L., HPLC Analysis of Phenolic Compounds. In Food Analysis by HPLC; Nollet, L.M.L. Ed., CRC Press, 2000, 796. Stecher G., Huck C. W., Stoeggl. W. M., Bonn G. K., Phytoanalysis: a challenge in phytomics. TrAC (Trends in Analytical Chemistry). 2003, 22, 1-14. Goldberg D. M., Tsang E., Karumanchiri A., Diamandis E. P., Soleas G. J., Ng E., A method to assay the concentrations of phenolic constituents of biological interest in wines. Anal. Chem. 1996, 68, 1688-1694. Makarov A., Denisov E., Kholomeev A., Balschun W., Lange O., Strupat K., Horning S., Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer. Anal. Chem. 2006, 78, 2113-2120. Breitling R., Pitt A. R., Barrett M.P., Precision mapping of the metabolome. Trends in Biotech. 2006, 24, 543-548. Kussmann M., Rezziand S., Daniel H., Profiling techniques in nutrition and health research. Curr. Opin. Biotech. 2008, 19, 83-99. Soleas G. J., Tomlinson G., Diamandis E. P., Goldberg D. M., The relative contributions of polyphenolic constituents to the antioxidant status of wines: development of a predictive model. J. Agric. Food Chem. 1997, 45, 3995-4003. Dourtoglou V., Mamalos A., Makris D. P., Kefalas P. Storage of olives (Olea europaea L.) under CO2 atmosphere: liquid chromatography-mass spectrometry characterization of indices related to changes in polyphenolic metabolism. J. Agric. Food Chem. 2006, 54, 22112217.
10
11
12
13
Acknowledgements
The authors are grateful to David Kusel for insightful comments to the manuscript.
www.thermo.com
2009 Thermo Fisher Scientic Inc. All rights reserved. Mass Frontier is a trademark of HighChem; SIMCA is a trademark of Umetrics AB, Umea, Sweden; Charmes des Kirwan is a trade name of Maison Schrder & Schyler, Bordeaux, France; Margaux is a protected designation of origin. All other trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
AN30173_E 02/09C
Pesticides Pesticides
t
454: Analysis of Haloacetic Acids in Drinking Water by IC-MS/MS 391: Analysis of Regulated Pesticides in Drinking Water Using Thermo Scientific Accela and EQuan 434: Analysis of Triazine Pesticides in Drinking Water Using LC-MS/MS (EPA Method 536.0)
t t t t t t t
387: Multi-residue Analysis of Pesticides in Food using GC/MS/MS with the Thermo Scientific TSQ Quantum GC 378: Quantitation-Enhanced Data-Dependent (QED) Scanning of Drinking Water Samples Using the Thermo Scientific EQuan System for Pesticide Analysis on a Triple Stage Quadrupole 323: Simultaneous Detection of 88 Pesticides on the Thermo Scientific TSQ Quantum Discovery using a Novel LC/MS/MS Method 373: Testing LC-MS System Robustness with Automated Sample Cleanup Using Red Wine as a Matrix 453: UHPLC Separation of Triazine Herbicides at Elevated Temperature 355: Utility of H-SRM to Reduce Matrix Interference in Food Residue Analysis of Pesticides by LC/MS/MS using the Thermo Scientific TSQ Quantum Discovery 351: Zero Cross-talk on the Thermo Scientific TSQ Quantum
t t t
425: Determination of Different Classes of Pesticide Residues in Processed Fruits and Vegetables by LC-MS Using the Thermo Scientific TSQ Quantum Ultra According to EU Directive 91/414 EEC 437: LC-MS/MS Analysis of Herbicides in Drinking Water at Femtogram Levels Using 20 mL Thermo Scientific EQuan Direct Injection Techniques 395: LC-MS/MS Determination of Malachite Green and Leucomalachite Green in Fish Products
Analysis of Haloacetic Acids in Drinking Water by IC-MS/MS

Charles Yang1 and Stacy Henday2 1 Thermo Fisher Scientific, San Jose, CA; 2Dionex Corporation, Sunnyvale, CA
Introduction
Key Words TSQ Quantum Access EPA Ion chromatography Water analysis Haloacetic acids (HAAs) are formed as disinfection byproducts when water is chlorinated to remove microbial content. The chlorine reacts with naturally occurring organic and inorganic matter in the water, such as decaying vegetation, to produce by-products that include HAAs. Of the nine species of HAAs, five are currently regulated by the EPA (HAA5): monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), monobromoacetic acid (MBAA), and dibromoacetic acid (DBAA). The remaining four HAAs are unregulated: bromochloroacetic acid (BCAA), bromodichloroacetic acid (BDCAA), dibromochloroacetic acid (DBCAA), and tribromoacetic acid (TBAA). According to the U.S. Environmental Protection Agency (EPA), there might be an increased risk of cancer associated with long-term consumption of water containing levels of HAAs that exceed 0.6 mg/L.1 EPA Methods 552.1, 552.2, and 552.3, are used to determine the level of all nine HAAs in drinking water.2,3,4 These methods require derivatization and multiple extraction steps followed by gas chromatography (GC) with electron capture detection (ECD). In comparison to the conventional EPA methods using GC with ECD, the combination of ion chromatography and mass spectrometry (IC-MS and IC-MS/MS) offers sensitive and rapid detection without the need for sample pre-treatment. Ion chromatography is a form of liquid chromatography that uses ion-exchange resins to separate atomic and molecular ions. The retention time in the column is predominantly controlled by the interactions of the ions of the solute with the resin. Coupling IC with the highly selective detection of a triple quadrupole mass spectrometer allows unambiguous identification of substance peaks. Matrix interference effects are greatly reduced, which improves the sensitivity and lowers the detection limits. In the method described here, water samples can be injected directly into an ion chromatography system that is coupled to a Thermo Scientific TSQ Quantum Access triple stage quadrupole mass spectrometer. The separation of all nine HAAs addressed in the EPA methods is achieved with an anion-exchange column using an electrolytically formed hydroxide gradient.
Goal
To develop a simple, rapid, and sensitive IC-MS/MS method for analyzing haloacetic acids in water.
Ion Chromatography
IC analysis was performed on a Dionex ICS 3000 system (Dionex Corporation, Sunnyvale, CA). Samples were directly injected and no sample pre-treatment was required. The IC conditions used are shown in Table 1.
Column Set: Suppressor: Column Temperature: Injection Volume: Flow Rate: Dionex IonPac AG24 (2 50 mm), IonPac AS24 (2 250 mm) ASRS 300, 2 mm 15 C 100 L 0.3 mL/min KOH gradient, electrolytically generated (Table 2)
Table 1. Ion chromatography system conditions

Retention Time (min) [KOH] mM
0.00 15.1 30.8 31.0 46.8 47.0
7.0 7.0 18.0 60.0 60.0 7.0
Table 2. Electrolytically formed hydroxide gradient details
The separation performed on the IonPac AS24 column used a hydroxide gradient. It is known that hydroxide is not a recommended eluent for mass spectrometers. The addition of an ASRS 300 anion self-regenerating suppressor is critical. This suppressor is placed in line after the column and electrolytically converts the hydroxide into water, making the separation compatible with mass spectrometric detection. See Figure 1.
Figure 1: Flow schematic of the IC-MS/MS system
In addition, a matrix diversion valve was placed in line prior to the mass spectrometer. This valve functions to divert the high sample matrix waste from the MS source, prolonging the time in between cleanings. Acetonitrile was teed into the eluent stream after the matrix diversion valve. The acetonitrile had two main purposes: to assist in the desolvation of the mobile phase and to act as a makeup flow when the IC eluent was diverted to waste.
Mass Spectrometry
Ion source polarity: Spray voltage: Sheath gas pressure: Auxiliary gas pressure: Capillary temperature: Table 3. Mass spectrometer conditions
Positive ion mode 4000 V 40 units 15 units 270 C
MS analysis was carried out on a TSQ Quantum Access triple stage quadrupole mass spectrometer with a heated electrospray ionization (H-ESI) probe. The MS conditions used are shown in Table 3.
Individual standards were infused into the mass spectrometer to determine optimum tube lens settings and collision energies for the product ions. Table 4 describes the MS conditions for specific HAAs and internal standards.
Analyte MCAA MBAA DCAA DBAA BCAA TCAA BDCAA DBCAA TBAA MCAA-ISTD MBAA-ISTD DCAA-ISTD TCAA-ISTD
Q1 (m/z) 93.01 136.99 127.02 214.80 171.00 161.06 79.00 206.74 250.70 94.01 138.00 128.01 162.06
Q3 (m/z) 35.60 79.09 83.20 79.20 79.20 117.10 79.00 79.13 79.10 35.60 79.09 84.20 118.10
CE (V) 10 12 11 24 35 10 15 15 25 10 12 11 10
Tube Lens (V) 26 33 26 33 44 69 30 30 26 26 33 26 69
Skimmer Offset (V) 0 0 0 0 0 0 0 0 0 0 0 0 0
Scan Time (s) 1.25 1.25 1.25 1.25 1.25 1.60 1.60 2.50 2.50 1.25 1.25 1.25 1.60
Table 4. MS conditions for the various HAAs and internal standards
The status of the ion chromatography system was monitored at the same time as the MS data acquisition, as shown in Figure 2.

The separation of the nine HAAs is shown in Figure 3. The selectivity of the IC-MS/MS system allows separation of the HAAs from common inorganic matrix ions. This allows matrix peaks of chloride, sulfate, nitrate, and bicarbonate to be diverted to waste during the analytical run and avoids premature fouling of the ESI-MS/MS instrument source. An internal standard mixture of 13C labeled MCAA, MBAA, DCAA, and TCAA was spiked into each sample at 3 ppb. The calibration curves were generated using internal standard calibrations for all of the HAA compounds in water. Excellent linearity results were observed for all compounds as shown in Figures 4, 5, and 6. Analytes were run at levels of 250 ppt to 20 ppb. It must be noted that the TCAA analyte could not be
TSQ Quantum Access
Figure 2: These chromatograms show the progress of the pump pressure and front end detector data along with the TSQ Quantum Access MS data. The left side of the screen shows the status of the ion chromatography system and the status of the TSQ Quantum Access.
detected at levels below 2.5 ppb. TCAA sensitivity is very strongly correlated with the source temperature of the mass spectrometer. To improve the TCAA detection, the temperature was lowered. However, lowering the temperature impacted the detection of the other eight analytes. This phenomenon of TCAA temperature sensitivity has been reported in studies with other MS instrumentation configurations.5 To test the recoveries of all nine HAAs, spiked matrix samples were run in a matrix of 250 mg/L of each of chloride and sulfate, 150 mg/L of bicarbonate, 30 mg/L of
1. 2. 3. 4. 5.
Monochloroacetic acid (MCAA) Monobromoacetic acid (MBAA) Dichloroacetic acid (DCAA) Bromochloroacetic acid (BCAA) Dibromoacetic acid (DBAA)
6. 7. 8. 9.
Trichloroacetic acid (TCAA) Bromodichloroacetic acid (BDCAA) Dibromochloroacetic acid (DBCAA) Tribromoacetic acid (TBAA)
Figure 3: Separation and detection of the nine haloacetic acids using an ISC-3000 system with an IonPac AS24 column, coupled to a TSQ Quantum Access MS/MS system. Analyte levels were 2.5 ppb each in deionized water with an injection volume of 100 L.
Figure 4: Calibration curve overlay of the HAA compounds in water by IC-MS/MS
nitrate, and 100 mg/L ammonium chloride preservative, for a total chloride concentration of 316 mg/L. The results are shown in Table 5. Excellent recoveries and reproducibility were achieved for most of the samples. However, difficulty was observed when quantitating low levels of DBCAA in matrix. DBCAA does not ionize as strongly as the other analytes in the method and is very susceptible to temperature changes in the column. Method detection limits (Table 6) were calculated by
seven replicate injections of 1.0 ppb of each analyte and the equation MDL=t99%xS(n-7), where: t is Students t at 99% confidence intervals (t99%, n=7 = 3.143) and S is the standard deviation. Table 6 compares these results to the calculated MDL values of EPA Method 552.2, which uses liquid-liquid extraction and methylation of the carboxylic acids before determination by GC-ECD. The results obtained by the IC-MS/MS method were comparable to those achieved in EPA Method 552.2.
Figure 5: Overlay of calibration curves of dibromochloroacetic acid and tribromoacetic acid in water by IC-MS/MS
Figure 6: Overlay of calibration curves of dibromoacetic acid and bromodichloroacetic acid in water by IC-MS/MS
Analyte MCAA MBAA DCAA BCAA DBAA TCAA BDCAA DBCAA TBAA
Average RT 12.59 14.06 24.44 26.88 30.09 39.05 45.13 43.55 47.44
%RSD RT 0.00 0.27 0.02 0.18 0.16 0.24 0.04 0.07 0.25
Average Area 764439 1627886 11236488 2468467 731710 4855405 1212887 1064 1333
%RSD Area 2.34 2.91 3.98 4.85 3.26 10.98 4.78 22.20 17.60
Table 5. Reproducibility of area and retention time in the TSQ Quantum Access for seven injections of 2 ppb concentration in simulated matrix
Analyte MCAA MBAA DCAA BCAA DBAA TCAA BDCAA DBCAA TBAA
Calculated MDL (L/L) 0.203 0.392 0.097 0.136 0.100 0.403 0.159 0.459 0.407
EPA Method 552.2 MDL (L/L) 0.273 0.204 0.242 0.251 0.066 0.079 0.091 0.468 0.820
Table 6. Calculated MDL response of HAA9 on the TSQ Quantum Access
Conclusion
IC-MS/MS is a powerful tool used in the quantitation of haloacetic acid samples. When compared to the conventional EPA methods using GC with electron capture, using IC-MS/MS to analyze for haloacetic acids saves analysts several hours of sample preparation because there is no requirement for sample pretreatment. The resolution between the matrix peaks and haloacetic acids is excellent, which allows for minimum interference in detection. Excellent recoveries and reproducibility were achieved when samples were spiked into a simulated matrix containing 250 mg/L of each of chloride and sulfate, 150 mg/L bicarbonate, 30 mg/L of nitrate and 100 mg/L ammonium chloride preservative for a total chloride concentration of 316 mg/L. Results are comparable to those achieved in EPA Method 552.2.
References
1. U.S. Environmental Protection Agency, Microbial Health Effects Tables: Potential Adverse Health Effects from High/Long-term Exposure to Hazardous Chemicals in Drinking Water, 2002. 2. U.S. Environmental Protection Agency, Method 552.1, Determination of Haloacetic Acids and Dalapon in Drinking Water by Ion Exchange Liquid-Solid Extraction and Gas Chromatography with Electron Capture Detection, Rev. 1.0, 1992. 3. U.S. Environmental Protection Agency, Method 552.2, Determination of Haloacetic Acids and Dalapon in Drinking Water by Liquid-Liquid Extraction, Derivatization, and Gas Chromatography with Electron Capture Detection, Rev 1.0, 1995. 4. U.S. Environmental Protection Agency, Method 552.3, Determination of Haloacetic Acids and Dalapon in Drinking Water Liquid-Liquid Microextraction, Derivatization, and Gas Chromatography with Electron Capture Detection, Rev 1.0, 2003. 5. Slignsby, R.; Saini, C.; Pohl, C.; Jack, R. The Measurement of Haloacetic Acids in Drinking Water Using IC-MS/MSMethod Performance, Presented at the Pittsburgh Conference, New Orleans, LA, March 2008.
www.thermo.com
Legal Notices 2009 Thermo Fisher Scientic Inc. All rights reserved. IonPac, ASRS, and Dionex are registered trademarks of Dionex Corporation. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientic Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

AN62963_E 01/09S
Analysis of Regulated Pesticides in Drinking Water Using Accela and EQuan

Jonathan R. Beck and Charles Yang; Thermo Fisher Scientic, San Jose, CA
Introduction
Key Words TSQ Quantum Accela LC System EQuan LC-MS/MS Pesticide Analysis Water Analysis Pesticides are used throughout the world to control pests that are harmful to crops, animals, or people. Because of the danger of pesticides to human health and the environment, regulatory agencies control their use and set pesticide residue tolerance levels. The limits of detection (LODs) for many of these substances are at the partsper-trillion (ppt) level. In order to achieve this level of detection, ofine sample pre-concentration is often performed. However, these sample preparation procedures can be time consuming, adding as much as one to two days to the total analysis time. Therefore, a method for online sample pre-concentration that bypasses the ofine sample pre-concentration provides a signicant time savings over conventional methods. We describe a method for online sample cleanup and analysis using the Thermo Scientic EQuan system. This method couples a Fast-HPLC system with two Thermo Scientic Hypersil GOLD LC columnsone for preconcentration of the sample, the second for the analytical separationand an LC-MS/MS instrument. Large volumes of drinking water samples (1 mL) can be directly injected onto the loading column for LC-MS/MS analysis, thus eliminating the need for ofine sample pre-concentration and saving overall analysis time. Using this conguration, run times of six minutes are achieved for the analysis of a mixture of pesticides. For separation prior to analysis using an LC-MS/MS instrument, Fast-HPLC allows for signicantly shorter run times than conventional HPLC.
No other sample treatment was performed prior to injection. The mass transitions and collision energies for each compound are listed in Table 1.
HPLC Fast-HPLC analysis was performed using the Thermo Scientic Accela High Speed LC System. A 1 mL water sample was injected directly onto a 20 mm 2.1 mm ID, 12 m Hypersil GOLD loading column in a high aqueous mobile phase at a ow rate of 1 mL/min (see Figure 1a). After approximately one minute, a 6-port valve on the mass spectrometer was switched via the instrument control software. This enabled the loading column to be back ushed onto the analytical column (Hypersil GOLD 50 2.1 mm ID, 1.9 m), where the compounds were separated prior to introduction into the mass spectrometer (Figure 1b). After all of the compounds were eluted from the analytical column at a ow rate of
Analyte Tricyclazole Thiodicarb Carbofuran Carbaryl Diuron Bensulfuron-methyl Flazasulfuron Siduron Azoxystrobin Halosulfuron-methyl Daimuron Precursor Mass (m/z) 190.09 355.06 222.10 202.14 233.05 411.13 408.08 233.19 404.16 435.11 269.21 Product Mass (m/z) 106 88 165 145 72 149 182 137 372 182 151 Collision Energy (eV) 10 14 14 10 20 22 24 20 15 24 14
Goal
To demonstrate the use of Fast-HPLC and a large volume injection to analyze sub-ppb concentrations of regulated pesticides in drinking water samples.
Table 1: List of mass transitions and collision energies for each compound analyzed.
Sample Preparation Bottled drinking water was spiked with a mixture of the following pesticides: carbofuran, carbaryl, diuron, daimuron, bensulfuron-methyl, tricyclazole, azoxystrobin, halosulfuron-methyl, azasulfuron, thiodicarb, and siduron. Concentrations were prepared at the following levels: 0.5, 1, 5, 10, 50, 100, 500, and 1000 pg/mL (ppt).
Figure 1a: 6-port valve position one (load position), for loading the sample onto the loading column.
Figure 1b: 6-port valve position two (inject position), for eluting the compounds trapped on the loading column onto the analytical column.
850 L/min, the 6-port valve was switched back to the starting position. The loading and analytical columns were cleaned with a high organic phase before being reequilibrated to their starting conditions. The total run time for each analysis was six minutes. The mobile phases for the analysis were water and acetonitrile, both containing 0.1% formic acid. The gradient prole for each pump is shown in Figure 2. The pressure at the beginning of the gradient was monitored. At a ow rate of 850 L/min (at the initial gradient conditions with the ow going through only the Hypersil GOLD 50 2.1 mm, 1.9 m column), the backpressure for the Fast-HPLC system was approximately 450 bar. For comparison, an earlier method which used a Hypersil GOLD 50 2.1 mm, 3 m column had a backpressure of approximately 150 bar at a ow rate of 200 L/min.
Calibration curves for all 11 compounds were generated using Thermo Scientic LCQUAN 2.5 software. Excellent linearity was achieved for all of the compounds analyzed in this experiment. Figure 4 shows a representative calibration curve for the compound azoxystrobin over the concentration range 0.5 to 1000 pg/mL (ppt). The calibration curve t parameters and the limits of detection for the analytes are summarized in Table 2. The nal column in the table lists the Minimum Performance Reporting Limit (MPRL) for these compounds as set by the Japanese Ministry of Health, Labour, and Welfare1. All of the compounds were detected and quantied at levels well below these regulatory requirements.
MS MS analysis was carried out on the Thermo Scientic TSQ Quantum Access triple stage quadrupole mass pectrometer with a heated electrospray ionization (H-ESI) probe. The MS conditions were as follows: Ion source polarity: Positive ion mode Spray voltage: 4000 V Vaporizer temperature: 450C Sheath gas pressure (N2): 50 units Auxiliary gas pressure (N2): 50 units Ion transfer tube temperature: 380C Collision Gas (Ar): 1.0 mTorr Q1/Q3 Peak Resolution: 0.7 u Scan Width: 0.002 u
Figure 3: Chromatograms showing the SRMs for each of the components in the mixture. Two different HPLC conditions are shown: the Fast-HPLC run and the standard HPLC run. All compounds in the Fast-HPLC run are eluted in less than three minutes (circled in green). Those in the standard HPLC run are eluted much later (circled in red). These chromatograms represent a calibration level of 500 pg/mL (ppt).

Chromatograms for the calibration standard at a concentration of 500 pg/mL are shown in Figure 3. In the Fast-HPLC run, all 11 of the individual analytes were eluted before three minutes. In contrast, none of the analytes in the standard HPLC run were eluted until nearly eight minutes into the run. Further optimization of the chromatography for the Fast-HPLC would produce even shorter run times.
Figure 4: Calibration curve for the compound azoxystrobin. This calibration curve covers the range from 0.5 to 1000 pg/mL (ppt)
Figure 2: Gradient proles for the two LC pumps used in this experiment. The Fast-HPLC pump gradient is shown on the left, and the loading pump gradient is show on the right.
Analyte Tricyclazole Thiodicarb Carbofuran Carbaryl Diuron Bensulfuron-methyl Flazasulfuron Siduron Azoxystrobin
R2 0.9972 0.9930 0.9928 0.9345 0.9978 0.9933 0.9944 0.9973 0.9974
Limit of Detection (ppt) 0.5 5 1 100 100 0.5 1 0.5 0.5
MPRL (ppt) 800 800 50 500 200 4000 300 3000 5000
Table 2: List of calibration curve t parameters, limits of detection, and Minimum Performance Reporting Levels (MPRL) for each compound from the Japanese Ministry of Health, Labour and Welfare. All calibrations were carried our using a linear curve t and a weighting factor of 1/X.
Conclusion
The implementation of Fast-HPLC, coupled with the online pre-concentration and sample preparation technique EQuan, yielded analysis of 11 pesticides in drinking water in less than one-third the time of conventional HPLC analysis. All of the compounds eluted within three minutes, which included a one-minute loading time for the sample to be pre-concentrated on the loading column. The total run time for the analysis was six minutes. The Fast-HPLC method can be further shortened to produce faster chromatographic run times. The use of large volume injections achieved results below the MPRL regulatory requirements for each of the 11 pesticides. Because the limits of detection were much lower than the MPRL values, the integrated peaks yielded excellent signal-to-noise ratios and allowed for condence in reporting the results.
Reference
1
http://www.mhlw.go.jp/index.html (Japanese language version), http://www.mhlw.go.jp/english/index.html (English language version)

www.thermo.com
Legal Notices 2007 Thermo Fisher Scientic Inc. All rights reserved. All other trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientic Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

AN62449_E 10/08S
Analysis of Triazine Pesticides in Drinking Water Using LC-MS/MS (EPA Method 536.0)
Jonathan Beck, Thermo Fisher Scientic, San Jose, CA, USA
Introduction
Key Words Drinking Water Analysis Herbicides Hypersil GOLD Columns Triazines TSQ Quantum Access The US EPA has recently issued a draft form of a proposed method for the analysis of triazine compounds in drinking water.1 This method uses a simple method to directly analyze triazine compounds using LC-MS/MS without requiring any solid phase extraction (SPE) or other lengthy sample preparation steps. This application note demonstrates the analysis of these compounds over the concentration range 0.25 5.0 ng/mL (ppb) using the Thermo Scientic TSQ Quantum Access triple stage quadrupole mass spectrometer and the Thermo Scientic Accela HPLC system.
HPLC Conditions
Column: Solvent A: Solvent B: Flow Rate: Injection Volume: HPLC Gradient: Thermo Scientic Hypersil GOLD 100 x 2.1 mm, 3 m 5 mM Ammonium Acetate Methanol 400 L/min 100 L Time %A %B 0:00 98 2 10:00 98 2 20:00 10 90 25:00 10 90 25:06 98 2 30:00 98 2
The following triazine and triazine degradates were analyzed: Atrazine, Atrazine-desethyl, Atrazine-desisopropyl, Cyanazine, Propazine, and Simazine, purchased from Sigma-Aldrich, St. Louis, MO, and Ultra Scientic, North Kingstown, RI. The following internal standards were used: Atrazine-d5, Atrazine-desethyl-d7, Atrazine-desisopropyl-d5, Cyanazine-d5, Propazine-d14, and Simazine-d10, purchased from C/D/N Isotopes, Inc., Pointe-Claire, Quebec, Canada. Standards and internal standard stocks were prepared in solutions of methanol and diluted to their appropriate concentrations prior to analysis.
Mass Spectrometer Conditions

Ionization Source: Sheath Gas: Auxiliary Gas: ESI Voltage: Ion Transfer Tube Temperature: Collision Gas: Q1/Q3 Peak Resolution: Scan Width: Positive Electrospray 30 arbitrary units 10 arbitrary units 3.5 kV 350 C 1.5 mTorr 0.7 Da 0.01 Da
Sample Preparation
While no SPE was required for this method, samples were treated as per the EPAs draft method. The method calls for the addition of ammonium acetate at 20 mM for pH adjustment and dechlorination and sodium omadine at 64 mg/L to prevent microbial degradation, both purchased from Sigma-Aldrich, St. Louis, MO. All samples were prepared in reagent water. All samples were spiked with the internal standard solution, resulting in a nal concentration of 5 ng/mL (ppb) for each internal standard. Calibration standards were prepared at the following levels: 0.25, 0.5, 1, 2, 2.5 and 5 ng/mL.
MS Parameters
Compound Precursor Mass Product Mass Collision Energy Tube Lens
Atrazine-desisopropyl Atrazine-desethyl Simazine Atrazine Propazine Cyanazine Atrazine-desisopropyl-d5 Atrazine-desethyl-d7 Simazine-d10 Atrazine-d5 Propazine-d14 Cyanazine-d5
174 188 202 216 230 241 179 195 212 221 244 246
132 146 124 174 124 214 137 147 137 179 196 219
17 16 17 16 17 15 17 17 19 17 18 16
90 95 80 85 80 100 85 95 95 95 95 100

The triazine compounds eluted from the LC column in 20 minutes. A chromatogram of each compound and the internal standards is shown in Figure 1. All peaks are chromatographically resolved from one another. Calibration curves were generated for each compound over the range 0.25-5 ppb. All calibration curves exhibited excellent linearity, ranging from 0.9964 for Atrazine-desethyl to 0.9982 for Atrazine. The calibration curve for Simazine is shown in Figure 2. The other compounds exhibit similar linearity, and are not shown in this application note.
Figure 2: Calibration curve for Simazine, 0.25-5 ppb
Conclusion
The TSQ Quantum Access LC-MS/MS is an excellent choice for the analysis of triazine compounds and their degradates. Linearity over the entire calibration range of 0.25 to 5 ppb is observed. Separation of all the analytes is achieved with the Hypersil GOLD column allowing for unambiguous identication and quantitation of all of the compounds in this application note.
References
1. Smith, G.A., Pepich, B.V., Munch, D.J. Determination of Triazine Pesticides and their Degradates in Drinking Water by Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC/ESI/MS) Draft 5.0, April 2007.
Figure 1: Chromatogram of the triazine compounds at 2 ppb, and their internal standards
www.thermo.com
AN62880_E 09/08M
Determination of Different Classes of Pesticide Residues in Processed Fruits and Vegetables by LC-MS Using the TSQ Quantum Ultra According to EU Directive 91/414 EEC
Eleni Botitsi, Anastasios Economou, Spyros Antoniou, and Despina Tsipi; General Chemical State Laboratory, Pesticide Residues Laboratory, Athens, Greece
Introduction
Key Words TSQ Quantum Ultra Surveyor HPLC System H-SRM Food Safety Pesticide Residues Sensitivity A diet rich in fruits and vegetables is thought to reduce the risk of some types of cancer, atherosclerosis, and heart disease. However, commercially grown produce often contains high levels of pesticide residues that can lead to serious health problems when consumed. Due in large part to growing public concern over the amount of pesticide residues in foods, the European Union (EU) has enacted several directives to x Maximum Residue Limits (MRLs) for different pesticide residues in food of plant origin. MRLs represent the maximum amount of pesticide residues that might be expected in a commodity produced under conditions of good agricultural practice and typically range between 0.01 mg/kg and 10 mg/kg1. Although MRLs are not maximum toxicological limits, care is taken to ensure that these maximum levels do not generate toxicological concerns. Thus far, MRLs have been set for approximately 250 active substances. To cover the full variety of agricultural raw commodities (approximately 260 products of plant and animal origin), MRLs must be established for more than 260,000 pesticide/commodity combinations1,2 . In the EU, pesticides are regulated principally by Directive 91/414/EEC concerning the placing of plantprotection products on the market3. According to this legislation, chemical substances or micro-organisms in pesticides are approved for use only if they have undergone a peer-reviewed safety assessment. All foodstuffs intended for human consumption or animal feed in the EU are now subject to a maximum residue limit for pesticides to protect human and animal health. Regulation (EC) 396/20054 consolidates in a single act all the limits applicable to various types of food and feed. It establishes MRLs for products of plant and animal origin at the Community level, taking into account good agricultural practices. It was based on several substantial amendments in the Council Directives: 76/895/EEC5, which relates to the xing of maximum levels for pesticide residues in and on specic fruits and vegetables 86/362/EEC6 for cereals and cereal products 86/363/EEC for products of animal origin
7
Additionally, more stringent legislation has been established concerning pesticides in baby food. Since 1999, the EU has introduced the Commission Directives 1999/39/EC9 and 1999/50/EC10 which limit all pesticide , residues to an MRL value of 0.010 mg/kg in processed cereal-based foods and in fruit and vegetables intended for the production of baby foods. MRLs below 0.010 mg/kg have been established for a few pesticides of higher toxicity, while the use of certain very toxic pesticides has been completely prohibited in the production of baby foods, as underlined in Commission Directives 2003/13/EC11 and 2006/125/EC12 . Newactiveingredients entering the market to replace compounds banned by Directive 91/414/ EEC possess considerably different physicochemical properties, and thus demand the development of multi-residue analytical methods. Analytical methodologies used to determine pesticide residues in foods must be capable of quantifying very low levels of residues as well as conrming their identity. This task becomes more difcult as MRLs are decreased and the number of target pesticides and metabolites increases. Therefore, the challenge is to develop a sensitive, cost-effective, multiresidue analytical method that can quickly identify and conrm pesticide residues belonging to various chemical classes in food products. At the same time, the method must accurately quantify these residues at low levels, thus fullling the performance criteria described inMethod Validation and Quality Control Procedures for Pesticide Residues Analysis in Food and Feed, European Commission Document SANCO 2007/313113 .
Goal
To develop a multi-residue liquid chromatographyelectrospray ionization tandem mass spectrometry (LCESI-MS/MS) method for the detection and quantication of 45 pesticides, including parent compounds and their transformation products from different chemical classes, in various food matrices.
LC-ESI-MS/MS is the analytical technique of choice to assay environmental and food matrices with high sensitivity and selectivity. The technique is especially well-suited for the identication and quantication of polar and thermally labile pesticides and metabolites down to mg/kg levels. The pesticides included in this study are listed in Table 1.
90/642/EEC8 for plant products
Compound Acephate Aldicarbb (Na+) Aldicarb-sulfoxide (Na+) Aldicarb-sulfone (Na+) Acetamiprid Azoxystrobin Carbaryl Carbofuran 3-hydroxy-carbofuran (-H2O) Chlorpropham Carbendazim + benomylb Cyprodinil Demeton-S Demeton-S-methyl Demeton-S-methyl-sulfone Demeton-S-methyl-sulfoxide Dimethomorph Ac / Bc Disulfoton Disulfoton-sulfone Disulfoton-sulfoxide Ethoprofos Fenhexamidd Flusilazole Imazalil Imidachloprid Kresoxim-methyl Metalaxyl Methiocarb Methiocarb sulfoxide (Na+) Methomyl Myclobutanil Oxamyl (Na+) Penconazole Pirimicarb Propiconazole Propoxur Pyrimethanil Tetraconazoled Thiabendazole Thiachloprid Thiodicarb Thiophanate-methyl Triadimefon Triadimenol Ac/ Bc Triazophos
a b
tR (min)a 2.5 19.7 2.7 4.1 17.3 28.8 24.0 23.1 15.8 29.6 2.5 25.3 26.0 22.8 7.0 3.3 26.8 27.3 33.8 27.1 24.2 29.2 29.0
29.8 21.4 15.3 31.8 24.8 27.6 6.6 5.0 28.7 4.2 30.0 7.3 30.8 22.7 21.2 29.4 2.5 21.0 23.3 22.5 28.9 27.1/27.5 30.4
Parent ion (m/z) 184 213 229 245 223.0 404.2 202.1 222.1 220 214.0 192.0 226.1 259.0 253.0 263.0 247.0 388.1 275.0 307.0 291.0 243.0 302.0 304.0 316.1 297.0 256.1 314.0 280.1 226.0 185.0 163.0 289.0 242 284.0 239.1 342.0 210.1 200.0 372.0 374.0 202.0 253.0 355.0 343.0 294.1 296.1 314.1
Quantier ion (m/z) 143 (10V) 89 (22 V) 166 (9V) 109 (25V) 126 (21 V) 372 (16 V) 145 (25 V) 123 (24 V) 135 (15V) 172 (12 V) 160 (22 V) 93 (40 V) 89 (22 V) 61 (40 V) 109 (30 V) 169 (17 V) 301 (23 V) 89 (15 V) 125 (20 V) 185 (15 V) 131 (21 V) 97 (22 V) 247 (21 V) 159 (24 V) 209 (22 V) 222 (14 V) 220 (15 V) 169 (11 V) 122 (23 V) 106 (12 V) 125 (35 V) 70 (20V) 159 (35 V) 182 (15 V) 159 (31 V) 111 (17 V) 182 (35 V) 159 (38 V) 131 (36 V) 99 (45 V) 88 (20 v) 151 (23 V) 197 (19 V) 70 (16 V) 162 (19 V)
Quantier ion (m/z) 125 (10V) 116 (22 V) 109 (15V) 166 (25V) 90 (35 V) 344 (26 V) 127 (25 V) 165 (24 V) 163 (15V) 154 (19 V) 132 (31 V) 77 (46 V) 116 (22 V) 89 (20 V) 169 (20 V) 109 (29 V) 165 (35 V) 61 (30 V) 153 (20 V) 157 (25 V) 173 (21 V) 97 (26 V) 165 (31 V) 255 (25 V) 175 (22 V) 116 (19 V) 192 (25 V) 121 (19 V) 170 (23 V) 88 (12 V) 70 (25 V) 121 (20V) 70 (35 V) 72 (30 V) 69 (31 V) 168 (10 V) 168 (35 V) 161 (31 V) 175 (36 V) 126 (25 V) 108 (20 V) 311 (15 V) 225 (19 V) 99 (16 V) 119 (33 V)
Retention time Benomyl was measured as carbendazim 14 c Dimethomorph and triadimenol exist as two isomers with different retention times d For fenhexamid and tetraconazole, the isotopic parent ions were selected due to the lack of a second sound transition
Table 1: Retention times and compound-specic ESI(+)-MS/MS parameters
Sample Preparation A stock mix solution of all the pesticides was prepared at a concentration of 1 mg/L. Calibration solutions in the concentration range 0.5-100 g/L were prepared by serial dilution of the stock solution. Samples were prepared for analysis using extraction with ethyl acetate. Individual samples of fruits and vegetables were rst homogenized. After homogenization, a 10.0 g sample was extracted using ethyl acetate and anhydrous sodium sulfate. The mixture was ultrasonicated for 20 minutes. The mixture was ltrated through a thin layer of anhydrous sodium sulfate and the ltrate was evaporated. The extracts were then reconstituted in 5 mL of methanol. The solution was diluted with water and then ltered through a 0.45 m syringe lter14 . HPLC HPLC analysis was performed using the Thermo Scientic Surveyor HPLC System. Each 20 L sample was injected onto a 150 2.1 mm, 3.5 m, C18 HPLC column equipped with a 10 2.1 mm, 3.5 m, C18 HPLC guard column. A gradient LC method used mobile phases A (0.1% formic acid) and B (0.1% formic acid in acetonitrile) at a ow rate of 0.2 mL/min. The gradient was: 03 min A:B = 90:10 (v/v), 3 31 min A:B = 90:10 (v/v) to A:B = 10:90 (v/v), 3136 min A:B = 10:90 (v/v), 36 36.5 min A:B = 10:90 (v/v) to A:B = 90:10 (v/v), 36.5 45 min A:B = 90:10 (v/v).
MS MS analysis was carried out on a Thermo Scientic TSQ Quantum Ultra triple stage quadrupole mass spectrometer with an electrospray ionization source.
The MS conditions were as follows: Ion source polarity: Positive Spray voltage: 4000 V Sheath gas pressure (N2): 40 units Auxiliary gas pressure (N2): 10 units Ion transfer tube temperature: 350C Collision gas pressure (Ar): 1.0 mTorr Q1 resolution: 0.2 FWHM (H-SRM) Q3 resolution: 0.7 FWHM Scan Type: H-SRM Dwell time: 2050 ms The LC-MS/MS method was developed according to the scheme shown in Figure 1. The run was divided into four time segments based on the retention times of the target compounds. Multiple scan events were included in each time segment. For each target compound, the protonated molecule [M+H]+ was usually investigated, except in the cases of compounds where the adduct [M+Na]+ was the base peak in the ESI(+) spectra. Two transitions were selected per compound in order to perform quantication and identication simultaneously. The SRM transitions that were monitored are summarized in Table 1. Identication criteria for the target compounds were based on the LC retention time (tR) and on the ratio of the two monitored transitions for each compound.13,14
Figure 1: LC-ESI-MS/MS method

Although LC-MS/MS is a selective technique, interferences due to isobaric compounds can appear in chromatograms. These isobaric interferences increase the chemical background and can make it difcult to integrate the desired analyte peak reproducibly. Among the compounds included in this study were three sets of isobaric compounds and one set of compounds that share the same fragment ions, which increases the likelihood of cross-talk. Therefore, to eliminate the noise and lower the detection limits, all of the assays in this study were run in the Highly Selective Reaction Monitoring (H-SRM) mode with the Q1 FWHM peak width set at 0.214 . The H-SRM chromatograms of a mix solution of certain pesticides at a concentration of 1 g/L are shown in Figure 2. Linearity of the method was proven for all cases because the R2 values were usually greater than 0.99 for the linear regression equations (1/x weighted) in the
concentration ranges tested. The instrumental detection limits (IDLs) were, in most cases, below 0.5 g/L. Figure 3 displays the linearity plots of selected compounds. Linearity data for certain compounds are summarized in Table 2. Using the H-SRM mode reduced the matrix effects by minimizing the chemical noise caused by co-eluting isobaric compounds. Consequently, the signal-to-noise ratio was enhanced in the complicated food matrices. This effect can be observed in the chromatograms in Figure 4, which show the analysis of a peas sample in the SRM and H-SRM modes. The top two SRM chromatograms illustrate the background in a blank peas extract whereas the bottom two SRM chromatograms show the peaks for methomyl in a peas extract spiked with 1 ppb of methomyl. The narrower window of the Q1 set at 0.2 FWHM in the H-SRM mode improves the selectivity of the analysis and increases the signal-to-noise ratio.
Figure 2: SRM chromatograms for certain pesticides of the standard mix solution
1400000
10000000
Imidachloprid y = 12773x - 5101 R2 = 0.9957
Acephate y = 77684x + 11680 R2 = 0.9967
700000
H+ CH2-N Cl N NH N NO2
5000000
H+ OCH3 CH3O - P - NHCOCH3 O

0
m/z 256
0
m/z 184
100
50 g/L
100
50 g/L
6000000
Thiabendazole y = 53717x - 4547 R2 = 0.9933
Carbaryl y = 46572x + 36911 R2 = 0.9944 H+ O
3000000
400000
NH N N
H+
HN O
m/z 202
0 0
m/z 202
100 0 50
g/L
50
g/L
100
4000000
180000
Demeton-S y = 37613x - 9921 R2 = 0.9972
Disulfoton y = 1646x - 11944 R2 = 0.9903 H+ OCH2CH3 CH3CH2O - P - SCH2CH2SCH2CH3 O

90000
2000000
H+ OCH2CH3 CH3CH2O - P - SCH2CH2SCH2CH3 S

0
m/z 259
0
m/z 275
100
50
g/L
100
50
g/L
Figure 3: Linearity plots for certain compounds
Figure 4: H-SRM and SRM chromatograms of methomyl in pea sample matrix
The matrix-matched calibration curves of methomyl at different Q1 settings are shown in Figure 5 and data of the calibration curves are listed in Table 3. The signal itself is reduced by a factor of two when the Q1 FWHM peak width is changed from 0.7 to 0.2, yet the linearity and accuracy are improved (as demonstrated by the correlation coefcients and back-calculated values of the matrix-matched standards at the low concentration levels, in Table 3).
Linear regression equations Y=116806 +77683.7 X Y=-590.6 +1115.4 X Y=-363884 + 213698 X Y=36911 + 46572 X Y=10192 + 211684 X Y=11107 + 161251 X Y=-5289 + 6893.5 X Y=-57425 + 30565.4 X Y=-9921 + 37615 X Y=-11944 + 1646.2 X Y=40274 + 141033 X Y=-1633.2 + 8994 X Y=-10106 + 40922 X Y=-5101.1 +12773.2 X Y=-7877.4 + 3056.8 X Y=28427.5 +117245 X Y=4861 + 48380.4 X Y=-2440.7 +13847.8 X Y=-16905.7 +10101.5 X Y=23403 +168260 X Y=9181 + 151300 X Y=-4723.7 + 9197.2 X Y=-4546.8 + 53716.7 X Y=-18350 +134057 X
Some samples were found to contain pesticide residues. Figure 6 displays SRM chromatograms of a sample of frozen peas that contained residues of triazophos and myclobutanil. The conrmation of identity was based on the ion ratio of monitored transitions in the sample and in the standard solution according to the EU 13 Guidelines for pesticide residues monitoring. The concentrations of the residues found in the sample were below the Maximum Residue Limits (MRLs)1,2.
IDLs (g/L) 0.5 0.7 0.2 0.3 0.1 0.2 0.6 0.3 0.3 1.5 0.4 0.4 0.3 0.3 1.0 0.3 0.3 0.4 0.4 0.2 0.2 0.4 0.3 0.3
Compound Acephate Aldicarb Azoxystrobin Carbaryl Carbendazim Carbofuran Chlorpropham Cyprodinil Demeton-S Disulfoton Disulfoton Sulfoxide Disulfoton Sulfone Ethoprofos Imidachloprid Kresoxim-methyl Metalaxyl Methiocarb Methomyl Myclobutanil Pirimicarb Propoxur Pyrimethanil Thiabendazole Triazophos
Concentration range (g/L) (1-100) (1-100) (0.5-100) (0.5-100) (0.5-100) (0.5-100) (1-100) (0.5-50) (0.5-100) (5-100) (0.5-100) (0.5-100) (0.5-100) (0.5-100) (2-100) (0.5-100) (0.5-100) (0.5-100) (0.5-100) (0.5-100) (0.5-100) (0.5-100) (0.5-100) (0.5-100)
R2 0.9967 0.9900 0.9912 0.9903 0.9964 0.9920 0.9954 0.9931 0.9972 0.9903 0.9961 0.9904 0.9940 0.9957 0.9900 0.9964 0.9921 0.9990 0.9953 0.9953 0.9947 0.9900 0.9933 0.9954
Table 2: Linearity data and instrumental detection limits (IDLs) for certain pesticides
1/x R2 1 g/L 5 g/L 10 g/L
Peas Matrix 0.1 g/mL Q1: 0.2 FWHM Y = -2469.2 + 10863 X 0.9966 0.91 g/L (91%) 4.89 g/L (97%) 9.78 g/L (97%)
Peas Matrix 0.1 g/mL Q1: 0.7 FWHM Y = 4631.3 + 18381.3 X 0.9851 0.68 g/L (68%) 4.74 g/L (94%) 11.5 g/L (85%)
Peas Matrix 0.2 g/mL Q1: 0.2 FWHM Y = -3845.1 + 8212 X 0.9945 0.87 g/L (87%) 5.26 g/L (105%) 10.5 g/L (95%)
Peas Matrix 0.2 g/mL Q1: 0.7 FWHM Y = 10244 + 19142 X 0.9861 1.24 g/L (124%) 4.56 g/L (91%) 9.05 g/L (90%)
Accuracy of Matrix-Matched Calibration Curves (1/x)
Table 3: Linearity and accuracy data for methomyl in pea matrix
2.50E+05
2.00E+05
0.2 g/mL pea matrix at Q1 0.2 (FWHM) 0.2 g/mL pea matrix at Q1 0.7 (FWHM) 0.1 g/mL pea matrix at Q1 0.2 (FWHM) 0.1 g/mL pea matrix at Q1 0.7 (FWHM)
1.50E+05
Q1: 0.7 Da
1.00E+05
Q1: 0.2 Da
5.00E+04
0.00E+00
0 1 2 3 4 5 6 7 8 9 10
g / L
Figure 5: Matrix-matched calibration curves of methomyl in pea extract at Q1: 0.2 (FWHM) and 0.7 (FWHM)
A. standard solution of pesticides TRIAZOPHOS TIC: 314.1 162, 119 SRM 1: 314.1 119 [119] / [162] = 0.16* SRM 2: 314.1 162
NL: 2.8E4
B. Pea sample extract

NL: 1.3E4
TIC: 314.1
NL: 4.2E3
162, 119
SRM 1: 314.1 119 [119] / [162] = 0.17
NL: 1.8E3
NL: 2.6E4
SRM 2: 314.1
162
NL: 1.8E4
MYCLOBUTANIL TIC: 289 125, 70
NL: 1.9E4
TIC: 289
125, 70
NL: 3.3E3
NL: 9.5E3
SRM 1: 289
70
[70] / [125] = 0.91*

NL: 1.1E4
SRM 1: 289
70
NL: 1.8E3
[70] / [125] = 0.92*

NL: 1.8E3
SRM 2: 289
125
SRM 2: 289
125
Figure 6: LC-ESI-SRM chromatograms of frozen pea sample extract, with residues of triazophos and myclobutanil
Conclusion
A multi-residue LC-ESI-MS/MS method was developed for the reliable conrmation and quantication of pesticides from different chemical classes at low ppb levels in food matrices. The method uses the Highly Selective Reaction Monitoring (H-SRM) mode of the TSQ Quantum Ultra triple quadrupole mass spectrometer to effectively reduce the background interference and improve the signal-tonoise ratios. For the pesticides investigated, satisfactory precision and accuracy were achieved and Limit of Quantitation (LOQ) values of 0.010 mg/kg were established. The method can be expanded to include more pesticides and their metabolites to improve the range of pesticide residues monitored in food commodities.
References
1 2 3
http://www.europa.eu.int/comm/food/plant/protection/pesticides/index_en.htm European Food Safety Authority (EFSA), www.efsa.europa.eu Commission Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market. Ofcial Journal of the European Communities L 230: 1-32. European Parliament and Council, Regulation (EC) 396/2005 of 23 February 2005, Maximum Residue Levels of Pesticides in Products of Plant or Animal Origin. Ofcial Journal of the European Communities L 70: 1-16. Commission Directive 76/895/EEC of 23 November 1976, relating to the xing of maximum levels for pesticide residues in and on fruit and vegetables. Ofcial Journal of the European Communities L 340: 26-31. Commission Directive 86/362/EEC of 24 July 1986 on the xing of maximum levels for pesticide residues in and on cereals. Ofcial Journal of the European Communities L 221: 37-42. Commission Directive 86/363/EEC of 24 July 1986 on the xing of maximum levels for pesticide in and on foodstuffs of animal origins. Ofcial Journal of the European Communities L 221: 43-47. Commission Directive 90/342/EEC of 27 November 1990 on the xing of maximum levels for pesticide residues in and on certain products of plant origin, including fruit and vegetables. Ofcial Journal of the European Communities L 350: 71-79. Commission Directive 1999/39/EC of 6 May 1999 Amending Directive 96/5/EC on processed cereal-based foods and baby foods for infants and young children. Ofcial Journal of the European Communities L 124: p.8-10.
10
Commission Directive 1999/50/EC of 25 May 1999 Amending Directive 91/321/EEC on infant formulae and follow-on formulae. Ofcial Journal of the European Communities L 139: 29-31. Commission Directive 2003/13/EC of 10 February 2003 Amending Directive 96/5/EC on processed cereal-based foods and baby foods for infants and young children. Ofcial Journal of the European Communities L 41: 33-35. Commission Directive 2006/125/EC of 5 December 2006 on processed cereal-based foods and baby foods for infants and young children. Ofcial Journal of the European Communities L 33: 16-35. Method Validation and Quality Control Procedures for Pesticide Residues Analysis in Food and Feed European Commission Document No. SANCO/2007/3131. Helen Botitsi, Anastasios Economou and Despina Tsipi. Development and validation of a multi-residue method for the determination of pesticides in processed fruits and vegetables using liquid chromatographyelectrospray ionization tandem mass spectrometry. Anal BioAnal Chem 2007, 389, 1685-1695.
11
12
13
14
www.thermo.com
AN62726_E 05/08S
LC-MS/MS Analysis of Herbicides in Drinking Water at Femtogram Levels Using 20 mL EQuan Direct Injection Techniques
Jonathan R. Beck, Charles Yang, Thermo Fisher Scientic, San Jose, CA, USA
Introduction
Key Words TSQ Quantum Access EQuan System Herbicides QED Water Analysis As concerns grow over the toxic effects of herbicides and other chemicals in our environment, the need to accurately monitor these substances in drinking water and foods becomes even more critical. LC-MS/MS is routinely used by the environmental and food industries to identify and quantify pesticide and herbicide residues. However, this method typically requires extensive ofine sample preconcentration methods, which can be expensive and time-consuming, to meet the stringent requirements and low limits of detection set forth by federal and international regulatory authorities. An online preconcentration and cleanup method has been developed that improves both sensitivity and precision and yields unmatched throughput. The Thermo Scientic EQuan system for online sample cleanup and analysis consists of a triple quadrupole mass spectrometer with an electrospray ionization source (ESI), two LC quaternary pumps, an autosampler, and two LC columns having C18 selectivity one for preconcentration of the sample, the second for analytical separation. A 6-port valve switches between the columns and is controlled by the instrument software. In addition to quantitative information, qualitative full scan product ion spectra are collected in the same analytical run and data le, using a technique called Reverse Energy Ramp (RER). This full scan spectrum provides additional conrmatory information for the compounds being analyzed. The resulting product ion spectra can be library searched for positive identication, or ion ratios can be used to conrm the presence of a particular compound, helping to eliminate false positive samples. This method uses drinking water for direct injection onto the loading column, with no sample preparation or ofine concentration. This application note provides a comparison of the online sample preconcentration of 1 mL, 5 mL, and 20 mL injections of drinking water samples spiked with herbicide compounds.
Goal
To compare different large volume injections using a loading column and an analytical column with two HPLC pumps.
Sample Preparation Drinking water containing 0.1% formic acid was spiked with a mixture of the following herbicides: ametryn, atraton, atrazine, prometon, prometryn, propazine, secbumeton, simetryn, simazine, terbuthylazine, and terbutryn (Ultra Scientic, North Kingstown, RI). The concentrations of the herbicides in the spiked water ranged from 0.1 pg/mL to 10 pg/mL. Calibration standards were prepared at the following concentrations: 0.1, 0.5, 1.0, 5.0, and 10.0 pg/mL. HPLC Spiked water samples and blank water samples (1 mL, 5 mL, or 20 mL) were injected directly onto a loading column (Thermo Scientic Hypersil GOLD 20 mm x 2.1 mm ID, 12 m) using an HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland). After the sample was completely transferred from the sample loop to the loading column, a 6-port valve was switched to enable the loading column to be back ushed onto the analytical column (Hypersil GOLD 50 mm x 2.1 mm ID, 3 m), where the compounds were separated prior to introduction into the mass spectrometer. After all of the compounds were eluted, the valve was switched back to the starting position. The loading and analytical columns were cleaned with a high organic phase before being re-equilibrated to their starting conditions (Figure 1a and 1b). Control and timing of the 6-port valve was through the computer data system, Thermo Scientic LCQUAN.
Figure 1: a) 6-port valve in position 1 (load position), for loading the sample onto the loading column. b) 6-port valve in position 2 (inject position), for eluting the compounds trapped on the loading column onto the analytical column.
Slightly different LC programs were used in each method, depending on the volume of the sample injected. The loading pump ow rates ranged from 1 mL/min for 1 mL samples to 5 mL/min for 20 mL samples. This allowed the run times at the higher injection volumes to be shortened because the time to transfer the sample from the sample loop to the loading column depends on the ow rate. The same LC program was used for the analytical column. Two HPLC pumps were used for the analysis: one for transferring the sample from the injection loop to the loading column, and one for back ushing the compounds off of the loading column and separating them on the analytical column. The loading pump was a Thermo Scientic Surveyor Plus LC pump and the analytical pump was a Thermo Scientic Accela U-HPLC pump. The HTC autosampler was equipped with a 5 mL syringe. To accommodate larger injection volumes (> 5 mL), a CTC macro sequence was programmed to allow for multiple syringe lls and deliveries to the sample loop from a 10 mL vial. For 20 mL samples, two 10 mL vials were used and the macro allowed sampling from adjacent vials lled with the same sample. The macro is shown in Figure 2. Because this multi-sampling scheme can be quite time consuming, the ability to perform look-ahead injections allows for signicant time savings. The loop can be switched to an ofine position during a run, and subsequent samples can be prepared and injected while a sample is being run.
MS MS analysis was carried out on a Thermo Scientic TSQ Quantum Access triple stage quadrupole mass spectrometer with an electrospray ionization (ESI) source. The MS conditions were as follows:
Ion Source Polarity: Spray Voltage: Ion Transfer Tube Temperature: Sheath Gas Pressure: Auxiliary Gas Pressure: Collision Gas (Ar): Q1/Q3 Peak Resolution: Scan Width: Positive ion mode 4000 V 300 C 30 arbitrary units 5 arbitrary units 1.5 mTorr 0.7 Da 0.002 Da
Quantitative and qualitative data were collected in the same run and data le.

Chromatograms of the herbicide simazine at three different injection volumes are shown in Figure 3. A very small peak can be seen for the 1 mL injection volume; however, the integration is not shown in the chromatogram. Injections at higher volumes show superior signal-to-noise ratios and intensity, which allow for analysis of very low concentration samples (pg/mL and sub pg/mL). To test the reproducibility of the multiple syringe ll method with a 20 mL loop, eight replicate injections were performed using the 1 pg/mL calibration standard. The results of this study are shown in Table 1. No internal standard was used in this analysis; however, if one were to be included, the % Relative Standard Deviations (RSD) values would likely improve. Table 1 also shows the peak areas and calculated difference in peak areas between the 1, 5, and 20 mL injections.
FPO
Number of syringe lls from the rst vial.
Number of syringe lls from the second vial (optional). Volume to be pulled per syringe injection
Figure 2: The method setup screen for the CTC Autosampler, showing the capability to perform multiple injections from the same vial. The red box highlights the parameters used to control the number of syringe lls from two consecutive vials. In this example, a total of 20 mL will be injected.
Compound
Area, 1 mL
Area, 5 mL
Area, 20 mL
Factor 1 mL to 5 mL
Factor 5 mL to 20 mL
%RSD (n = 8)
Atraton Simetryn Prometon/Secbumeton Ametryn Simazine
ND ND 3.26E+06 4.34E+06 3.18E+05
1.16E+07 4.27E+06 1.07E+07 1.42E+07 1.28E+06
5.42E+07 1.94E+07 4.80E+07 5.99E+07 5.70E+06
N/A N/A 3.30 3.27 4.03
4.69 4.56 4.47 4.22 4.44 4.02 3.49
11.15 8.93 9.89 11.59 5.32 3.99 4.97
Prometryn/Terbutryn 6.19E+06 1.89E+07 7.61E+07 3.05 Atrazine 1.26E+06 4.45E+06 1.55E+07 3.53 Table 1: Reproducibility for 20 mL injections (n = 8) at a 1 pg/mL concentration level, without an internal standard.
In addition to quantitative data, qualitative data was collected for each analyte using Quantitation-Enhanced Data-Dependent MS/MS (QED-MS/MS) scanning with the Reverse Energy Ramp (RER) scan function. The reverse energy ramp allows the collision energy in Q2 to be ramped from a high energy to a lower energy as Q3 is scanning the product ions from Q2 from low mass to high mass. This provides a rich product ion spectrum that can be used for library searching or ion ratio calculations to help eliminate false positive results. The RER provides a much richer product ion when compared to a Q3 product ion scan collected with a static collision energy. For this experiment, the collision energy for the RER was set to 25 eV and the ramp value was set to 20 eV. This results in a ramp from 45 eV at the low mass range of Q3. As Q3 scans to higher masses, the collision energy in Q2 is ramped lower and ends at a collision energy of 25 eV. Figure 4 shows the full scan Q3 spectrum that was collected during the analytical run for the calibration standard at a level of 1 pg/mL. It also shows a ramp illustrating the collision energy ramp applied to Q2.
Figure 3: Chromatograms showing the injection of simazine with 1, 5, and 20 mL injection volumes. The concentration of simazine is 1 pg/mL for all three injections.
Figure 4. QED-MS/MS Q3 spectrum for a 1pg/mL injection of atrazine. The collision energy was 25 eV and the ramp was 20 eV.
Conclusion
Using a preconcentration column in tandem with an analytical HPLC column allowed for the quantitation of a triazine herbicide mixture over the concentration range 0.1 10.0 pg/mL. Direct 20 mL injections were performed with the two HPLC columns. The large injection volume capabilities of the EQuan system eliminated the need for laborious and expensive ofine preconcentration using solid phase extraction. Injection volumes ranging from 1 mL to 20 mL are possible using this conguration, thus offering exibility for laboratories based on their sensitivity and reporting requirements.
Acknowledgements
We would like to thank Scott Harrison from LEAP Technologies (Carrboro, NC) for assistance with the CTC macro for multiple syringe lls and Mark Harrison from Thermo Fisher Scientic (Hemel-Hempsted, UK) for further assistance with the macro and for technical discussions.
www.thermo.com
Legal Notices 2008 Thermo Fisher Scientic Inc. All rights reserved. CTC is a trademark of CTC Analytics AG. All other trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientic Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
AN62858_E 10/08M
LC-MS/MS Determination of Malachite Green and Leucomalachite Green in Fish Products

Tao Ding 1, Jingzhong Xu1, Bin Wu 1, Huilan Chen1, Chongyu Shen1, Fei Liu2, and Kefei Wang 3
1 2
Food Laboratory, APFIC, Jiangsu Entry-Exit Inspection and Quarantine Bureau (JSCIQ), Nanjing, China Thermo Fisher Scientic, Shanghai, China 3 Thermo Fisher Scientic, San Jose, CA, USA
Introduction
Key Words Hypersil GOLD Surveyor HPLC TSQ Quantum Discovery MAX Food Safety
Reduction
Experimental
Chemicals and Reagents
All chemicals were of reagent grade or better. MG oxalate salt and LMG were from Sigma-Aldrich (St Louis, MO, USA), and d6-LMG from WITEGA (Berlin, Germany).
Malachite Green (MG, see Figure 1), a triphenylmethane dye, is an effective and inexpensive fungicide used in aquaculture, particularly in Asian countries. During metabolism MG reduces to Leucomalachite Green (LMG), which has been shown to accumulate in fatty sh tissues.
Sample Preparation
Extraction: 1. To 5.00 g of homogenized roasted eel meat, add 50 L 1 g/mL of d6-LMG as internal standard (ISTD), 1 mL 0.25 g/L hydroxylamine hydrochloride (NH2OH-HCl), 1 mL 0.05 mol/L p-toluenesulfonic acid, 2 mL 0.1 mol/L NH4Ac-HAc buffer (pH 4.5), and 40 mL acetonitrile. 2. Homogenize for 2 min. 3. Centrifuge the mixture at 3000 rpm for 3 min. 4. Collect the supernatants into a 250-mL separation funnel. 5. Extract the meat once more with 20 mL acetonitrile.
Liquid-Liquid Extraction:
H-SRM
H3C N CH3 N CH3
Oxidation
H3C
N CH3
CH3
CH3
CH3
Malachite Green (MG)
Leucolmalachite Green (LMG)
Figure 1: Structure and Conversion of Malachite Green and Leucomalachite Green
Both MG and LMG have demonstrated putative carcinogenic activity, and thus have been banned for use in aquaculture by both the U.S. FDA and European Union (EU). But trace levels of MG and LMG residues continue to be found in sh products. In a 2005 report,[1] malachite green was found in 18 out of 27 live eel or eel products imported from China to Hong Kong local market and food outlets, resulting in a government recall of all remaining products to be destroyed. Based on European Commission decision 2002/657/EC, an analytical test method to detect MG and LMG must have a Minimum Required Performance Limit (MRPL) of 2 g/kg of total malachite greens (MG+LMG) in sh muscle. Detection of MG and LMG has been reported by using UV-Vis, uorescence spectrometry and mass spectrometry coupled to HPLC separation. Among these detection techniques, the sensitivity and selectivity are poor with UV-Vis, and the uorescence detection requires a post-column oxidation (e.g. with lead oxide) to convert LMG to MG. Only mass spectrometry allows for detection of both LMG and MG without post-column oxidation, and with superior sensitivity and selectivity.[2] In this work, we report an LC-MS/MS method to detect MG and LMG in roasted eel meat using a triple quadrupole mass spectrometer operated in highly selective reaction monitoring (H-SRM) mode. The method is sensitive and selective, and has been validated for routine detection of < 0.5 g/kg of MG+LMG. Moreover, we demonstrate the capability of using H-SRM to reduce the chemical noise in complex sample matrices to improve detection of ultra-low level MG and LMG.
1. To the acetonitrile crude extract in the separation funnel, add 30 mL of dichloromethane (DCM) and 35 mL DI water, shake for 2 min. 2. Collect the DCM. 3. Extract the aqueous phase one more time with 20 mL DCM. 4. Evaporate the combined DCM solvent to dryness, and reconstitute in 3 mL of formic acid/acetonitrile (2:98).
Solid Phase Extraction (SPE):
1. Condition the Oasis 60 mg/3 cc MCX cartridge (Waters, Milford, MA, USA) with 3 mL acetonitrile, and 3 mL 2% v/v formic acid aqueous solution. 2. Load the sample (at ~0.2 mL/min). 3. Wash with 2 mL formic acid:acetonitrile (2:98) and 6 mL of acetonitrile. 4. Elute with 4 mL NH4Ac (5 mol/L and pH 7)/MeOH (5:95). 5. Evaporate the MeOH at 45C under reduced pressure 6. Dilute to 1.0 mL with initial mobile phase of water (0.1% v/v formic acid)/MeOH (70:30) 7. Filter with a 0.45 m syringe lter before injection to LC-MS.
Note: For very fatty roasted eel tissues, prior to the SPE with the MCX, the extracts after liquid-liquid extraction were cleaned up with a Superclean 60 mg/3 cc LCAlumina N cartridge (Waters, Milford, MA, USA): 1. Condition the cartridge with 3 mL acetonitrile 2. Load the sample, collect the elute 3. Wash with 3 mL acetonitrile, collect the elute to be combined with elute in (2) 4. Add 120 L of formic acid to the combined elute.

Eel meat tissues are in general fatty, and the roasted eel meat contains additional cooking oil and avor chemicals, making the sample matrix complicated. The extraction of MG and LMG from roasted eel meat involves sample extraction, liquid-liquid extraction, and one or two steps of SPE clean up. A similar method using the liquid-liquid extraction (with DCM) and one step SPE clean up (with SCX) were also reported for analysis of MG and LMG in both the raw and the processed eel products. [3] For extraction of MG and LMG in other raw sh meats, the procedure could be simplied. For example, Roudaut et al. have recently reported the following method without using the SPE for salmon, trout, tilapia and catsh:[4] Use 2 g of homogenized sample Add 200 L standard solution (for spike experiment only) Add 200 L ISTD solution 20 ng/mL Add 600 L Water (800 L for unknown) Add 2 mL Hydroxylamine HCl 5 g/L Stir the mixture for 10 min Add 8 mL acetonitrile Stir 10 min at 100 rpm Centrifuge for 5 min Filter on 0.45 um Inject 20 L to a TSQ Quantum Figure 2 shows the comparison of SRM and H-SRM chromatograms of a matrix matched standard (i.e., standard spiked into a blank roasted eel extract sample after sample preparation) containing 0.02 pg/L (0.2 pg on-column) MG and 0.1 pg/L (1 pg on-column) LMG and with 1 pg/L ISTD. As shown, with H-SRM, signal-to-noise (S/N) ratios have improved signicantly from 2-5 to 20-25. Note that the S/N improved despite the absolute signal (measured by the peak areas) decreasing by approximately half, indicating that the gains in S/N are from eliminating noise (isobaric interferences) in the sample matrix. The instrument detection limit in the current matrix is thus estimated to be 0.1 pg for MG and 0.5 pg for LMG with H-SRM based on 10 S/N. These detection limit values, corresponding to 0.004 g/kg and 0.02 g/kg for MG and LMG in meat tissues, respectively, have far exceeded our current requirement to detect <0.5 g/kg of MG+LMG in roasted eel meat. The response linearity was evaluated over the range of 0.05-8.0 g/kg using matrix matched standard solutions. The correlation coefcients obtained are >0.99 (weight factor = 1/X). Figure 3 shows the representative calibration curves. The analytical method was validated by analyzing fortied roast eel samples at 1, 2 and 5 g/kg levels for both LG and LMG, corresponding to 0.5, 1, and 2.5 MRPL, respectively. Seven replicates were performed at each level. The results are summarized in Table 2. Excellent recovery values of 90-106% were obtained with RSD% ranging from 3.7 to 11%.
Chromatography Conditions
Thermo Scientic Surveyor HPLC Thermo Scientic Hypersil GOLD CN 50 2.1 mm 5 m Mobile Phase: A: Methanol B: Water with 0.1% v/v Formic Acid Gradients: Time (min) A% 0.0 30% 2.0-6.0 90% 6.1 30% 10.0 30% Flow Rate: 220 L/min Injection volume: 10 L HPLC: Column:
Mass Spectrometry Conditions

Mass Spectrometer: Source: Sheath Gas: Auxiliary Gas: Capillary Temperature: Source CID: Q1 Peak Width (FWHM): Q3 Peak Width (FWHM): Collision Gas: SRM Transitions: Scan Time: Thermo Scientic TSQ Quantum Discovery MAX ESI+, 4000 V 40 unit 5 unit 350C -10 V 0.7 Da (0.2 Da for H-SRM) 0.7 Da Ar (1.5 mTorr) See Table 1 0.1 s
MG (M*) LMG (MH+) d6-LMG (MH+)
Precursor Ion 329.1 331.3 337.2
Product Ion (Collision Energy) 313 (33)* 208 (48) 239 (31)* 316 (18) 240 (30)
Table 1: SMR Transitions and Collision Energy Values for MG and LMG
MG (329.1>313)
LMG (331.3>239)
D6-MG (337.2>240)
Figure 2: Comparison of SRM (left) and H-SRM (right) Chromatograms of a Matrix Matched Standard Containing 0.02 pg/L MG and 0.05 pg/L LMG (10 L injection).
Figure 3: Representative calibration curves for MG and LMG with matrix matched standard solutions.
Spike (g/kg) MG LMG
1.0 95 (5.8) 106 (7.0%)
2.0 101 (3.7) 94 (11)
5.0 90 (7.5) 92 (4.7)
Table 2: Recovery% (RSD%) of MG and LMG (ISTD Corrected) in Roasted Eel Meat (n=7)
Conclusions
A highly sensitive and selective LC-MS/MS method using the TSQ Quantum Discovery MAX has been developed for determining Malachite Green and Leucomalachite Green in roasted eel meat. The method shows excellent linearity (0.05 to 8.0 g/kg), accuracy (90-106% recovery) and reproducibility (4-11% RSD), far exceeding the EUs requirement of MRPL of 2 g/kg of MG+LMG. The method has been implemented at the JSCIQ lab for routine monitoring of <0.5 g/kg (MG+LMG) in roasted eel and other sh products (with variations of sample preparation procedures). Highly selective reaction monitoring (H-SRM) has been shown to reduce the chemical noise effectively in the complicated sample matrix, which should be useful to further improve the method sensitivity and specicity (i.e., to eliminate both false positive and false negative) in support of enforcement of a zero tolerance policy toward the use of MG and LMG for aquaculture.
References
1. http://news.gov.hk/en/category/healthandcommunity/050819/html/ 050819en05002.htm 2. DR Doerge, MI Churchwell, TA Gehring, YM Pu and SM Plakas, Analysis of Malachite Green and Metabolites in Fish Using Liquid Chromatography Atmospheric Pressure Chemical Ionization Mass Spectrometry Rapid Communications in Mass Spectrometry, 12, , 1625-1634 (1994). 3. Hubert Tang PO, Twinnie Tso. S.C. and Clare Ho, Determination of Malachite Green and Leucomalachite Green in Fish Products 5th , International Symposium on Hormone and Veterinary Drug Residue Analysis, Antwerp, Belgium, May 16-19, 2006. 4. B Roudaut, B Delepine, M Bessiral and P Sanders, Malachite Green and Leucomalachite Green in Fish Flesh by Liquid Chromatography Tandem Mass Spectrometry (Validation of A Conrmatory Method) 2nd AOAC , International Symposium on Recent Advances in Food Analysis, Prague, Czech Republic, November 2-4, 2005.
www.thermo.com
AN62296_E 03/07S
Multi-residue Analysis of Pesticides in Food using GC/MS/MS with the TSQ Quantum GC
Kuniyo Sugitate, Michiko Kanai, Thermo Fisher Scientic, Yokohama, Japan Masahiro Okihashi, Division of Food Chemistry, Osaka Prefectural Institute of Public Health, Osaka, Japan Dipankar Ghosh, Thermo Fisher Scientic, San Jose, CA, USA
Introduction
Key Words TSQ Quantum GC H-SRM Pesticide Residues in Food Positive List System QED SRM Food safety concerns are on the rise amongst consumers worldwide. In 2006, sweeping changes were made to the Food Hygiene Law in Japan regarding residues of agricultural chemicals, including pesticides, in foods. As a result, standard residue values were established for approximately 800 pesticides. All food items produced in or imported into Japan are required to meet the standards established by this law. If pesticide residues in any food items exceed these standards, then the distribution and sale of the food is prohibited. This Positive List System has had a signicant effect not only on the Japanese domestic production, but also on much of the food exported to Japan from various foreign countries such as China, the United States, and Taiwan. There are numerous types of pesticides regularly used in the agricultural industry, including insecticides, fungicides, herbicides, and growth regulators. Because each type has different physicochemical properties, there are limitations on simultaneous analysis. Among the pesticides for which standard values are currently set, GC/MS/MS can analyze approximately 300 compounds. The superior selectivity of this technique allows interference-free quantication, even with peak coelution, and provides positive conrmation of various pesticides in a single analytical run. To accurately monitor pesticide residues, a high throughput multi-residue screening method that can quantitate a large number of pesticide residues during a single analytical run is needed.
graphite carbon/PSA dual layer solid phase extraction column and eluted with 50 mL of acetonitrile/toluene (3:1). After the eluate was concentrated under reduced pressure, it was dissolved (1 g/mL) in 10 mL of acetone/n-hexane to give the test solution.
GC
GC analysis was performed using the Thermo Scientic TRACE GC Ultra System. The GC conditions were as follows: Column: Rxi-5MS 30 m x 0.25 mm I.D., 0.25 m df (Restek Corp., Bellefonte, PA) Injection mode: Splitless with surge injection (200 kPa, 1 min) Injection temperature: 240 C Oven temperature: 80 C (1 min) 20 C/min 180 C 5 C/min 280 C (10 min) Flow rate: Constant ow 1.2 mL/min Transfer line temperature: 280 C
AS
The samples were injected through the Thermo Scientic TriPlus autosampler. The autosampler conditions were as follows: Injection volume: 1 L Injection mode: Hot needle Syringe: 80 mm
MS
Goal
To simultaneously analyze 103 pesticides using the Thermo Scientic TSQ Quantum GC system, using SRM and H-SRM. Additionally, to show the utility of QED MS/MS for structural conrmation of the analytes undergoing quantication.
MS analysis was carried out on a TSQ Quantum GC triple stage quadrupole mass spectrometer. The MS conditions were as follows: Ionization mode: EI positive ion Ion volume: Closed EI Emission current: 25 A Ion source temperature: 220 C Scan type: SRM and H-SRM Scan width: 0.002 a.m.u. Scan time: 0.01 s Peak width: Q1, 0.7 Da; Q3, 0.7 Da FWHM Peak width for H-SRM: Q1, 0.4 Da; Q3, 0.7 Da FWHM Collision gas (Ar) pressure: 1.2 mTorr A total of 103 pesticides were analyzed to determine the product ion to be used for quantitation. Table 1 lists the SRM transitions and the optimum collision energy for each of the compounds and a summary of the calibration range, linearity, and the reproducibility of each individual compound at 5 ppb (ng/mL).
Page 1of 6
Sample Preparation
Green pepper, carrot, grapefruit and banana samples were prepared for analysis using a method based on the simple and quick QuEChERS approach.1 A 10 g sample of food was homogenized in a food processor and placed in a polypropylene centrifuge tube. The sample was extracted with 20 mL of acetonitrile in a homogenizer. Then, 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride were added and the resulting mixture was centrifuged. After centrifugation, the supernatant was loaded onto a
R.T.
Precursor Ion (m/z)
Product Ion (m/z)
Collision Energy
R2
Range
CV(%) n=5
R.T.
Precursor Ion (m/z)
Product Ion (m/z)
Collision Energy
R2
Range
CV(%) n=5
Mevinphos XMC Tecnazene Ethoprpphos Ethaluralin Benuralin Monocrotophos -BHC Dicloran Simazine Propazine -BHC -BHC Cyanophos Pyroquilon Diazinon Phosphamidon-1 -BHC Benoxacor Propanil Phosphamidon-2 Dichlofenthion Dimethenamid Bromobutide Paration-methyl Tolclofos-methyl Ametryn Mefenoxam Bromacil Quinoclamine Diethofencarb Cyanazine Chlorpyrifos Parathion Triadimefon
6.44 7.52 8.03 8.22 8.42 8.62 8.62 9.03 9.25 9.30 9.50 9.57 9.73 9.78 9.90 4.94 10.06 10.26 10.7 10.95 10.97 10.99 11.06 11.09 11.24 11.38 11.43 11.57 11.98 12.18 12.34 12.52 12.57 12.59 12.67
192 122 261 200 316 292 192 219 206 201 214 219 219 243 173 304 264 184 219 264 259 262 264 279 230 232 263 265 227 249 205 305 207 225 225 314 291 208 301 236 272 195 239 262 367 123 145 274 146 236 241 237 256 286 271
127 107 203 114 276 264 127 183 176 172 172 183 183 109 130 179 127 83 183 127 120 202 127 223 154 176 109 250 170 190 188 276 172 125 189 258 109 111 223 148 243 103 167 200 213 81 112 121 118 125 206 160 144 202 128
10 10 15 10 10 10 10 15 10 10 10 15 15 10 20 15 10 20 15 10 15 10 10 15 10 10 10 15 10 10 15 10 10 15 10 15 15 25 20 15 10 10 10 15 25 10 10 10 10 15 15 10 20 15 5
0.9999 0.9999 0.9996 0.9981 0.9997 0.9989 0.9754 0.9999 0.9994 0.9999 0.9998 1.0000 0.9998 0.9996 0.9996 0.9995 0.9989 0.9992 0.9994 0.9972 0.9999 0.9993 0.9970 0.9994 0.9996 0.9990 0.9982 0.9998 0.9999 0.9995 0.9988 0.9995 0.9989 0.9985 0.9994 0.9991 0.9962 0.9986 1.0000 0.9974 0.9993 0.9956 0.9997 0.9979 0.9991 0.9991 0.9987 0.9987 0.9984 0.9961 0.9996 0.9998 0.9932 0.9958 0.9989
0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 5-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 5-100 0.1-100 0.2-100 0.1-100 5-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 2-100 0.1-100 0.1-100
4.03 2.55 5.41 7.91 4.14 1.86 19.47 4.51 2.30 4.33 1.99 3.51 6.57 3.56 2.95 4.40 10.31 7.39 5.17 17.11 3.30 3.65 8.77 2.21 2.51 5.91 3.74 2.52 0.90 5.81 3.87 4.08 4.24 4.64 3.41 3.37 9.76 6.10 1.23 5.53 4.32 6.29 4.67 4.54 3.49 3.79 3.74 1.82 1.96 7.41 4.54 5.26 12.09 4.66 8.96
Flutlanil Hexaconazole Profenofos Uniconazole-P Pretilachlor Flamprop-methyl Oxyuorfen Azaconazole Bupirimate Thiuzamide Fenoxanil Chlorbenzilate Pyriminobacmethyl-Z Oxadixyl Triazophos Fluacrypyrim Edifenphos Quinoxyfen Lenacil Trioxystrobin Pyriminobacmethyl-E Tebuconazole Diclofop-methyl Mefenpyr-diethyl Pyributicarb Pyridafenthion Acetamiprid Bromopropylate Piperophos Fenpropathrin Etoxazole Tebufenpyrad Anilofos Phenothrin-1 Tetradifon Phenothrin-2 Mefenacet Cyhalofop-buthyl Cyhalothrin-1 Cyhalothrin-2 Pyrazophos Bitertanol Pyridaben Cafenstrole Cypermethrin-1 Halfenprox Cypermethrin-2 Cypermethrin-3 Cypermethrin-4 Fenvalerate-1 Flumioxazin Fenvarelate-2 Deltamethrin+ Tralomethrin Tolfenpyrad Imibenconazole
15.06 15.06 15.28 15.38 15.37 15.66 15.69 15.79 15.82 15.84 16.25 16.43 16.76 16.86 17.30 17.38 17.72 17.74 17.78 18.01 18.19 18.39 18.51 19.15 19.24 19.46 19.39 19.64 19.84 19.98 20.06 20.10 20.31 20.49 20.54 20.66 21.22 21.23 21.30 21.66 22.06 22.80 22.97 23.18 24.03 24.72 24.79 24.92 25.06 25.13 26.47 26.50 26.91 28.15 29.11 30.35
173 214 337 234 162 276 361 217 316 449 293 251 302 163 257 189 310 272 153 222 302 250 253 253 165 340 152 341 320 265 300 333 226 183 356 183 192 357 181 181 373 170 147 100 181 263 181 181 181 167 354 167 181 383 375
145 172 267 137 132 105 300 173 208 429 155 139 256 132 162 129 173 237 136 162 256 125 162 189 108 199 116 185 122 210 270 171 157 165 229 165 136 229 152 152 232 141 117 72 152 235 152 152 152 125 176 125 152 171 260
15 15 15 15 15 10 10 15 10 10 20 15 15 10 10 10 10 10 15 10 15 20 15 20 10 10 20 15 10 10 20 20 15 10 10 10 15 10 20 20 10 20 20 5 20 15 20 20 20 10 20 10 20 20 15
0.9986 0.9924 0.9968 0.9966 0.9982 0.9986 0.9980 0.9981 0.9982 0.9972 0.9989 0.9976 0.9986 0.9998 0.9941 0.9988 0.9927 0.9993 0.9979 0.9966 0.9982 0.9907 0.9991 0.9992 0.9973 0.9940 1.0000 0.9956 0.9939 0.9973 0.9969 0.9978 0.9948 0.9967 0.9998 0.9968 0.9955 0.9967 0.9975 0.9984 0.9963 0.9873 0.9958 0.9958 0.9983 0.9979 0.9982 0.9985 0.9948 0.9977 0.9937 0.9979 0.9967 0.9968 1.0000
0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.5-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.2-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.1-100 0.2-100 0.1-100 0.1-100 0.1-100 0.2-100 50-100 0.1-100 0.2-100 0.1-100 0.1-100 0.5-100 0.2-100 5-100 0.2-100 0.1-100 0.1-100 0.1-100 0.2-100 0.2-100 0.1-100 0.1-100 0.1-100 0.1-100 2-100 0.1-100 2-100 2-100 2-100 0.1-100 0.1-100 0.1-100 0.2-100 2-100 50-100
1.93 8.98 6.61 11.37 6.72 3.93 6.07 7.07 4.65 2.75 3.73 0.81 2.70 3.72 6.72 2.15 7.95 4.50 5.19 8.47 2.12 13.03 2.14 3.35 2.00 4.71 3.72 7.51 6.87 8.84 13.35 5.56 16.13 4.17 3.79 4.90 5.52 3.21 6.67 10.46 6.76 1.29 9.77 9.29 10.25 6.91 16.27 13.79 3.11 9.66 3.26 8.20 4.84
Prohydrojasmon-1 10.12 Prohydrojasmon-2 10.66
Pirimiphos-methyl 12.00
Chlorthal-dimethyl 12.73 Nitrothal-isopropyl 12.78 Fthalide Fosthiazate Diphenamid Pyrifenox-Z Fipronil Allethrin Dimepiperate Phenthoate Quinalphos Paclobutrazol Endosulfan- Butachlor 13.04 13.05 13.12 13.1 13.64 13.79 13.67 13.87 13.87 13.88 14.45 14.67 14.73
Imazamethabenz- 14.81 methyl Butamifos Napropamide 15.00 15.01
Table 1: Retention times, SRM conditions, calibration range, linearity, and the reproducibility of each individual pesticide residue compound Page 2 of 6

Figure 1 shows an example calibration curve for Propazine at 0.1-100 ppb with a corresponding chromatogram at 1 ppb, showing excellent reproducibility (r2 = 0.9998). Figure 2 shows examples of GC/MS/MS chromatograms of various pesticides in which 1 ppb of each pesticide was added to green pepper. Even at this extremely low concentration (1/10 of the uniform standard value for pesticides), it was possible to make measurements with remarkably high sensitivity with the TSQ Quantum GC.
Figure 3 shows the chromatograms for cypermethrin, fenvalerate and deltamethrin (+ tralomethrin). Cypermethrin is a synthetic pyrethroid compound with a high detection ratio in agricultural produce. In addition to having a slow elution time in the GC, it has 4 peaks that are due to different isomers that must be resolved. As the chromatograms show, measurements with good sensitivity were obtained even at the low concentration of 5 ppb.
Figure 1: Calibration curve (0.1-100 ppb) and SRM chromatogram (1 ppb) for Propazine
Figure 2: GC/MS/MS chromatograms of various pesticides at 1 ppb in green pepper samples
Page 3 of 6
Figure 3: Chromatograms for cypermethrin, fenvalerate and deltamethrin
Figure 4: Comparison of SRM Mode with H-SRM Mode. (a) Flamprop-methyl in grapefruit (1 ppb). (b) Parathion in green-pepper (1 ppb).
Page 4 of 6
Advantages of H-SRM
H-SRM is an acronym for Highly-Selective Reaction Monitoring, which is a more advanced form of Selective Reaction Monitoring (SRM). H-SRM can eliminate chemical noise, lower detection limits, and reduce the likelihood of generating false positives. For many pesticides that are subject to matrix-dependent interference, the measurements can be successfully carried out using the H-SRM mode. With H-SRM, the precursor ion is selected with a smaller peak width. The more stringent tolerance accounts for the higher selectivity, which can lower LOQs and increase precision and accuracy at the limits of detection. The effects of H-SRM over SRM are illustrated for amprop-methyl in grapefruit and parathion in green-pepper in Figure 4.
Structural Conrmation with QED

QED MS/MS stands for Quantication Enhanced by Data Dependent MS/MS. A QED scan on a triple quadrupole instrument delivers an information rich mass spectrum that can be used for structural conrmation of analytes while undergoing quantication by SRM (or H-SRM). The specicity provided by H-SRM followed by QED MS/MS provides uncompromised quantitation performance at low levels followed by a fast, highly-specic full MS/MS scan for conrmation. Figure 5 shows the QED scan results obtained from a carrot test sample spiked with 10 ppb diazinon.
Figure 5: Chromatogram from a carrot test sample (upper row) and the MS/MS spectrum obtained with QED (lower row) Page 5 of 6
Zero Cross-talk
Cross-talk can potentially occur when fragment ions from one SRM transition remain in the collision cell while a second SRM transition takes place. This can cause signal artifacts in the second SRM transitions chromatogram. It can be especially problematic when different SRM events have the same product ions formed from different precursor ions. However, the orthogonal design of the collision cell in the TSQ Quantum eliminates cross-talk. Figure 6 shows the absence of cross-talk between two different SRM transitions of paclobutrazol and thiuzamide. Both yield a product ion of m/z 125, but no artifacts are seen in either chromatogram with a scan time of 10 ms. Similarly, the SRM transitions of triszophos and diclofop-methyl 5 also show no evidence of cross talk, even though they both yield product ions at m/z 162.
Conclusion
Simultaneous analysis was carried out on multi-component pesticide residues in food products using a quadrupole GC/MS/MS system, the TSQ Quantum GC. Results obtained indicated excellent sensitivity (0.1 ppb), reproducibility (10% at 5 ppb) and linearity (R2 > 0.995) in the range of 0.1-100 ppb. No cross-talk was observed for the analysis of closely eluting multi-component mixtures. Using H-SRM, interferences from the sample matrix background were substantially reduced, leading to improved LOQs. In addition, QED provided MS/MS structural conrmation of the analytes undergoing quantication.
References
1. Okihashi, M.; Kitagawa, Y.; Akutsu, K.; Obana, H.; Tanaka, Y. Rapid method for the determination of 180 pesticide residues in foods by gas chromatography/mass spectrometry and ame photometric detection; J Pestic Sci 2005, 30(4), 368-77. 2. The Japanese Ministry of Health, Labour, and Welfare: www.mhlw.go.jp/english/topics/foodsafety/positivelist060228/index.html(English) www.mhlw.go.jp/topics/bukyoku/iyaku/syoku-anzen/zanryu2/index.html (Japanese)
www.thermo.com Figure 6: No cross-talk was observed in the SRM transitions of paclobutrazol and thiuzamide or in the SRM transitions of triszophos and diclofop-methyl.

AN387_E 02/08M
Quantitation-Enhanced Data-Dependent (QED) Scanning of DrinkingWater Samples Using EQuan for Pesticide Analysis on a Triple Stage Quadrupole
Jonathan Beck, Thermo Fisher Scientic, San Jose, California, USA Mihoko Yamaguchi and Kaori Saito, Thermo Electron K.K., Yokohama, Japan
Overview
Key Words TSQ Quantum EQuan LC-MS/MS Pesticides QED RER Water Analysis In recent years, the use of LC-MS/MS for pesticide analysis has become increasingly popular due to the amenability of electrospray ionization (ESI) for polar compounds. This application note describes the multi-residue assay of a group of pesticides in drinking water by using the Thermo Scientic EQuan system. The method utilizes SRM for quantication, followed by Data Dependent QED-MS/MS and library searching for the structural conrmation of these analytes. Traditionally, water samples are extracted in a very time consuming manner, using 1 L of sample that is concentrated 100-1000 times before analysis. This extraction can take several hours or even days of preparation time. Also, the added expense of extraction cartridges leads to more expense per sample for labs. EQuan is a solution that allows for automated online preconcentration of samples without the need for extensive sample preparation or manual intervention.
Many pesticide samples are regulated at a very low level (ppt, or ng/L levels), and in order to detect compounds at these low levels, time consuming extraction and concentration of samples is required before analysis. EQuan utilizes two LC pumps, a large volume autosampler, two HPLC columns, and a Thermo Scientic TSQ Quantum Mass Spectrometer to reduce sample preparation time and to analyze the samples at the concentration levels that are required.
SamplesDrinking water was spiked with tricyclazole (0.8), carbaryl (0.5), carbofuran (0.05), asulam (2.0), diruon (0.2), siduron (3.0), daimuron (8.0), carpropamid (0.4), thiodicarb (0.8), azoxystrobin (5.0), azasulfuran (0.3), bensulfuron methyl (4.0), and halosulfon methyl (3.0) at concentration levels from 0.5 ppt (pg/mL) to 1000 ppt. These compounds are all regulated by the Japanese Ministry of Health Labour and Welfare. The reporting level for each compound, as set by the Japanese Ministry of Health, Labour, and Welfare, in ppb (g/L) is given in parenthesis after each compound. HPLC ConditionsTwo pump systems were used, a Thermo Scientic Surveyor L-Pump for loading the 1 mL sample onto the loading column (Thermo Scientic Hypersil GOLD 20 2.1 mm 12), and a Surveyor MS Pump Plus for eluting the compounds off of the loading column and separation on the analytical column (Hypersil GOLD 50 2.1 mm 3). The mobile phase for both pumps was Water with 0.1% Formic Acid (A), and Acetonitrile with 0.1% Formic Acid (B). For the loading L Pump, the gradient used is shown in Table 1, and the gradient used for the analysis pump is shown in Table 2. A divert valve on the mass spectrometer is programmed by the data system to control the loading and elution of the two LC columns. In this experiment, the valve is in the load position from 0 to 1.5 minutes to allow for the entire 1 mL sample to collect on the loading column before switching to the analysis position until all of the analytes are eluted, 12.5 minutes in this case. After switching back to the loading position, the loading column can be rinsed and re-equilibrated by the loading L pump. A schematic of the EQuan system is shown in Figure 1.
Introduction
There is an increasing emphasis for using multiple SRMs (selected reaction monitoring) and the use of Ion Ratio Conrmation (IRC) to positively conrm the presence of banned or controlled substances in samples. For example, the 2002/657/EC European Commission Decision dictates Identication Points (IPs) that must be met for a sample to be deemed positive. These criteria can include the number of MS/MS transitions, ion ratios, or the type of mass spectrometer used (high resolution Vs. low resolution). The Quantitation Enhanced Data-Dependent (QED) scan on a regular triple quadrupole instrument delivers an information rich MS/MS which can be used to conrm the existence of compounds while they are being quantied. When using QED, a full scan MS/MS mass spectrum is obtained by Data Dependent scanning for conrmatory analysis during the single reaction monitoring experiment (SRM), which is used for routine quantitation. Once a particular SRM transition reaches a user set intensity threshold, the instrument automatically triggers QED, using an innovative new technique called Reversed Energy Ramp (RER) which produces the high sensitivity product ion spectrum. The RER function linearly ramps the collision energy from a high to low value, while scanning Q3. The RER scan generates a highly sensitive, fragment-rich MS/MS spectrum that can be used to positively conrm the existence of a compound.
Figure 1: Diagram showing the EQuan setup used in this experiment.
Time 0 1.5 2 12.5 12.6 14.5 14.6 17
%A 95 95 95 95 5 5 95 95
%B 5 5 5 5 95 95 5 5
Flow Rate (mL/min) 1.0 1.0 0.0 0.0 1.0 1.0 1.0 1.0
MS Conditions Thermo Scientic TSQ Quantum Discovery Ion source and polarity: ESI, Positive ion mode Spray Voltage: 4500 V Sheath Gas: 45 units (N2) Auxiliary Gas: Not Used Transfer Tube Temperature: 330C Collision Gap Pressure: 1.0 units (Ar) MS Scan Functions Two different scan functions, a SRM (selected reaction monitoring) followed by a data dependent QED scan function were selected in the method. The SRM transitions can be seen in Figure 2a, and the QED scan function can be seen in Figure 2b.
Table 1: Gradient program for the loading pump. The ow is turned off from 2 to 12.5 minutes to conserve mobile phase, and the column is rinsed from 12.6 to 14.5 minutes with a high organic phase, before re-equilibrating to starting conditions.
Time 0.00 1.50 10.0 12.0 12.1 17.0
%A 95 95 0 0 95 95
%B 5 5 100 100 5 5
Table 2: Gradient program for the analysis pump. The ow rate for this analysis is 0.2 mL/min.
Page 2 of 8
Figure 2a: SRM transitions monitored in the experiment.
Figure 2b: QED scan function. A RER from 55 to 10 eV triggered by a SRM transition greater than 5.0104 counts. Dynamic exclusion was used to allow only one QED spectrum to be collected for each SRM.
Page 3 of 8

Figures 3 and 4 show the chromatogram of the 10 ppt standard from 5 to 11.5 minutes for all of the analytes listed in the Experimental Conditions section. This level is ve times lower than the lowest MRL for the mixture of compounds (Carbofuran, 50 ppt). Asulam, the peak show-
ing the lowest S/N in at this concentration level is 200 times lower than the MRL. Excellent linearity is obtained for all of the compounds over the concentration range 1 ppt to 500 or 1000 ppt at the high end. Figure 5 shows the calibration curve for Asulam. Table 3 summarizes the calibration data for each compound, individually.
100 50 0 100 50 0 100 50 Relative Abundance 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0
5.0 5.5 6.0 6.5 7.0 7.5 5.39
7.77
Tricyclazole
8.84
Carbaryl
8.55
Carbofuran Asulam
9.50
Diuron
9.88
Siduron Daimuron
9.99
10.14
10.86
Carpropamide
8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5
Time (min.)
Figure 3: A chromatogram at a concentration level of 10 ppt for the rst eight compounds (in order of increasing precursor ion mass) analyzed in the mixture, from 5 to 11.5 minutes.
100
10.86
50
Thidicarb
9.74
0 100
50
Azoxystrobin
9.69
Relative Abundance
0 100
50
Flazasulfuron
9.63
0 100
50
Bensulfuron-methyl
10.50
0 100
50
Halosulfuron-methyl
5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time (min.) 9.0 9.5 10.0 10.5 11.0
Figure 4: A chromatogram at a concentration level of 10 ppt for the last ve compounds (in order of increasing precursor ion mass) analyzed in the mixture, from 5 to 11.5 minutes.
Page 4 of 8
While excellent linearity and quantitative results were gathered using SRM transitions, additional QED data were collected during each run for each compound. An example of a QED full scan MS/MS spectrum is shown in Figure 6 for the compound Carbofuran. This QED scan function fragmented the precursor ion m/z 222 for Carbofuran over a reverse energy ramp of 10 to 55 eV, as specied in the second scan function (Figure 2b). Using the built in environmental compound library available for the TSQ Quantum that includes over 1000 compounds, Carbofuran was the #1 hit in the list of
possible compounds (Figure 7). This feature allows for additional positive conrmation data for compounds that are analyzed, providing the required number of IPs needed for the positive presence of a compound. Library searching for all of the compounds in the experimental mixture yielded matches for either the rst or second compound in the list of possible compounds. Furthermore, the library searches of the QED scans for the two compounds Diuron and Siduron, which both have precursor masses of m/z 233, correctly identied each unique compound, based on differences in their QED spectra.
5500000 5000000 4500000 4000000 3500000

Area
3000000 2500000 2000000 1500000 1000000 500000 0

0 20 0 400 60 0 ppt 800 1000
Figure 5: Calibration curve for the analyte Asulam from 1 to 1000 ppt.
Compound Tricyclazole Carbaryl Carbofuran Asulam Diuron Siduron Daimuron Carpropamide Thidicarb Azoxystrobin Flazasulfuron Bensulfuron-methyl Halosulfuron-methyl
Retention Time (min) 7.7 8.7 8.5 5.3 9.4 9.8 10.0 10.8 8.8 9.6 9.6 9.5 10.4
High Concentration 500 ppt 500 ppt 500 ppt 1000 ppt 500 ppt 500 ppt 500 ppt 500 ppt 500 ppt 1000 ppt 1000 ppt 1000 ppt 500 ppt
Equation y = 21149 + 112678x R2 = 0.9993 y = 36518 + 78565x R2 = 0.9964 y = 54509 + 290697x R2 = 0.9977 y = -726 + 5356x R2 = 0.9992 y = 758 + 32087x R2 = 0.9988 y = 51461 + 88505x R2 = 0.9994 y = 144173 + 285515x R2 = 0.9963 y = 10377 + 37079x R2 = 0.9999 y = 16505 + 35334x R2 = 0.9959 y = 45456 + 198901x R2 = 0.9978 y = -3499 + 86802x R2 = 0.9989 y = -2657 + 65708x R2 = 0.9945 y = 1944 + 40565x R2 = 0.9981
Table 3: Results of pesticide calibration curves. All curves were a linear curve t with a weighting factor of 1/x.
Page 5 of 8
100 95 90 85 80 75 70 65 60 Relative Abundance 55 50 45
221.97
122.81
164.96
40 35 30 25 20 15 10 5 0
205.27 57.58 30.66 43.16

40
56.74 67.16
60
87.57 77.24 94.88 80.46

80 100
105.45 116.58
120
136.96 141.18 133.62

140 m/z
186.93 154.43
160
173.10
180
193.34
200
218.03
220
Figure 6: QED spectrum of Carbofuran at the 5ppt calibration level. Searching against the standard library available on the TSQ Quantum instrument platform yields a positive conrmation.
Sample Spectrum
Library Spectrum
Figure 7: Library search result for the QED spectrum generated at the 5 ppt calibration level. Carbofuran, highlighted in blue is the #1 hit in the list of possible compounds.
Page 6 of 8
Conclusions
The Quantitation Enhanced Data Dependent scan function, standard on all TSQ Quantum mass spectrometers, allows the user to obtain conrmatory data following quantitative analysis. This is of particular signicance when analyzing environmental pollutants in water samples. EQuan, with its large injection volume, allows for signicant time savings over traditional SPE concentration methods, and allows for detection and quantitation of compounds at levels well below the regulatory requirements. The built-in library of over 1000 compounds in the industry standard NIST format can help users to positively identify compounds based on EU regulations. Additionally, users have the ability to add to or replace the spectra in the library to increase their positive hit probabilities when searching the library.
References
The 2002/657/EC European Commission Decision can be found on the World Wide Web at: http://ec.europa.eu/food/food/chemicalsafety/residues/lab_analysis_en.htm The Japanese Ministry of Health, Labour, and Welfare can be found on the World Wide Web at: http://www.mhlw.go.jp/index.html (Japanese) http://www.mhlw.go.jp/english/index.html (English)
Page 7 of 8

www.thermo.com
2007 Thermo Fisher Scientic Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries.
Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
AN62265_E 01/07S
Simultaneous Detection of 88 Pesticides on the TSQ Quantum Discovery Using a Novel LC-MS/MS Method
Dipankar Ghosh1, Lutz Alder2, Mark Churchill1, Wilhelm Gebhardt1, Eric Genin1, and Jeanette Klein2
1 2
Thermo Fisher Scientic, San Jose, CA, USA; Federal Institute for Health Protection of Consumers & Veterinary Medicine, POB 330013, D-14191 Berlin, Germany
Overview
Key Words TSQ Quantum Discovery Cross-talk EPA LC-MS/MS Pesticides Pesticide residues in food are strictly regulated according to the provisions of US Environmental Protection Agency (EPA) CFR Title 40. Several hundred sections in Part 180 detail the maximum pesticide residue (tolerance) for a wide variety of foods. A pesticides allowable tolerance (measured in ppm) can span several orders of magnitude, depending upon the food source. For example, the tolerance for captan in cattle fat is 0.05 ppm, while 100 ppm of captan is acceptable in lettuce and spinach. To analyze the large numbers of samples whose pesticide treatment history is usually unknown, the US Food and Drug Administration (FDA) uses analytical methods capable of simultaneously determining a number of pesticide residues. These cost-effective multi-residue methods (MRMs) can determine about half of the approximately 400 pesticides and their metabolites with EPA tolerances. Most commonly, residues in extracts are separated by GC or HPLC, and then detected using UV absorbance, nitrogen phosphorus detection, or electron capture detection. Due to its specicity in identifying compounds, LC-MS/MS is emerging as the technique of choice for identifying and quantifying pesticides. The most commonly used MRMs can also detect many metabolites, impurities, and alteration products of pesticides. Conventional MS/MS methods generally require extensive optimization of operating parameters for each target analyte or even for compounds belonging to the same chemical class, signicantly impacting analytical throughput. The objective of this work was to demonstrate the use of the Thermo Scientic TSQ Quantum Discovery in developing an automated, generic, high-throughput LC-MS/MS screening method to simultaneously detect and quantitate nearly 100 pesticides following minimal separation using an HPLC.
Goals
Develop a multi-residue LC-MS/MS screening method to detect 88 analytes using a single, automated experiment with a short chromatographic time scale Demonstrate the utility of using different time segments and scan events Illustrate the large linear dynamic range for pesticide analysis in a multi-residue context Exhibit the absence of cross-talk between co-eluting components
Chemicals and Reagents Water, methanol, and acetic acid were HPLC grade and purchased from J. T. Baker Chemicals, France. Samples Pesticides listed in Table 1 were purchased from Sigma unless otherwise noted. Standards solutions of 0.1, 0.5, 1, 5, 10 and 50 pg/L were prepared in methanol. Sample Analysis HPLC analysis was performed on the Thermo Scientic Surveyor HPLC System, using a Thermo Scientic AQUASIL C18 50 2.1 mm column. Mobile phase A was water/methanol 80/20 (v/v) and mobile phase B was methanol/water 90/10 (v/v) both contained 0.05% acetic acid. Solvent was pumped at 200 L/min and analytes eluted using a linear gradient of 100% A to 100% B over 11 minutes, holding at 100% B for 12 minutes, then returning to 100% A in 2 minutes. Mass Spectrometry Instrument: TSQ Quantum Discovery Source: ESI Ion polarity: Positive Spray voltage: 3.5 kV Sheath/Auxiliary gas: Nitrogen Sheath gas pressure: 50 (arbitrary units) Auxiliary gas pressure: 15 (arbitrary units) Ion transfer capillary temperature: 350 C Scan type: SRM CID conditions: Ar at 1.5 mTorr
100
Pesticide Bendiocarb
Structure
O
Mwt (da) Use
80
223
O O O HN
Relative Intensity
60
Insect control against public health, industrial & storage pests
40
20
NH
0 10 20 30 40 50 60
N N
Pyrimethanil
199
Collision Energy (V)
Fungal control on vine/fruits, ornamentals
COMPOUND
PARENT M/Z
PRODUCT M/Z
COLLISION ENERGY V
NH
Bendiocarb Bendiocarb Pyrimethanil Pyrimethanil Thiabendazol Thiabendazol Cyprodinil Cyprodinil
224.1 224.1 200.1 200.1 202.0 202.0 226.1 226.1
167.1 109.0 107.1 182.1 175.0 131.1 93.1 77.1
10 22 28 28 30 36 40 46
Thiabendazol
N N
201
Fungal control on cotton, barley, bananas
NH
N N
Cyprodinil
225
Fungal control in cereals, grapes, strawberries
Figure 1a: Simultaneous multicomponent optimization of MS/MS parameters
Figure 1b: Structures of the four pesticides used to generate the optimization graph of Figure 1a
Multi-residue Optimization One of the most time consuming parts in the development of a large multicomponent assay is the optimization of MS/MS parameters for each analyte. The TSQ Quantum Discovery allows multicomponent optimization of MS/MS parameters to be carried out automatically, thus allowing for faster method development. Up to eight SRM transitions can be optimized simultaneously, either from a single parent component or from multiple components. In effect, this means the ability to carry out the optimization procedure 11 times for 88 pesticides (instead of 88 times if they were carried out singly), thus saving a signicant amount of time in method development. An example of this is given in Figure 1a, displaying the simultaneous optimization of eight SRM transitions from four pesticides. The structures of these compounds are shown in Figure 1b. MS Instrument Method To accommodate such a large number of components over a short time range, the acquisition time was divided into two segments, each containing three scan events. Allowing for analyte overlap between the time segments, a total of 59 SRM transitions were performed in segment one and 56 SRM transitions in segment two, with dwell times of 20 ms for each transition. A graphical representation of the actual instrument method is shown in Figure 2.
Figure 2: Splitting the acquisition time into two time segments and three scan events improves instrument performance for complex screening analyses
Page 2 of 6

Figure 3a shows the LC-MS/MS chromatogram generated from the pesticide mix eluting over a chromatographic time scale of 16 minutes. The complexity of the chromatogram can be seen by expanding the area from 8 to 11 minutes (Figure 3b), where several different pesticides can typically be observed to co-elute.
Figure 3a: LC-MS/MS chromatogram of 88 pesticides at 50 pg/L
Figure 3b: Detection of minor components under the larger peaks
Page 3 of 6
Component Name Daminozoid Methamidophos Acephate Omethoat Propamocarb Aldicarb-sulfoxid Butocarboxim-sulfoxid Butoxycarboxim Aldoxycarb Pymetrozin Carbendazim Methomyl Demeton-S-methyl-sulfon Oxidemeton-methyl Monocrotophos Ethiofencarb-sulfon 3-hydroxy-carbofuran Ethiofencarb-sulfoxid Thiabenzadol Dimethoat Vamidothion Imidacloparid Metamitron Quinmerac Clethodim-imin-sulfon Pirimicarb Clethodim-imin-sulfoxid Butocarboxim Aldicarb PyridateXX Thiacloprid Propoxur Thiophanat-methyl Bendiocarb Carbofuran Cinosulfuron Triasulfuron 5-hydroxy-clethodim-sulfon Ethiofencarb Metsulfuron-methyl Nicosulfuron Carbaryl Chlorosulfuron Isoxaflutole Amidosulfuron Metalaxyl Imazalil Atrazin 3,4,5-Trimehacarb Clethodim-sulfon Desmedipham Phenmedipham Pyrimethanil Isoproturon Fenpropimorph Thiodicarb Flazasulfuron Bensulfuron-methyl Clethodim-sulfoxid Diuron Prosulfuron Azoxystrobin Methiocarb Promecarb Iprovalicarb Fenhaxamid Linuron Triflusulfuron-methyl Cyprodinil Spiroxamine Metolachlor Tebufenzoid Thiofanox Fenoxycarb Fentin-hydroxide Diflubenzuron Tebuconazol Rimsulfuron Haloxyfop-methyl Indoxacarb Triflumuron Clethodim Fluzifop-P-butyl Haloxyfop-ethoxyethyl Flurathiocarb Quizalofop-ethyl Flufenoxuron Pyridate
RT 1.04 1.27 1.37 1.87 2.13 2.45 2.45 2.62 2.55 2.48 3.63 3.56 3.71 4.17 4.30 4.97 4.87 5.17 5.28 5.40 5.59 5.52 5.58 5.67 5.73 6.12 6.41 6.50 7.02 7.20 7.21 7.53 7.44 7.44 7.58 7.62 8.13 8.09 8.10 8.18 8.21 8.45 8.63 8.58 8.72 8.64 8.84 9.08 9.14 9.42 9.43 9.13 9.22 9.38 9.39 9.65 9.57 9.60 9.75 9.93 9.85 9.95 9.95 10.09 10.25 10.26 10.19 10.45 10.39 10.64 10.83 10.93 11.08 11.02 11.30 11.38 11.83 11.88 11.81 12.15 12.26 12.27 12.44 12.52 13.73 15.28
Area 4674516 3829646 2535282 1516561 4988264 15089 92079 30232 210393 6401121 67750507 298259 3006988 1366861 277016 292068 1110901 24648309 3611384 472217 1506719 2252454 7922156 2327412 20969231 5154573 3903019 2151719 8244302 5497747 2762590 1420464 917258 19195703 794473 440622 356038 1655866 534961 55407 665994 490049 2483727 44104 2766611 7172377 22304226 5876561 606123 1513363 1349750 11739201 7708543 41217540 131141 275363 337368 673868 2269026 166599 5924744 1164890 1530917 2190910 2163320 1668513 5971 14661158 75041736 15042434 1226295 118596 409691 4603640 700297 7987902 8823982 370334 1448162 856888 8028262 3551906 1424774 9664875 40186 66084
Specified Calculated Amount Amount 50.000 48.743 50.000 49.984 50.000 47.557 50.000 51.212 50.000 49.355 50.000 90.324 50.000 59.539 50.000 63.008 50.000 49.163 50.000 50.687 50.000 49.534 50.000 57.081 50.000 49.419 50.000 N/F 50.000 52.403 50.000 48.786 50.000 51.737 50.000 48.253 50.000 49.962 50.000 50.512 50.000 51.537 50.000 52.085 50.000 49.143 50.000 50.826 50.000 48.892 50.000 50.280 50.000 49.645 50.000 48.852 50.000 49.942 50.000 49.736 50.000 50.557 50.000 49.906 50.000 50.642 50.000 49.829 50.000 49.441 50.000 47.468 50.000 48.954 50.000 49.817 50.000 51.049 50.000 52.066 50.000 43.245 50.000 53.046 50.000 49.618 50.000 50.356 50.000 51.395 50.000 50.460 50.000 51.281 50.000 49.756 50.000 49.047 50.000 47.974 50.000 48.104 50.000 49.328 50.000 49.761 50.000 50.569 50.000 50.340 50.000 46.936 50.000 51.509 50.000 44.644 50.000 47.765 50.000 50.361 50.000 42.400 50.000 50.076 50.000 49.681 50.000 51.120 50.000 50.988 50.000 51.113 50.000 48.797 50.000 54.571 50.000 50.662 50.000 50.560 50.000 49.975 50.000 55.610 50.000 -129.926 50.000 49.579 50.000 50.016 50.000 49.001 50.000 49.908 50.000 N/F 50.000 48.905 50.000 43.918 50.000 50.860 50.000 59.816 50.000 51.498 50.000 52.226 50.000 48.972 50.000 51.959 50.000 44.371 50.000 40.512
Equation Y = 56652 + 94739.2 R2 = 0.9988 Y = 27105.9 + 77160.3 R2 = 0.9992 Y = 51553.4 + 52226.8 R2 = 0.9955 Y = 20184.5 + 30007.4 R2 = 0.9985 Y = 31459.9 + 101707 R2 = 0.9997 Y = 1383.55 + 151.735 R2 = 0.4642 Y = 5384.2 + 1636.97 R2 = 0.9372 Y = 761.85 + 491.892 R2 = 0.8296 Y = 5547.18 + 4392.29 R2 = 0.9977 Y = 40420.7 + 127084 R2 = 0.9995 Y = 61584.2 + 1.369e + 006 R2 = 0.9999 Y = 9692.33 + 5055.42 R2 = 0.9934 Y = 13010.2 + 61109.5 R2 = 0.9995 Y = 38265.8 + 97760.8 R2 = 0.9989 Y = 16543.9 + 26399.4 R2 = 0.9964 Y = 1760.21 + 5714.23 R2 = 0.9983 Y = 3310.44 + 5709.23 R2 = 0.9871 Y = 24680.6 + 23533.9 R2 = 0.9964 Y = 58379.8 + 494513 R2 = 0.9999 Y = 18998 + 71871.1 R2 = 0.9997 Y = 5505.94 + 9269.53 R2 = 0.9982 Y = 9529.36 + 29111.3 R2 = 0.9969 Y = 1941.62 + 45795.4 R2 = 0.9993 Y = 21139.4 + 156284 R2 = 0.9995 Y = 1206.9 + 47627.9 R2 = 0.9992 Y = 50637 + 418055 R2 = 0.9999 Y = 4532.14 + 103738 R2 = 0.9998 Y = 2919.87 + 79954.2 R2 = 0.9992 Y = 11575.3 + 42852.7 R2 = 0.9998 Y = 6792.58 + 165899 R2 = 0.9999 Y = 37790.7 + 109491 R2 = 0.9997 Y = 7780.97 + 55512 R2 = 0.9996 Y = 731.145 + 28034.9 R2 = 0.9989 Y = 4657.68 + 18501.5 R2 = 0.9991 Y = 84755.6 + 389971 R2 = 0.9996 Y = 5092.19 + 16844.4 R2 = 0.9956 Y = 7849.1 + 9161.13 R2 = 0.9973 Y = 1197.11 + 7170.92 R2 = 0.9995 Y = 2896.19 + 32493.3 R2 = 0.9989 Y = 8787.37 + 10443.5 R2 = 0.9964 Y = 7332.14 + 1450.79 R2 = 0.9559 Y = 8256.25 + 12710.6 R2 = 0.9937 Y = 7636.28 + 10030.3 R2 = 0.9977 Y = 30166.7 + 49922.5 R2 = 0.9996 Y = 1474.67 + 829.433 R2 = 0.9605 Y = 19113 + 55207.1 R2 = 0.9998 Y = 146813 + 142726 R2 = 0.9951 Y = 79337.7 + 449862 R2 = 0.9998 Y = 31771.1 + 120463 R2 = 0.9994 Y = 6241.14 + 12764.5 R2 = 0.9963 Y = 38755.8 + 32266.2 R2 = 0.9944 Y = 31936.5 + 28010.3 R2 = 0.9982 Y = 72893 + 237379X R2 = 0.9996 Y = 36587.8 + 153161 R2 = 0.9996 Y = 1.58954e + 006+850358 R2 = 0.9877 Y = 1721.31 + 2830.73 R2 = 0.9925 Y = 8454.51 + 5510.05 R2 = 0.9964 Y = 60.8872 + 7558.17 R2 = 0.9836 Y = 9675.27 + 14310.6 R2 = 0.9951 Y = 9647.22 + 45246.5 R2 = 0.9996 Y = 2469.03 + 3987.42 R2 = 0.9581 Y = 109126 + 120494 R2 = 0.9973 Y = 8196.01 + 23612.5 R2 = 0.9991 Y = 20134.2 + 30341.5 R2 = 0.9990 Y = 17552.3 + 43313.4 R2 = 0.9994 Y = 21459.1 + 42744.1 R2 = 0.9990 Y = 26949.6 + 34745.1 R2 = 0.9980 Y = 2451.06 + 64.4984 R2 = 0.6233 Y = 222152 + 293778 R2 = 0.9982 Y = 2.9315e + 006 + 1.54218e + 006 R2 = 0.9880 Y = 148631 + 303973 R2 = 0.9992 Y = 14352.5 + 22309.7 R2 = 0.9985 Y = 110983 58.591 R2 = 0.0058 Y = 27661.2 + 8821.35 R2 = 0.9956 Y = 89525.3 + 93833.1 R2 = 0.9963 Y = 7945.49 + 14453.7 R2 = 0.9879 Y = 170977 + 163478 R2 = 0.9964 N/A Y = 344151 + 187469 R2 = 0.9885 Y = 28514.8 + 9081.59 R2 = 0.9678 Y = 72372.4 + 29896.2 R2 = 0.9902 Y = 5040.92 + 14409.7 R2 = 0.9991 Y = 365417 + 162992 R2 = 0.9866 Y = 131575 + 70529.2 R2 = 0.9887 Y = 65299.8 + 30427.4 R2 = 0.9828 Y = 380173 + 193325 R2 = 0.9886 Y = 10159.7 + 1134.66 R2 = 0.9607 Y = 18878.2 + 2097.21 R2 = 0.8556
All compounds were mixed at the same concentration and the SRM method allows even those with low responses to be detected under other analytes. A summary of the results for these pesticides at 50 pg/L is tabulated in Table 1. As is clearly evident, excellent linearity was observed with the coefcient of correlations of most components varying from 0.9900 to 0.9998.
Table 1: Results of pesticide analysis at 50 pg/L
Page 4 of 6
2,500,000
5,500,000 5,000,000
250,000
2,000,000
4,500,000 4,000,000 200,000
1,500,000 Area Area
3,500,000 3,000,000 2,500,000 2,000,000 1,500,000 100,0000 Area 150,0000
1,000,000
500,000
1,000,000
500,000
Metamitron
0 0 10 20 30 40 50
500,000 0 0 10 20
Clethodim-sulfoxide
0 30 40 50 0 10 20
Isoxaflutole
30 40 50
2,200,000 2,000,000 1,800,000 1,600,000 1,400,000 Area 1,200,000 1,000,000 800,000 600,000 400,000 200,000 0 0 10 20 30 40 50 Area
1,200,000 1,000,000 800,000 600,000 400,000 200,000
Iprovalicarb
0 0 10 20
Methiocarb
30 40 50
Figure 4. Linearity of response for the ve components: metamitron, clethodim-sulfoxide, isoxautole, iprovalicarb, methiocarb
Linearity
Peak areas were used for quantitation and the resultant linearity of responses are plotted in Figure 4 for ve different compounds. Although no internal standard was used during the assay, excellent correlation coefcient values were observed between 0.9990 and 0.9998 for the ve components metamitron, clethodimin-sulfoxide, isoxautole, iprovalicarb, and methiocarb.
This was demonstrated during the pesticide assay by monitoring the SRM transitions of three components: triasulfuron, metasulfuron-methyl and chlorosulfuron. These compounds have different precursor ions but all generate a product ion at m/z 167 (see Table 2). The chromatograms in Figure 5 show the transitions for these compounds, with no evidence of any cross-talk.
Pesticide Retention time (min) SRM transition (m/z)
Absence of Cross-talk
High-throughput characterization of very complex mixtures requires rapid analysis of coeluting analytes. An effective way to accomplish this is by reducing the dwell and interscan times. However, cross-talk can occur in triple quadrupole instruments when short scan times are employed because the fragment ions from one SRM transition are often scanned out during another transition. This is due to some fragment ions from one transition still residing in the collision cell when the next transition starts, resulting in signal artifacts. However, the patented design of the orthogonal collision cell of the TSQ Quantum Discovery virtually eliminates cross-talk.
Triasulfuron Metasulfuron-methyl Chlorosulfuron
7.62 8.10 8.45
402 > 167 382 > 167 358 > 167
Table 2: Characteristics of the three pesticides triasulfuron, metasulfuron-methyl, and chlorosulfuron used to demonstrate zero cross-talk on the TSQ Quantum Discovery
Page 5 of 6
Conclusions
An LC-MS/MS screening assay to monitor 88 pesticides using minimal LC separation was developed using the TSQ Quantum Discovery. It was possible to detect all components within a chromatographic time scale of 16 minutes by performing SRM transitions during two userdetermined time segments. Even with dwell times of only 20 ms, no cross-talk interference was observed during the analysis. Typical food monitoring applications require screening for tens to hundreds of pesticides. Although conventional detection is accomplished using UV absorbance, nitrogen phosphorus, or electron capture detection, LC-MS/MS provides superior sensitivity, and more importantly, specicity of identication as compared with these other commonly used techniques. The LC-MS/MS-based method described here, with its speed, sensitivity, and specicity, is highly applicable to both the environmental monitoring and agrochemical industries operating within EPA and FDA criteria.
100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 Relative Abundance 60 50 40 30 20 10 0 100 90 80
7.62
offices, Thermo Fisher Scientific maintains

Triasulfuron
a network of representative organizations throughout the world.
8.10
Metasulfuron-methyl
8.45
CD-ROM The data generated for this application note, along with the instrument and processing methods, are available on a CD-ROM from Thermo Fisher Scientic at www.thermo.com/quantum.
70 60 50 40 30 20 10 0
Chlorosulfuron
References
U.S. Environmental Protection Agency website at www.epa.gov: US Environmental Protection Agency Code of Federal Regulations 40. Chapter I, Subchapter E, Part 180 details the tolerances and exemptions from tolerances for pesticide chemicals in food. Pesticide Analytical Manual Volume 1, Sections 605-606 (describes MS applications and benets). Transmittal No. 94-1 (1/94), Form FDA 2905a (6/92). Available on the FDA website at www.cfsan.fda.gov. Chapter 3 describes multi-class multi-residue methods, while Chapter 4 provides selective multiresidue methods.
6.5
7.0
7.5
8.0
8.5 Time (min)
9.0
9.5
10.0
10.5
Figure 5: No cross-talk was observed when the pesticides triasulfuron, metsulfuron-methyl, and chlorosulfuron were detected
www.thermo.com
AN62488_E 11/07S
Testing LC-MS System Robustness with Automated Sample Cleanup Using Red Wine as a Matrix
Jonathan Beck, Thermo Fisher Scientic, San Jose, CA, USA
Introduction
Key Words TSQ Quantum Ultra EQuan System Hypersil GOLD Columns Pesticides Achieving low limits of detection (LODs) of pesticides, antibiotics and veterinary residues in food residues and drinking water is of paramount importance in order to monitor the regulatory levels as stated by the US, Japanese and EU directives. These substances pose a signicant health threat and therefore, need to be accurately detected at the lowest levels, typically at part per trillion (ppt). Traditionally, LC-MS/MS has been used by the environmental and food industries for the identication and quantitation of these residues. However, this methodology typically requires extensive ofine sample preparation, which can be time consuming and expensive. The Thermo Scientic EQuan environmental quantitation system consists of a Thermo Scientic TSQ Quantum series mass spectrometer, two Thermo Scientic Surveyor HPLC pumps with a preconcentration column, an analytical column, and a CTC autosampler. The unique capabilities of EQuan for online preconcentration and cleanup of samples result in improved sensitivity and precision, as well as unmatched throughput. In previous experiments, using the EQuan system for online sample preconcentration and detection of pesticides in ground water yielded lower limits of detection compared to standard injection techniques. See Table 1. Typically, when red wine is analyzed using LC-MS/MS, some form of sample preparation and/or extraction is necessary prior to injection. In this application note, the EQuan system was tested for robustness using a matrix of neat red wine spiked with a mixture of pesticides using large volume (1000 L) injections.
1 mL Injection Area Propham 100 L Injection Area
Goal
To test the robustness of an LC-MS system for an automated online preconcentration system using a dirty matrix.
Sample Preparation Red Burgundy wine was spiked with a mixture of nine herbicides and six fungicides at a level of 500 pg/mL (500 ppt). The following herbicides were analyzed: atrazine, cyanazine, simazine, propazine, trietazine, metazachlor, propachlor, pendimethalin, and propyzamide. The following fungicides were analyzed: utriafol, triadimefon, epoxiconazole, usilazole, tebuconazole, and propiconazole. No other sample treatment was performed prior to injection. HPLC HPLC analysis was performed using an HTC PAL Autosampler with two LC quaternary pumps and two LC columns, the rst for preconcentration of the sample and the second for the analytical analysis. A sample of 1000 L of the spiked neat wine was injected directly onto the Thermo Scientic Hypersil GOLD 20 2.1 mm, 12 m loading column in a high aqueous mobile phase (see Figure 1a). After 1 minute, a six-port valve on the mass spectrometer was switched by Thermo Scientic LCQUAN 2.5 instrument control software. This enabled the load column to be back ushed onto the analytical column (Hypersil GOLD 50 2.1mm, 3 m), where the com-
Gain Factor
1 mL 100 L 1 mL Injection Injection Gain Injection Area Area Factor Area Isoproturon Diuron
100 L Injection Area
1 mL Gain Injection Factor Area Linuron
100 L Injection Gain Area Factor
1 ppt 5 ppt 10 ppt 50 ppt 100 ppt 500 ppt 1000 ppt 5000 ppt
2.17E+04 2.71E+04 5.09E+04 6.51E+04 2.47E+05 5.29E+05 2.59E+06
3.00E+04 5.69E+04 2.82E+05
8 9 9
5.53E+04 3.35E+05 6.68E+05 3.33E+06 6.54E+06 3.11E+07 5.81E+07 2.58E+08
NA 3.17E+04 4.90E+04 2.82E+05 5.24E+05 2.60E+06 5.23E+06 2.44E+07
11 14 12 12 12 11 11
1.97E+04 4.15RE+04 8.25E+04 4.47E+05 8.83E+05 4.65E+06 9.39E+06 4.95E+07
1.73E+03 5.65E+03 1.18E+04 3.72E+04 7.60E+04 3.80E+05 7.63E+05 3.68E+06
11 7 7 12 12 12 12 13
6.96E+03 1.99E+04 5.91E+04 1.34E+05 7.36E+05 1.43E+06 9.49E+06
7.98E+03 2.50E+04 1.28E+05 2.47E+05 1.25E+06
7 5 6 6 8
Table 1: Calculations demonstrating the gain in peak areas due to larger injection volumes in ground water samples
pounds were separated prior to introduction into the mass spectrometer (see Figure 1b). After all of the compounds were eluted from the analytical column, the 6-port valve was switched back to the starting position, and the loading and analytical columns were cleaned with a high
organic phase before being re-equilibrated to their starting conditions. The total run time for each analysis was 22 minutes. The mobile phases for the analysis were water and methanol, both with 0.1% formic acid.
Preconcentrate
Waste LC Pump
400 L/min
b
Waste
Preconcentration Column
Analyze
LC Pump
Preconcentration Column
Load Pump and CTC

1 mL/min
Analytical Column
MS
Load Pump/CTC
Analytical Column
MS
PEEK Tubing Internal Flow Routing
Surveyor LC Pump Plus
Autosampler
Surveyor MS Pump Plus
6-Port Valve
3 m Hypersil GOLD Analytical Column TSQ Quantum Series MS
12 m Hypersil GOLD Preconcentration Column
Figure 1: The schematic of the EQuan system used for this assay
Figure 2: Ion sweep cap after several hundred injections, showing contamination from red wine
Figure 3: Electrospray ionization source with the electrospray probe removed, showing the main spray pattern directed towards the drain
MS MS analysis was carried out on a Thermo Scientic TSQ Quantum Ultra triple quadrupole mass spectrometer with an electrospray ionization source. The MS conditions were as follows: Electrospray ionization: Positive Spray voltage: 3.0 kV Ion transfer tube temperature: 350 C Sheath gas pressure: 45 arbitrary units Auxiliary gas pressure: 5 arbitrary units Ion sweep gas pressure: 3 arbitrary units Collision gas (Ar): 1.0 mTorr Q1/Q3 peak resolution: 0.7 Da Scan width: 0.002 Da The source of the mass spectrometer was adjusted so that the ESI probe was off axis to prevent contamination of the ion transfer tube. The position of the probe was set so that the main spray pattern of the electrospray hit the Thermo Scientic Ion Sweep cone below the center line and off to the left by about 0.5 cm. The probe depth was set to position C on the electrospray probe. An ion sweep gas of three arbitrary units was set to prevent any large droplets from entering the ion transfer tube of the mass spectrometer.

The back pressure of the loading column and the analytical column were monitored over the course of the wine injections to determine if the columns were becoming clogged with any particulates from the wine. Over 600 injections, the back pressure on the 12 m loading column remained at approximately 20 bar under the starting conditions of the analytical run, while the back pressure on the 3 m
analytical column remained at approximately 72 bar. The resulting spray pattern of the electrospray can be seen in Figure 2. A thick deposit of red wine residue is clearly visible from just below the center of the sweep cone to the outside radius. The red wine spray can also be seen on the inside of the electrospray housing in Figure 3. In the picture, the drain is dark purple in color, illustrating that the main excess spray of the red wine was directed to the bottom of the ion source and away from the main orice of the mass spectrometer. Additionally, the ESI probe can be adjusted to be closer to the ion transfer tube, which increases robustness by allowing less side scatter from the electrospray beam, thus focusing the main spray pattern lower on the ion sweep cap. The reproducibility of the method is shown in Figure 4. The graph plots the peak area for metazachlor for 164 injections of red wine. The rst four injections were excluded from the %RSD calculation. Because the loading column was new at the beginning of the runs, several injections were required to condition the column before a stable peak area was achieved. A representative chromatogram is shown in Figure 5. As shown in Figure 6, after several hundred injections of the spiked red wine matrix, no degradation in column performance or source robustness was observed. In total, over 600 injections were made on the system with no loss in column performance.
Conclusion
This application note demonstrates the robustness of the TSQ Quantum Ultra triple quadrupole mass spectrometer and an online extraction and preconcentration method. The described sample cleanup technique improves signalto-noise ratios by a factor of 10 to 100 (based on injection volume) for low concentration samples in red wine matrices. Preliminary results using onion and tobacco matrices have yielded similar results in terms of column performance and mass spectrometer robustness. Further studies will be conducted in other matrices, as well as with other pesticides, herbicides, and insecticides.
References
Metazachlor Peak Area

12000000 10000000 8000000
Area
6000000 4000000 2000000 0 0 50 100
150
200
Wang, Y.; Catana, F.; Yang, Y.; Roderick, R.; van Breemen, R.B. An LC-MS Method for Analyzing Total Resveratrol in Grape Juice, Cranberry Juice, and in Wine; J Agric. Food Chem. 2002, 50(3), 431-435.
Injection Number
Figure 4: Scatter plot of the peak area for 164 injections (1000 L) of metazachlor spiked in red wine. The %RSD is 9% when the rst four points are excluded.
Simazine 202 > 124 Propyzamide 256 > 190 Tebuconazole 308 > 125
Propachlor 212 > 170
Metazachlor 282 > 212
Flusilazole 316 > 247
Pendimethalin 282 > 212 Propazine/ Trietazine 230 > 188 Epoxyconazole 330 > 121
Cyanazine 241 > 214
Triadimefon 294 > 197 Propiconazole 342 > 159
Simazine 202 > 124
Flutriafol 302 > 123
Figure 5: Example chromatograms for a 1000 L injection of spiked red wine

RT: 8.13-13.32 100
90 80 70 60 50 40 30 20 10 0 9
9TH Injection
321ST Injection
10
11
12
13
Time (min)
Figure 6: Different injections of metazachlor (retention times have been offset for greater visibility)
www.thermo.com
Legal Notices 2007 Thermo Fisher Scientic Inc. All rights reserved. HTC PAL is a trademarks of CTC Analytics AG, Zwingen, Switzerland. All other trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientic Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
AN62495_E 11/07S
View addional Thermo Scientic LC/MS application notes at: www.thermo.com/appnotes
UHPLC Separation of Triazine Herbicides at Elevated Temperature

Dave Thomas, Thermo Fisher Scientific, San Jose, CA USA
Goal:
Key Words Accela UHPLC Environmental Analysis Herbicides High Temperature Hypercarb LC Column Triazines Increase throughput of the HPLC method for triazine herbicides by employing ultra high-speed liquid chromatography at elevated temperature on a heat stable Hypercarb column.
Chromatographic conditions
Column: Mobile phase: Gradient: Thermo Scientific Hypercarb 3.0 m, 1 x 100 mm (35003-101046) A: water B: acetonitrile Time %A %B 0.00 75 25 1.00 70 30 2.20 10 90 2.30 75 25 4.00 75 25 500 L/min PDA, 238 nm, 10-mm flow cell, 11nm bw, 20 Hz, 0s rise time 160 C (housed in Selerity temperature controller) 5 L sample loop, 2 L partial loop injection Syringe Speed: 4 L/sec Flush Speed: 100 L/sec Flush Volume: 400 L Wash Volume: 200 L Flush/Wash source: Bottle with 90:10 methanol:water
Introduction Temperature is a key variable in high performance liquid chromatography (HPLC), influencing solute diffusion rates, mobile phase viscosity, and solubility. For example, as column temperature increases, analyte diffusion increases. Increased analyte diffusion generally leads to an increase in the optimum linear velocity of the separation, so that equivalent chromatographic efficiency and resolution can be achieved at a higher flow rate. Furthermore, elevating the temperature reduces the operating backpressure. The net result is that separations can be performed faster without exceeding the pressure limitations of the instrument. This application uses a porous graphitic carbon stationary phase thermostatted in a high temperature column oven to separate triazine herbicides 5 to 10 times faster than is typical with conventional HPLC. The triazines and degradation products are separated on the Thermo Scientific Accela High Speed Liquid Chromatograph in 2 minutes on a Thermo Scientific Hypercarb 3 m, 1 x 100 mm column operated at 160 C. This application note also documents the performance of the high temperature liquid chromatographic method, including precision of retention time and peak area, resolution, and spike recovery from several environmental water matrices.
Flow rate: Detector: Column temp.: Injection:
Chemicals
Water, LC/MS-grade Acetonitrile, LC/MS-grade Methanol, LC/MS-grade Atrazine Ametryn Cyanazine Deisopropylatrazine, 1000 mg/L Desethylatrazine, 1000 mg/L Propanil, 1000 mg/L Propazine Prometryn Simazine Simetryn Fisher Scientific W6 Fisher Scientific A998 Fisher Scientific A456 Supelco 49085 ULTRA PST-024 ULTRA PST-1360 SPEX CertiPrep S-1135 SPEX CertiPrep S-1145 SPEX CertiPrep S-3155 ULTRA PST-850 ULTRA PST-840 ULTRA PST-1130 Chem Service PS-381
Experimental
Instrumentation
Thermo Scientific Accela HPLC system with PDA Detector Thermo Scientific ChromQuest 5.0 Chromatography Data System (CDS) Polaratherm Series 9000 Total Temperature Controller (Selerity Technologies)
Consumables
Autosampler vials, 1.8 mL glass, yellow septa Backpressure assembly Ferrules, high temperature Mixer, 50 L in-line static Mobile Phase Preheater, 0.005 x 70 cm Syringe filters, 0.45 m Nylon Sample Loop, 5 L Thermo Scientific A4954-010 Upchurch P-788 Selerity Technologies BM0054 Thermo Scientific 109-99-032 Selerity Technologies AD104 Thermo Scientific A5307-010 Thermo Scientific 109-99-007
Mobile Phase
Proportioned mobile phase: Filled Solvent Reservoir Bottle A of the Accela pump with fresh HPLC-grade water and purged the solvent line with at least 30 mL of the water. Connected a fresh bottle of HPLC-grade acetonitrile to Reservoir B and purged as above.
Results
Separation of seven triazine herbicides, two triazine degradation products often found in environmental samples, and propanil is shown in Fig 1. To optimize this separation, we adjusted the mobile phase composition to elute the first analyte with a capacity factor k > 2, thereby improving resolution of the target analytes from sample matrix junk. Analytes spanning a wide range of polarity are well resolved by the combination of high temperature, solvent gradient, and the selectivity of the Hypercarb stationary phase. Note that because of the reduced viscosity of the mobile phase at 160 C, this separation occurs at a linear flow rate of 15 mm/s equivalent to a flow rate of over 10 mL/min on a 4.6 mm i.d. column. The system backpressure under these conditions is less than 4000 psi (272 bar). Method performance is characterized by peak resolution, linear calibration range, limits of detection, and precision of retention time and peak area, as summarized in Table 2. MDLs for each analyte were determined by performing seven replicate injections of LC/MS-grade water fortified at a concentration of three to five times the estimated instrument detection limits, calculating the standard deviation of the measured concentration, and using the equation given in the figure caption. Note the good precision of retention time and peak area, as this reflects the temperature stability maintained by the high temperature oven. We analyzed several environmental water samples to demonstrate the efficacy of this method with real matrices. Samples of surface, ground and municipal drinking water were analyzed before and after fortification with a known amount of each target analyte. Spike recovery was calculated as the amount of each analyte found in the spiked sample divided by the amount expected (i.e., the amount determined in the blank plus the amount added in the spike). Even with the dirtiest matrix, Salinas River water, the target analytes are well separated from the early eluting matrix peaks (Figure 2) and recovery of the spiked analytes exceeds 80% (Table 3).
Calibration Standards
Individual Stock Solutions, 1000 mg/L: Accurately weighed 10 mg (0.010 g) of each neat compound into a 10-mL volumetric flask, added 5 mL acetonitrile, and sonicated to dissolve. Brought to volume with acetonitrile and mix. Used desethyl atrazine, deisopropyl atrazine and propanil, purchased as solutions of 1000 mg/L in methanol, as received. Combined Intermediate Standard 100 mg/L: Used a calibrated pipette to deliver 1000 L of each individual stock solution to a 10-mL volumetric flask. Brought to volume with acetonitrile and mix. Calibration standards: Used a calibrated pipette to dilute the intermediate standard with mobile phase in volumetric glassware to 30, 10, 3, 1, 0.3, 0.1, and 0.03 mg/L.
Samples
Samples of surface water (Salinas River, Monterey County, CA), ground water (domestic well, Santa Cruz county, CA), and drinking water (San Jose, CA tap water) were collected in accordance with established procedures, stored at 4 - 8 C, and were filtered through a 0.45 m nylon syringe filter into a glass autosampler vial before analysis.
System Preparation
To ensure good performance of this application, prepare the system as directed in Appendix A.
100
3 5 6
mAU
9 10 7 8 11 12
Column: Temperature: Flow rate: Detector: Injection: Solvents: Gradient:
1
50
Samples: Peaks:
0 0 1 Minutes 2 3
Figure 1: Separation of triazine herbicides, degradation products, and propanil on the Accela High Speed LC by reversed-phase chromatography with UV absorbance detection at 215 nm. Peaks: see Figure. Sample: overlay of 30 injections of triazines in HPLC-grade water with 20% acetonitrile. Conditions: see text for details.
Hypercarb 3 m, 100 x 1 mm 160 C 500 L/min Accela PDA at 215nm, 20Hz, 0s rise time 2 L partial loop from 5 L loop A: Water B: Acetonitrile Time (min) A% B% 0.00 75 25 1.00 70 30 2.20 15 85 2.10 75 25 4.00 75 25 triazines and propanil in 20% acetonitrile 1. Melamine 2. Unknown 3. Deisopropylatrazine 4. Desethylatrazine 5. Cyanazine 6. Propazine 7. Prometryn 8. Atrazine 9. Ametryn 10. Simazine 11. Simetryn 12. Propanil
100
mAU
1 50 2 3 10 4 5 6 7 8 9 11
12
A B C D
Minutes
Figure 2: Chromatograms of four environmental water samples spiked with 200 g/L each of triazines and propanil. Chromatograms obtained on the Accela High Speed LC by reversed phase chromatography with UV absorbance detection at 215 nm. Peaks: see Figure 1. Samples: top trace A, surface water (Salinas River); trace B, ground water (Simoes well); trace C, drinking water (San Jose tap); bottom trace D, HPLC-grade water. Conditions: see text for details.
Table 1. Useful properties of some triazine herbicides and degradation productsa 4-deethyl atrazine 6190-65-4
H 2N N N H N CH3
CAS#
6-deisopropyl atrazine 1007-28-9

Cl N H N CH2
Cyanazine 21725-46-2
H3C H N N Cl N N
Propazine 139-40-2
Cl
Prometryn 7287-19-6
S H3C N N N H N CH
3
CH3
N
HN
CH3
N
CH3
HN CH3
CH3
H3C
Cl
NH2
H3C
N H
N H
CH3
CH3
CH3
Formula MW (g/mol) pKa Log Po-w Water solubility, mg/L MeOH solubility, g/L
C6H10ClN5 187.633 1.51 3200
C5H8ClN5 173.606 1.15 670
C9H13ClN6 240.697 0.87 2.22 170 45 (ethanol)
C9H16ClN5 229.713 1.7 2.93 8.6 6.2 (toluene)
C10H19N5S 241.361 4.05 3.51 33 160
CAS#
Atrazine 1912-24-9
Cl
Ametryn 834-12-8
CH3 S N H N CH3
Simazine 122-34-9
Cl
Simetryn 1014-70-6
S H3C
N
Propanil 709-98-8
N H N CH3
Cl O
CH3
N CH3
H3C
N
N H CH3
N
CH3 Cl N H
NH
H3C
N H
N H
H3C
CH3
N H
HN
CH3
Formula MW (g/mol) pKa Log Po-w Water solubility, mg/L MeOH solubility, g/L
a
C8H14ClN5 215.687 1.7 2.61 34.7 18
C9H17N5S 227.334 4.1 2.98 209 510
C7H12ClN5 201.66 1.62 2.18 6.2 400
C8H15N5S 213.307 4 2.8 450
C9H9Cl2NO 218.082 2.29 3.07 152 540
http://toxnet.nlm.nih.gov
Table 2. Performance of high temperature method for triazines performed on Hypercarb 3 m, 1 x 100 mm column at 160 C. Precision, Retention Time % RSDc 0.25 0.22 0.16 0.13 0.10 0.09 0.08 0.04 0.04 0.02
Analyte deisopropylatrazine desethylatrazine cyanazine propazine atrazine simazine prometryn ametryn simetryn propanil
a b
ka 2.4 2.8 5.6 6.1 7.7 9.2 10.4 11.9 13.1 15.1
Ra 1.1 1.2 7.2 1.5 3.9 3.9 3.0 4.3 3.7 6.3
Linear range, mg/L 0.03 10 0.03 30 0.03 30 0.03 30 0.03 30 0.03 30 0.03 30 0.03 100 0.03 100 0.03 30
r2 0.9999 0.9999 0.9995 0.9996 0.9995 0.9994 0.9999 0.9995 0.9999 1.0000
MDLb g/L 6 16 40 23 14 30 32 8 16 25
Precision, Peak Area % RSDc 0.39 0.89 0.47 0.82 0.87 0.92 0.97 0.64 0.57 0.42
Capacity factor (k) and Resolution (R) calculated according to Reference 1. Detection limit MDL = ts,99 where t,99 = 3.14 for n = 7 replicates of the standard. c for n = 30 replicates.
Table 3. Percent recovery of analytes spiked into selected environmental water matrices. n = 3 replicates. Analyte deisopropylatrazine desethylatrazine cyanazine propazine atrazine simazine prometryn ametryn simetryn propanil HPLC water 94.0 93.0 92.5 97.0 94.5 98.0 104 97.0 99.0 101 Drinking water 104 110 102 106 104 102 117 104 98.5 103 Ground water 104 98.5 94.0 104 101 100 116 101 104 104 Surface water 93.0 85.0 97.0 101 98.5 103 110 98.5 100 80.0
Conclusion
A separation performed at 160 C on the Accela high speed chromatography system equipped with a heat stable Hypercarb column and high temperature column oven resolves 11 triazine herbicides in about two minutes with retention time and peak area precision better than 1% RSD for thirty replicates.
References 1. United States Pharmacopeia 30-National Formulary 25, United States Pharmacopeia, Rockville, Maryland 20852-1790, USA.
Suppliers
Chem Service, West Chester, PA, USA (http://www.chemservice.com) Selerity Technologies, Inc., Salt Lake City, UT, USA Sigma-Aldrich, St. Lois, MO, USA (http://www.sigmaaldrich.com) Supelco, Bellefonte, PA, USA (http://www.sigmaaldrich.com) Thermo Fisher Scientific, Waltham, MA, USA (http://www.thermofisher.com) ULTRA Scientific, No. Kingstown, RI, USA (http://www.ultrasci.com)
Appendix A.
System Preparation
Pump: Always plumb the Accela system with precut and polished 0.005 i.d. high-pressure tubing and high pressure fittings as shown in Figure 15 of the Accela Pump Hardware Manual (Document 60157-97000 Revision B). For all tubing connections that you make, ensure that the tubing end is square-cut and burr-free. Firmly push the tubing into the injection valve port as you tighten the high-pressure fitting in order to maximize peak efficiency. Prime the pulse dampener and purge the solvent lines as instructed in Chapter 4 of the Accela Pump manual. Verify that the pump is performing well by monitoring the pressure pulsation and by testing the pump proportioning accuracy as described in Chapter 5 of the pump manual. If your Accela pump does not include an inline 35 L dynamic mixer, then install a 50 L static mixer between the inline high pressure filter and the Accela AS mobile phase preheater. AS: Open the Instrument Configuration and verify that the Accela AS Configuration entry for Dead volume is correct (the calibrated volume in L written on the transfer tubing between the injection port and injection valve). Verify that the entry for Loop size is correct for the currently installed sample loop. Fill the Flush reservoir with 90:10 (v/v) methanol:water and flush the syringe with solvent to purge any air bubbles from the syringe and
tubing. Use the Wash/Flush conditions specified under Conditions to ensure low carryover between injections. Consult the Accela Getting Connected manual (Document 60057-97001 Revision A) for details.
Polaratherm Column Oven: Install the Total Temperature Controller according to the Polaratherm Series 9000 Installation and Operation Manual. Install the Hypercarb, 3 m 1 x 100 mm column, by using a 70-cm length of precut and polished 0.005 i.d. high-pressure tubing with mobile phase preheater. It is important to use a heat stable Hypercarb column as this does not contain any PEEK components that will degrade at the temperatures used in this method. Use the high temperature graphite/Vespel ferrules and fittings described in the Series 9000 manual. Ensure that the tubing is fully pushed into the column inlet when you tighten the high-pressure fitting. Detector: Use a 10 mm light-pipe flow cell. Install a 250 psi backpressure regulator after the flow cell outlet to suppress bubble formation in the flow cell. Verify that the deuterium lamp has been used for less than 2000 hours. Use Direct Control or a downloaded method to equilibrate the Accela system under the conditions shown above. Create a method based on these operating conditions and then create a sequence to make several injections of HPLC grade water. The system is ready to run standards and samples when the peak-to-peak baseline oscillation is between 50 200 AU/min (average of 10 1-min segments) and no significant peaks elute in the retention time window of the analytes.
www.thermo.com
Legal Notices 2009 Thermo Fisher Scientic Inc. All rights reserved. PolaraTherm is a trademarket of Selerity Technologies. Vespel is a registered trademark of E. I. du Pont de Nemours and Company. All other trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientic Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

AN62960_E 1/09S
Utility of H-SRM to Reduce Matrix Interference in Food Residue Analysis of Pesticides by LC-MS/MS Using the TSQ Quantum Discovery
Yoko Yamagishi, Thermo Fisher Scientic, C-2F 3-9 Moriya-cho Kanagawa-ku, Yokohama 221-0022, Japan
Introduction
Key Words TSQ Quantum Discovery Surveyor HPLC H-SRM Pesticides Zero Cross-talk With the recent trend of increased concern about food safety, the number of regulated pesticide residues in food has increased rapidly. In Japan, a new positive list system for monitoring pesticide residues will take effect in 2006. Consequently, an accurate high throughput multi-pesticide screening method which can quantitate high number of pesticide residues during a single analysis is required. LC-MS/MS is fast becoming the technique of choice for the identication and quantitation of pesticide residues. This is due, in part, to the ease of sample preparation and chromatographic conditions that LC-MS/MS allows, when compared to other techniques such as GC or HPLC with UV absorbance, nitrogen phosphorus detection, or electron capture detection. However, it can be extremely challenging to quantitate multi-pesticide residues in food because of interference from complex sample matrices. Although matrix-related interferences can be decreased by various sample clean up procedures, the analytical instrument used for the quantitation also has to be highly selective and sensitive. The unique Highly-Selective Reaction Monitoring (H-SRM) detection method available with the Thermo Scientic TSQ Quantum has proven to be very useful for this purpose. The analytical results of 35 pesticide residues in food with the H-SRM detection method are reported in this application note.
Sample Analysis
HPLC analysis was performed on the Thermo Scientic Surveyor HPLC System, using a Thermo Scientic HyPURITY C18 150 2.1 mm 5 m column. Mobile phase A was water, mobile phase B was methanol, and mobile phase C was water containing 10 mM ammonium acetate. Solvent was pumped at 200 L/min and analytes eluted using a linear gradient of 20% B to 99% B over 15 minutes, holding at 99% B for 3 minutes, and then returning to 20% B for 5 minutes. Mobile phase C was held at 1% throughout the run.
Mass Spectrometry Instrument: TSQ Quantum Discovery

Positive ESI
Spray Voltage: 5kV Sheath/Auxiliary gas: Nitrogen Sheath gas pressure: 40 (arbitrary units) Auxiliary gas pressure: 40 (arbitrary units) Ion transfer capillary temperature: 380 C Scan type: SRM or H-SRM CID conditions: Ar at 1.0 mTorr
Negative ESI
Goals
Illustrate the effectiveness of H-SRM for reducing background interference and improving s/n Develop a multi-residue LC-MS/MS screening method to detect 35 pesticides, and Exhibit the absence of cross-talk between co-eluting components.
Spray Voltage: 4.25 kV Sheath/Auxiliary gas: Nitrogen Sheath gas pressure: 50 (arbitrary units) Auxiliary gas pressure: 5 (arbitrary units) Ion transfer capillary temperature: 350 C Scan type: SRM or H-SRM CID conditions: Ar at 1.0 mTorr
MS Instrument Method
Thirty-ve pesticide residue compounds were analyzed to nd the product ion to be used for quantitation. Three of the compounds were ionized using negative electrospray, while the remaining 32 were ionized using positive electrospray in two different runs. A table of the compounds listing SRM transitions and the optimum collision energy are shown in Table 1.
Compound Name
Precursor Ion (m/z)
Product Ion (m/z)
Collision Retention Energy (V) Time (min)

Figure 1a shows the chromatogram of the 32 pesticides in positive ESI, and Figure 1b shows the three pesticides under negative ESI, all eluting over a chromatographic time scale of 18 minutes. While some compounds co-elute, the specicity of the H-SRM method allows for the individual quantitation and detection of each component, even at very low levels. A summary of the calibration range, linearity, and the reproducibility of each individual compound at 5 ppb (ng/mL) is tabulated in Table 2.
Oxamyl 237.17 Imidacloprid 256.12 Acetamiprid 223.12 Aldicarb 208.17 Propoxur 210.16 Carbofuran 222.16 Bendiocarb 224.14 Carbaryl 202.15 Ethiofencarb 226.13 Pirimicarb 239.22 Methabenzthiazuron 222.12 MIPC 194.17 Diuron 233.06 Azoxystrobin 404.17 BPMC 208.19 Siduron 233.20 Linuron 249.09 Methiocarb 226.14 Daimuron 269.21 Cumyluron 303.14 Tebufenozide 353.24 Iprodione 330.07 Diubenzuron 311.04 Etobenzanid 340.08 Cyprodinil 226.18 Phoxim 299.08 Bitertanol 338.21 Hexythiazox 353.13 Piperonyl butoxide 356.26 Flufenoxuron 489.09 Fenpyroximate 422.26 Chloruazuron 540.03 Teubenzuron 379.00 Hexaumuron 459.02 Lufenuron 509.00 (Positive in Black, Negative in Red)
72.0 209.1 126.0 116.0 111.0 165.1 167.0 145.0 107.0 182.1 165.0 95.0 72.1 372.1 152.0 137.0 182.0 169.1 151.1 185.0 133.0 245.1 158.0 121.0 93.0 129.0 269.2 228.0 177.1 158.0 366.1 382.9 339.0 439.0 326.0
15 16 23 8 14 14 10 10 14 16 17 20 19 15 10 17 18 10 14 14 19 15 14 36 38 12 10 16 13 20 15 20 12 12 18
3.9 6.3 7.3 9.0 10.3 10.4 10.4 11.0 11.3 11.5 11.9 11.9 12.4 12.8 13.1 13.2 13.2 13.4 13.7 13.9 14.7 14.7 14.8 15.2 15.2 15.4 15.6 16.8 17.2 17.4 17.6 17.8 17.08 16.04 16.77
Effect of H-SRM on Detection Limits

H-SRM is an acronym for Highly-Selective Reaction Monitoring (H-SRM), which is a more advanced form of Selective Reaction Monitoring (SRM). Although traditional SRM is a selective technique by itself, it still can not completely eliminate the interference from some food matrix components. Sometimes, it is possible to get incorrect qualitative results or the quantitative analysis can not reach the required detection limits of targeted compounds due to matrix-related interferences. The traditional SRM experiment, using a triple quadrupole instrument, is usually conducted with unit resolution (0.7 FWHM) for the precursor ion. With the more advanced H-SRM, the precursor ion is selected with a peak width of 0.1-0.2 FWHM. The more stringent tolerance accounts for the higher selectivity, which can lower LOQs and increase precision and accuracy at the limits of detection. This can also, in effect help reduce the overall bench time required for sample preparation.
Table 1: Summary of SRM transitions used for the analysis

3. 9 6. 3 7. 3 9. 0 10. 3 10. 4 10. 4 11. 0 11. 9
11. 3 11. 5 10 9.5 9 8.5 8 7.5 7 6.5 6
11. 9 6 12. 4 12. 8 13. 1 13. 2 13. 2 13. 4 13. 7 13. 9 14. 7 14. 7 14. 8 15. 2 15. 2 15. 4 15. 6 NL : NL : NL : NL : NL : NL : NL : 16. 8 NL : 17. 2 NL : NL : 17. 4 NL : 17. 6 17. 8 NL : NL : NL : NL : NL : NL : NL : NL : NL : NL : NL : NL : NL : NL : NL : NL : NL : NL : NL : NL : 17 18 5. 29E5 8. 68E5 2. 12E6 1. 25E5 2. 30E6 4. 57E6 3. 23E6 2. 90E6 2. 47E6 2. 75E6 2. 10E6 8. 19E5 6. 49E6 1. 75E6 1. 99E6 9. 56E5 3. 75E6 7. 47E6 5. 10E6 1. 59E6 9. 47E4 1. 18E6 4. 49E5 7. 01E5 1. 47E6 5. 71E5 7. 23E6 2. 02E6 1. 02E6 5. 31E6 5. 20E5 m s2 237- >72 m s2 256- >209 m s2 223- >126 m s2 208- >116 m s2 210- >111 m s2 222- >165 m s2 m s2 202- >145 m s2 226- >107 m s2 239- >182 m s2 194- >95 m s2 233- >72 m s2 404- >372 208->152 233->137 m s2 249- >182 m s2 226- >169 m s2 269- >151 m s2 303- >185 m s2 353- >133 m s2 330- >245 m s2 311- >158 m s2 340- >121 m s2 226- >93 m s2 299- >129 m s2 338- >269 m s2 356- >177 m s2 353- >228 m s2 489- >158 m s2 422- >366 m s2 540- >382
Relative Abundance
5.5 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 3 4 5 6 7 8 9 10 Time 11 12 13 14 15 16
Figure 1a: LC-MS/MS chromatogram of 32 pesticides at 10 ng/mL, positive ESI Page 2 of 6
Compound
R2
Range (ppb)
CV(%), n=5
Oxamyl Imidacloprid Acetamiprid Aldicarb Propoxur Carbofuran Bendiocarb Carbaryl Ethiofencarb Pirimicarb Methabenzthiazuron MIPC Diuron Azoxystrobin BPMC Siduron Linuron Methiocarb Daimuron Cumyluron Tebufenozide Iprodione Diubenzuron Etobenzanid Cyprodinil Phoxim Bitertanol Piperonyl butoxide Hexythiazox Flufenoxuron Fenpyroximate Chloruazuron Teubenzuron Hexaumuron Lufenuron
1.000 0.9994 0.9987 0.9993 0.9997 0.9996 0.9992 0.9999 0.9996 0.9995 0.9989 0.9987 0.9987 0.9989 0.9999 0.9989 0.9989 0.9997 0.9992 0.9993 0.9995 0.9979 0.9997 0.9997 0.9998 0.9997 0.9996 0.9996 0.9999 0.9997 0.9999 0.9987 0.9986 0.9973 0.9998
0.01-100 0.05-100 0.05-100 0.05-100 0.01-100 0.05-100 0.01-100 0.01-100 0.01-100 0.01-100 0.01-100 0.01-100 0.05-100 0.01-100 0.05-100 0.05-100 0.05-100 0.01-100 0.01-100 0.01-100 0.05-100 0.5-100 0.01-100 0.05-100 0.1-100 0.05-100 0.05-100 0.01-100 0.01-100 0.01-100 0.01-100 0.01-100 0.01-100 0.01-50 0.01-10
1.79 2.84 1.17 6.89 1.70 0.95 2.30 1.44 2.64 3.55 1.73 1.26 2.23 2.60 1.57 1.59 4.04 1.88 3.03 3.17 1.83 6.17 2.98 1.82 4.49 3.14 3.54 1.65 2.43 3.63 2.22 2.77 2.35 1.58 2.56
The effects of H-SRM over SRM are clearly illustrated for the three pesticides Iprodione, Biteranol and Etobenzanid in Figures 2 a, b, and c.
In order to quantitate mixtures of many compounds accurately, it is necessary to use short scan speed to ensure sufcient data points for integration. It is important that the system can maintain its sensitivity without cross-talk even at short scan speeds. Cross-talk occurs when ions from one scan event are still present in the collision cell when a second SRM transition is taking place. This leads to signal artifacts in the next transitions chromatogram. This can be especially problematic when different SRM events have the same product ions formed from different precursor ions. Thermo Fisher Scientics patented design of the orthogonal collision cell used on the TSQ Quantum product line eliminates cross-talk. Figure 3a shows the absence of cross-talk between two different SRM transitions, pirimicarb and linuron. Both yield a product ion of 182, and no artifacts are seen in either chromatogram, even when magnied 100-1000 times. The same effect is shown in Figure 3b for diubenzuron and ufenoxuron for a common product ion of 158 for dwell times of 20 msec.
Table 2: Calibration range and linearity of each compound, as well as the reproducibility of each compound at 5 ppb
16.44
16.77
100 95 90 85 80 75 70 65
17.08
Relative Abundance
60 55 50 45 40 35 30 25 20 15 10 5 0 3 4 5 6 7 8 9 10 11 T im e ( m in ) 12 13 14 15 16 17 NL : 2. 01E 5 m s2 379- >339 18 NL : 1. 01E 5 m s2 509- >326 NL : 2. 99E 5 m s2 459- >439
Figure 1b: LC-MS/MS chromatogram of 3 pesticides at 10 ng/mL, negative ESI Page 3 of 6
SRM Mode
100 95 90 85 80 75 70 65 60 Relative Abundance 55 50 45 40 35 30 25 20 15 10 5 0
H-SRM Mode
17. 0 RT : 14. 7
100 95
?
RT : 14. 7
90 85 80 75 70 65 Relative Abundance 60 55 50 45 40 35 30 25 20 15 10 5 0
10
11
12
13
14
15 16 Time (min)
17
18
19
20
10
11
12
13
14
15 16 Time (min)
17
18
19
20
Figure 2a: Comparison of SRM mode and H-SRM mode for the analysis of the fungicide Iprodione
SRM Mode
100 R T : 1 5 .6 4
H-SRM Mode
100 R T : 1 5 .6 1
90
90
80
80
70
70
Relative Abundance
50
Relative Abundance
60
60
50
40
40
30
30
20
20
10
10
0 13 14 15 Time (min) 16 17
0 13 14 15 Time (min) 16 17
Figure 2b: Comparison of SRM mode and H-SRM mode for the analysis of the fungicide Bitertanol
Page 4 of 6
SRM Mode
RT : 15. 2
100 95 90 85 80 75 70 65 60 Relative Abundance 55 50 45 40 35 30 25 20 15 10 5 0 Relative Abundance
H-SRM Mode
18. 0
RT : 15. 3
100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0
13
14
15
16 Time (min)
17
18
19
13
14
15
16 Time (min)
17
18
19
Figure 2c: Comparison of SRM mode and H-SRM mode for the analysis of the herbicide Etobenzanid
x 10
x 100 100 14. 84
10
11. 49
Pirim icarb
8
Dif lubenzuron
80
SRM 239 182

Relat ive Abundance
SRM 311 158
Relative Abundance
60 40 20 0 10. 56 10 12. 79 12 13. 94 14 16 18 20
No cr oss-t alk
No cr oss t alk
17. 24 17. 52
2
13 . 4 7. 69 9. 5 10 12 14 13 . 6 15 . 3 16
0
8
x 50
x 1000 13. 26 100 17. 43
10
Linuron
8
Fluf enoxuron
80
SRM 249 182

Relat ive Abundance
SRM 489 158
Relative Abundance
60 40 20 0 11. 08 10 12. 91 12 15. 53 14 T im e 15. 74 16 17. 90 18 20
4
10 . 9 8. 93 10 . 3 11 . 8 13 . 4 15 . 0 16
0
8 10 12 T im e ( m in ) 14
Figure 3a: No cross-talk is observed for the SRM transitions of primicarb and linuron
Figure 3b: No cross-talk is observed for the MRM transitions of diubenzuron and ufenoxuron
Page 5 of 6
Conclusions
An H-SRM LC-MS/MS method to monitor 35 pesticide residues was developed using the TSQ Quantum Discovery. All 35 pesticide residues were quantitated in 18 minutes. Using H-SRM, interferences from the sample matrix background were substantially reduced, leading to improved LOQs. Similarly, no cross-talk issues were detected for any of the tested analytes. Compared with traditional single pesticide analysis methods, the sample preparation procedures are usually simplied in multi-pesticide analysis methods. This means more interference from the sample matrix may be present making H-SRM the technique of choice for improving detection limits.
www.thermo.com
AN62487_E 11/07S
Zero Cross-talk on the TSQ Quantum

Dipankar Ghosh, Mark Churchill, Eric Genin, and Alan Schoen; Thermo Fisher Scientic, San Jose, CA
Introduction
Key Words TSQ Quantum Surveyor HPLC AQUASIL Environmental samples Quantitation SRM In assays involving closely eluting multi-target analytes, Selective Reaction Monitoring (SRM) is the most commonly used mass spectrometry technique for performing quantitative assays on triple quadrupole systems. Examples include DMPK assays, monitoring for drugs of abuse in urine extracts, and pesticide residues monitoring in environmental samples. The SRM experiment consists of three distinct events. First, precursor ions of a specic mass to charge (m/z) ratio are transmitted through the rst quadrupole, while all ions of different m/z values are ltered out. Secondly, the selected ions collide with a neutral gas present in the second quadrupole (collision cell) where they undergo collision induced decomposition (CID) reactions. Finally, product ions of specic m/z values are transmitted through the third quadrupole, after which they are detected. When performing LC-MS/MS analyses of multi component mixtures by SRM, it is often necessary to carry out such assays using very short dwell times of 1020 milliseconds. In certain instances, especially in environmental monitoring of pesticides, it is possible to get a large number of parent compounds of the same chemical class eluting very close to each other in a short chromatographic timescale. Oftentimes, these compounds also give rise to exactly the same product ions which are used to monitor the SRM transitions. For example, the compounds Triadimenol (mwt 295 amu), Tebuconazole (mwt 307 amu), Cyproconazole (mwt 291 amu), and Hexaconazol (mwt 314 amu) have different precursor ions, but all give rise to the same product ion at m/z 70. One potential scenario in such assays is that when SRM dwell times and inter scan times are very small, and sample concentrations are high, storage of product ions can take place in the collision cell. In other words, fragment ions from the one transition can still be in the collision cell when the next SRM transition is monitored. This can result in Cross talk, which is used to describe the phenomenon when the fragment ions from one SRM transition are scanned out during another transition. Even a 0.01% cross-talk effect can result in false positives being observed in the resulting quantitative data.
It is a well known fact that Thermo Scientic triple quadrupole systems, ranging from the earlier TSQ70 series to the current TSQ Quantum range, have never suffered from any cross-talk issues due to the advanced design of the collision cell. The purpose of this application note is to demonstrate the absence of cross-talk on the TSQ Quantum using examples from the analyses of pesticides.
Goal
To illustrate the absence of cross-talk in a screening assay for 88 different pesticides with closely eluting multi-component compounds belonging to the same chemical classes.
Chemicals and Reagents
Water, methanol and acetic acid were HPLC grade and purchased from J T Baker Chemicals, France.
Samples
Pesticides (3,4,5-Trimehacarb, 3-hydroxy-carbofuran, 5-hydroxy-clethodim-sulfon, Acephate, Aldicarb, Aldicarb-sulfoxid, Aldoxycarb, Amidosulfuron, Atrazin, Azoxystrobin, Bendiocarb, Bensulfuron-methyl, Butocarboxim, Butocarboxim-sulfoxid, Butoxycarboxim, Carbaryl, Carbendazim, Carbofuran, Chlorosulfuron, Cinosulfuron, Clethodim, Clethodim-imin-sulfon, Clethodim-imin-sulfoxid, Clethodim-sulfon, Clethodimsulfoxid, Cyprodinil, Daminozoid, Demeton-S-methylsulfon, Desmedipham, Diubenzuron, Dimethoat, Diuron, Ethiofencarb, Ethiofencarb-sulfon, Ethiofencarb-sulfoxid, Fenhaxamid, Fenoxycarb, Fenpropimorph, Fentinhydroxide, Flazasulfuron, Flufenoxuron, Flurathiocarb, Fluzifop-P-butyl, Haloxyfop-ethoxyethyl, Haloxyfopmethyl, Imazalil, Imidacloparid, Indoxacarb, Iprovalicarb, Isoproturon, Isoxautole, Linuron, Metalaxyl, Metamitron, Methamidophos, Methiocarb, Methomyl, Metolachlor, Metsulfuron-methyl, Monocrotophos, Nicosulfuron, Omethoat, Oxidemeton-methyl, Phenmedipham, Pirimicarb, Promecarb, Propamocarb, Propoxur, Prosulfuron, Pymetrozin, Pyridate, PyridateXX,
Pyrimethanil, Quinmerac, Quizalofop-ethyl, Rimsulfuron, Spiroxamine, Tebuconazol, Tebufenzoid, Thiabenzadol, Thiacloprid, Thiodicarb, Thiofanox, Thiophanat-methyl, Triasulfuron, Triumuron, Triusulfuron-methyl, Vamidothion) were purchased from Sigma unless otherwise noted. A standard solution containing the above 88 pesticides was prepared at 50 pg/mL in methanol.
MS Instrument Method
To accommodate the analysis of a large number of components over a short time range, the acquisition time was divided into two segments, each containing three scan events. Allowing for analyte overlap between the time segments, a total of 59 SRM transitions were performed in segment one and 56 SRM transitions in segment two, with dwell times of 20 msec for each transition.
Sample Analysis
HPLC analysis was performed on the Thermo Scientic Surveyor HPLC system, using a 50 2.1 mm Thermo Scientic AQUASIL C18 column. Mobile phase A was water/methanol 80/20 (v/v) and mobile phase B was methanol/water 90/10 (v/v)both contained 0.05% acetic acid. Solvent was pumped at 200 mL/min and analytes eluted using a linear gradient of 100% A to 100% B over 11 minutes, holding at 100% B for 12 minutes, then returning to 100% A in 2 minutes. MS analyses was carried out using a Thermo Scientic TSQ Quantum Discovery mass spectrometer.

Figure 1 shows the LC-MS/MS chromatogram generated from the pesticide mix eluting over a chromatographic time scale of 16 minutes.
A number of compounds from the list of pesticides belonging to the same class are highlighted in Tables 1, 2 and 3. Although they have different parent molecular weights, they belong to the same, or similar, chemical class and have a common basic carbon skeleton. This results in them giving the same structural product ions (i.e., identical mass product ions), which are being monitored for the SRM transition. This was demonstrated during the pesticide screening assay by extracting three examples of compound classes containing different precursor ions but all generating the same product ion mass.
Mass Spectrometry Instrument: TSQ Quantum Discovery Source: ESI Ion polarity: Positive Spray voltage: 3.5 kV Sheath/Auxiliary gas: Nitrogen Sheath gas pressure: 50 Auxiliary gas pressure: 15 Ion transfer capillary temperature: 350 C Scan type: SRM CID conditions: Ar at 1.5 mTorr
Figure 1 : LC-MS/MS chromatogram of 88 pesticides at 50 pg/L
Page 2 of 6
Example 1: Triasulfuron, Metsulfuron-methyl and Chlorosulfuron. These compounds have different precursor ions but all generate a product ion at m/z 167 (see Table 1 and Figures 1a and 1b). The chromatograms show the transitions for these compounds, with no evidence of cross-talk.
Retention Time (min)
O S
Compound
Parent ion
NH O O N N NH N
Predicted product ion fragment

NH N
Triasulfuron
7.62
O
Cl
O O
m/z 402
m/z 167
O
N
Metsulfuron-methyl
8.07
O S
O
NH NH
O
N
NH
m/z 382
m/z 167
Cl O S
NH NH
O N
NH
N O N
Chlorosulfuron
8.45
O O
N N N O
m/z 358
m/z 167
Table 1: Triasulfuron, Metsulfuron-methyl and Chlorosulfuron
100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 6.5 7.0 7.5
7.62
7.62
Triasulfuron m/z 402 >167
1.0 0.8 0.6 0.4 0.2 0.0
No cross-talk
Triasulfuron m/z 402 >167
8.07
8.07
1.0
Metsulfuron-methyl m/z 382 >167
0.8 0.6 0.4 0.2 0.0
No cross-talk
Metsulfuron-methyl m/z 382 >167
8.45
8.45
1.0
Chlorosulfuron m/z 358 >167
0.8 0.6 0.4 0.2 0.0 6.5 7.0 7.5 8.0 Time (min) 8.5
Chlorosulfuron m/z 358 >167
8.0 Time (min)
8.5
9.0
9.5
9.0
9.5
Figure 1a: Zero cross-talk was observed when the pesticides triasulfuron, metsulfuron-methyl and chlorosulfuron were detected. Peak height shown full scale. Arrows indicate position of potential cross-talk
Figure 1b: Zero cross-talk (indicated by red arrows) was observed when the pesticides triasulfuron, metsulfuron-methyl and chlorosulfuron were detected. Baseline magnied x100
Page 3 of 6
Example 2: Isoproturon and Diuron. These compounds have different precursor ions but both generate a product ion at m/z 72 (see Table 2 and Figures 2a and 2b). The chromatograms show the transitions for these compounds, with no evidence of cross-talk.
Retention Time (min) Predicted product ion fragment
Compound
Parent ion
NH N O
Isoproturon
9.22
m/z 207
Cl
N O
m/z 72
Diuron
9.75
Cl
NH N O
O
m/z 233
m/z 72
Table 2 : Isoproturon and Diuron
100 80 60 40 20 0 100 80 60 40 20 0 9.0
9.22 1.0
9.22
Isoproturon m/z 207 >72
0.8 0.6 0.4 0.2 0.0
Isoproturon m/z 207 >72
No cross-talk
9.75 1.0
9.75
Diuron m/z 233 >72
0.8 0.6 0.4 0.2 0.0
Diuron m/z 233 >72
9.5
10.0 Time (min)
10.5
11.0
11.5
9.0
9.5
10.0 Time (min)
10.5
11.0
11.5
Figure 2a : Zero cross-talk was observed when the pesticides Isoproturon and Diuron were detected. Peak height shown full scale. Arrow indicates position of potential cross-talk
Figure 2b : Zero cross-talk (indicated by red arrow) was observed when the pesticides Isoproturon and Diuron were detected. Baseline magnied x100
Page 4 of 6
Example 3: Nicosulfuron and Linuron. These compounds have different precursor ions but both generate a product ion at m/z 182 (see Table 3 and Figures 3a and 3b). The chromatograms show the transitions for these compounds, with no evidence of cross-talk.
Retention Time (min)
N O O NH S O N O O O N O N NH N O NH N O
Compound
Parent ion
Predicted product ion fragment
Nicosulfuron
9.59
m/z 411
Cl O OH N
m/z 182
Cl
Linuron
10.26
O N NH
Cl
NH
m/z 249
m/z 182
Table 3 : Nicosulfuron and Linuron
100 80 60 40 20 0 100 80 60 40 20 0 9.0 9.5
9.59
9.59
Nicosulfuron m/z 411 >182
1.0 0.8 0.6
Nicosulfuron m/z 411 >182
No cross-talk
0.4 0.2 0.0 1.0
10.26
10.26
Linuron m/z 249 >182
0.8 0.6 0.4 0.2 0.0
Linuron m/z 249 >182
10.0 Time (min)
10.5
11.0
9.0
9.5
10.0 Time (min)
10.5
11.0
Figure 3a: Zero cross-talk was observed when the pesticides Nicosulfuron and Linuron were detected. Peak height shown full scale. Arrow indicates position of potential cross-talk
Figure 3b: Zero cross-talk (indicated by red arrow) was observed when the pesticides Nicosulfuron and Linuron were detected. Baseline magnied x100
Page 5 of 6
Why is there Zero Cross-talk on the TSQ Quantum?

The reason for not observing any cross-talk on the TSQ Quantum can be ascribed to the way the collision cell (Q2) is operated. The ions are actively ejected radially by selecting Mathieu a and q operational parameters such that all ions of all masses become unstable. This instantaneously dumps all ions from the collision cell. The offset voltages are also operated to keep the cell empty while Q1 slews to the next precursor mass. Once Q1 is set, the cell (Q2) parameters are selected to accept and fragment the precursor ions for the next transition. As this procedure is not dependent upon ion residence times, each SRM transition becomes a discreet experiment, with no memory of the one before, thus totally eliminating any possibility of cross-talk. It should be noted that for SRM and MRM assays, this technique is superior to clearing a collision cell using axial ejection with a supplementary eld which is slower and less efcient. Indeed, axial techniques applied on this time scale always have some residual cross-talk.
Conclusions
An LC-MS/MS screening assay to monitor 88 pesticides using minimal LC separation was developed using the TSQ Quantum Discovery. It was possible to detect all components within a chromatographic timescale of 16 minutes by performing 88 SRM transitions. Even with dwell times of only 20 milliseconds no cross-talk interfered with the analysis. The absence of cross-talk was demonstrated by extracting and comparing the ion chromatogams of similar classes of compounds with different precursor ion masses but with the same product ion mass.
References U.S. Environmental Protection Agency website at www.epa.gov: US Environmental Protection Agency Code of Federal Regulations 40. Chapter I, Subchapter E, Part 180 details the tolerances and exemptions from tolerances for pesticide chemicals in food. Pesticide Analytical Manual Volume 1, Sections 605-606 (describes MS applications and benets). Transmittal No. 94-1 (1/94), Form FDA 2905a (6/92). Available on the FDA website at www.cfsan.fda.gov. Chapter 3 describes multi-class multi-residue methods, while Chapter 4 provides selective multi-residue methods.
www.thermo.com
AN62646_E 01/08S
Pesticides Pesticides, Mycotoxins, Plant toxins

t
30163: High Resolution and Precise Mass Accuracy: A Perfect Combination for Food and Feed Analysis in Complex Matrices
High Resolution and Precise Mass Accuracy: A Perfect Combination for Food and Feed Analysis in Complex Matrices
Markus Kellmann, Andreas Wieghaus, Helmut Muenster, Thermo Fisher Scientic, Bremen, Germany Lester Taylor, Dipankar Ghosh, Thermo Fisher Scientic, San Jose, CA, USA
Overview
Key Words Exactive Food Analysis Pesticide Screening High Resolution MS
Purpose: To demonstrate the analytical advantages of using high resolution (> 40,000) for the accurate screening of pesticides in complex matrices using a new benchtop Thermo Scientific Orbitrap detector. Methods: Use of ultra-high pressure liquid chromatography (U-HPLC) coupled with the Thermo Scientific Exactive mass spectrometer operating in high resolution mode. The Exactive detector is based on Thermo Scientific Orbitrap technology. Results: The combination of high resolution (15,000 50,000) accurate mass is required for the detections of pesticides and mycotoxins.
Ultra High Performance Liquid Chromatography (U-HPLC). In complex sample matrices (e.g. food, feed, hair, honey) this limited resolution leads to inaccurate mass measurements caused by unresolved background matrix interferences. In this work we show a full scan screening approach using a novel single stage Orbitrap mass spectrometer coupled to U-HPLC, capable of providing high mass accuracy at resolutions of up to 100,000. Additionally we will discuss two aspects of the analysis which also greatly benefit from very high resolution: resolving co-eluting, isobaric target compounds elemental composition determination
Methods
The Exactive, a new non-hybrid single-stage Orbitrap mass spectrometer coupled to the Thermo Scientific Accela U-HPLC chromatograph was used to evaluate a highly complex mixture of 116 pesticides, mycotoxins and plant toxins in different concentrations. A 12 min gradient was applied to a Thermo Scientific Hypersil GOLD RP 50 x 2 mm 1.9 m particle packed column with water/acetonitrile eluents. The method developed was evaluated with respect to sensitivity, selectivity and linearity in standard solutions and extracts from animal feed. Mass measurements were performed at different resolution settings (R = 15,000 and R = 50,000) to enable comparisons to data acquired by ToF instruments and to demonstrate the advantage of ultra high resolution. Orbitrap detection was carried out using automatic control of the number of ions entering the detector (AGC, target value = 106).
Introduction
Screening of pesticides, mycotoxins and veterinary drugs is of great importance in regulated environments such as food and animal feed analysis. Traditionally these type of experimentshave been carried out using triple quadrupole instruments. This approach has certain limitations: no post acquisition re-interrogation of data limited number of compounds per analysis cannot screen unidentified unknowns Because of these limitations, there is currently a trend towards full scan MS experiments in residue analysis. Current screening approaches are performed using high performance ToF instruments, with mass accuracies of < 5ppm and resolutions of about 15,000, coupled to
Figure 1: Schematic of the new Orbitrap benchtop mass spectrometer, including HCD collision cell for All Ion Fragmentation.
Results
Resolution of Isobaric Pesticides In cases where isobaric compounds co-elute, erroneous mass accuracy and elemental composition assignment will occur if the resolving power of the mass spectrometer is insufficient to separate these compounds. Figure 2 shows two pesticides Thiamethoxam (C8H10ClN5O3S) and Parathion (C10H14NO5PS), which have protonated molecular ions (MH+) at 292.02656 and 292.04031, respectively. A resolution higher than 40,000 is needed to resolve the protontated molecular ion of these two compounds completely. This is a pre-requisite for analysis of low concentration compounds in the presence of higher abundant ones. The example in Figure 2 shows an approximate 1:3 mixture of both pesticides measured and simulated. Influence on Elemental Composition Determination A limited resolution of 15,000 results in two major limitations. Firstly, the detection of unresolved doublets may result in significant mass errors which are outside the characteristic accuracy specification of the Exactive instrument. As a consequence, at lower resolution settings the mass windows for elemental composition determination have to be increased, resulting in much larger number of elemental composition proposals for the unknown or targeted compounds. This can be seen for the example (Figure 3) of Pirimicarb at m/z 239.1503. Due to the presense of an isobaric interference the peak at 239 shows a mass error of 6.5 ppm. At a resolution of 15,000 the underlying interference causes an apparent shift to higher mass, whereas at higher resolution (here 80,000) the doublet is clearly resolved and the mass accuracy is well within instrument specifications.
Figure 2: Mass chromatogram of two isobaric pesticides measured at a resolution of 15,000 (left) and 50,000 (right). Superimposed is the simulated mass trace at each resolution setting.
100 Relative Abundance 80 60 40 20 0 239.00 100 Relative Abundance 80 60 40 20 0 239.00 239.05 239.10 239.15 m/z 239.05 239.10 239.15 m/z Pirimicarb
R=15,000
Pirimicarb
239.15181 C11H19O2N4 6.50 ppm
239.20 239.15033 C11H19O2N4 0.32 ppm
239.25
R=80,000
Resolved interference
239.20
239.25
Figure 3: Improved mass accuracy simply by increasing the resolution in order to resolve the doublet. The peak at 15,000 resolution splits up into 2 by increasing the resolution to 80,000.
In order to limit the number of candidate elemental compositions to a single confident assignment, a sophisticated software algorithm is used. It takes into account the peak height and mass accuracy of the monoisotopic peak and its isotopes. However, in order to function correctly, all of the accurate mass values for the isotopic peaks must be within specified limits. The absence of interference peaks can only be assured by use of high resolution, (as can be seen in Figure 4). Here the fungicide Azoxystrobin is shown at resolutions of 15,000 and 80,000. The medium resolution spectrum shows very good mass accuracy for the monoisotopic peak, but gives unusually high mass errors for the A+1 and A+2 ions. This is due to an interference at m/z 405.1452, which cannot be resolved at medium resolution. Whereas the high resolution spectrum shows exellent mass accuracy for all three measured isotopes. Determining elemental compositions using data acquired at ~15,000 resolution will result in misleading or incorrect data. Only sufficient high resolution allows the determination of the accurate mass of the complete molecular ion cluster and therefore allows automated assignment of an elemental formula with a high degree of confidence.
Analyzing highly complex samples such as extracts from food or animal feed, and the screening of regulated substances including pesticides, mycotoxins and veterinary drugs is a major analytical challenge for mass spectrometry. On one hand the methodology must have a high intra scan dynamic range in order to detect low concentrated compounds in presence of high abundant matrix ions, on the other hand high selectivity and high sensitivity is needed to avoid false positive, or even worse, false negative results. In our procedure we analyzed an extract from horse feed as an example of extremely complex matrix, spiked with a mixture of 116 pesticides and mycotoxins. A dilution series ranging from 2 to 250 ppb (for each compound) was measured in duplicates at two different resolution settings. In addition a 100 ppb sample of the same mixture was analyzed at a resolution of 50,000 in order to determine the maximum number of detectable substances for this method.
Figure 4: The importance of resolution for the molecular ion AND its isotopes. At high resolution the complete molecular ion cluster is correctly detected.
LC-MS analysis of the extracted spiked samples showed the presence of 95 out of 116 compounds at 100 ppb in matrix. Figure 5 shows the overlaid ion chromatograms for all 116 compounds (3 ppm window) at 50 ppb (in matrix). The number of recovered pesticides in different concentrations is shown in Figure 5. The data illustrates that a greater number of detected compounds (higher sensitivity) with an extraction window of 3 ppm at higher resolution setting. This is exemplified in Figure 6, where extracted ion chromatograms of Sulcotrion at 50 ppb for R = 15,000 and R = 50,000 are shown. The higher resolved spectrum displays two peaks, of which the smaller one is Sulcotrion.
The lower resolved peak masks the pesticide signal completely. The only indication for the presence of Sulcotrion is a slightly broader peak or shoulder and a mass shift of the interfering ion towards higher masses. This would lead to a false negative result, if the analysis was only performed at a resolution of 15,000, and is the major reason, why the number of identified components in the case of R = 15,000 decreases disproportionately to the measurements at higher resolving power (diagram, Figure 5).
Figure 5: Overlaid extracted ion chromatograms from a mixture of 116 pesticides and mycotoxins at a 100 ppb level. Extraction was done with 3 ppm mass window. The inset chart shows the number of detected compounds at different concentrations (in matrix) at two different resolution settings.
Figure 6: Expanded view of the pesticide mixture at different resolution settings (top: 15,000 and bottom: 50,000). Pesticide Sulcotrion (m/z 328.02475) is masked under background ions at a resolution of 15,000 but is easily detected at 50,000 resolution (see also mass chromatogram inset).
For this reason, a dilution series was measured at higher resolution settings. One example (Metabenthiazuron) is shown in Figure 7. It demonstrates excellent linearity and sensitivity down to 2 ppb level (2 ng/mL).
Figure 7: Quantitation curve for Metabenthiazuron ranging from 2 to 250 ppb. The quantified peak for each concentration level demonstrates the high quality data even at the lowest level.
Conclusions
New benchtop Orbitrap mass spectrometer demonstrates superior mass resolving power compared to that obtained using TOF instruments. High resolving power (up to 100,000) provides precise mass accuracy for complex sample analysis High resolving power provides excellent sensitivity, linearity and selectivity in multi-residue screening of complex matrices. Fast scan speed (10 Hz) are fully compatible with the use of U-HPLC fast chromatography methods For the analysis of very complex samples it is advantageous to select the appropriate scan speed and resolution in order to avoid unresolved isobaric compounds (matrix ions from analyte ions) and still allow unambiguous detection of low abundant species.
Acknowledgements
We would like to thank Paul Zomer and Hans Mol from the RIKILT Institute for Food Safety in Wageningen, The Netherlands, for providing the samples.

www.thermo.com
AN30163_E 07/08C
Index BY COMPOUND CLASS
Index
BY COMPOUND CLASS
Acrylamide
Antibiotics
Analysis of (Fluoro)quinolones in Honey with Online Sample Extraction and LC-MS/MS Analysis of Sulfonamides in River Water using EQuan, an Online Concentration Analysis System Determination of Sulfonamide Antibiotics in Wastewater by Liquid ChromatographyTandem Mass Spectrometry Determination of Trace Level Nitrofuran Metabolites in Crawfish Meat by Electrospray LC-MS/MS on the TSQ Quantum Discovery MAX Highly Selective Detection and Identification of Nitrofurans Metabolites in Honey using LC-MS/MS LC-MS/MS Analysis of Malachite Green, Leucomalachite Green, Ciprofloxacin, and Tetracycline in Food Samples using a TurboFlow Method Multi-class Antibiotic Screening of Honey Using Online Extraction with LCMS/MS On-line Enrichment HTLC/MS/MS Assay for Multiple Classes of Antibiotics in Environmental Water Sources Simple and Rapid Analysis of Chloramphenicol in Milk by LC-MS/MS
Antimicrobials; Antibiotics
LC/MS/MS Analysis of Anti-Infectives In Raw and Treated Sewage
Flavanoids
Flavonoids
LC-MS Applications for Food, Beverage, and Water Testing Search | Contents | Index
Index-1
Index BY COMPOUND CLASS
Haloacetic Acids
Analysis of Haloacetic Acids in Drinking Water by IC-MS/MS
Melamine; cyanuric acid

Mycotoxins
Pesticides
Analysis of Haloacetic Acids in Drinking Water by IC-MS/MS Analysis of Regulated Pesticides in Drinking Water Using Accela and EQuan Analysis of Triazine Pesticides in Drinking Water Using LC-MS/MS (EPA Method 536.0) Determination of Different Classes of Pesticide Residues in Processed Fruits and Vegetables by LC-MS Using the TSQ Quantum Ultra According to EU Directive 91/414 EEC LC-MS/MS Analysis of Herbicides in Drinking Water at Femtogram Levels Using 20 mL EQuan Direct Injection Techniques LC-MS/MS Determination of Malachite Green and Leucomalachite Green in Fish Products Multi-residue Analysis of Pesticides in Food using GC/MS/MS with the TSQ Quantum GC Quantitation-Enhanced Data-Dependent (QED) Scanning of Drinking Water Samples Using EQuan for Pesticide Analysis on a Triple Stage Quadrupole Simultaneous Detection of 88 Pesticides on the TSQ Quantum Discovery using a Novel LC/MS/MS Method Testing LC-MS System Robustness with Automated Sample Cleanup Using Red Wine as a Matrix UHPLC Separation of Triazine Herbicides at Elevated Temperature Utility of H-SRM to Reduce Matrix Interference in Food Residue Analysis of Pesticides by LC/MS/MS using the TSQ Quantum Discovery Zero Cross-talk on the TSQ Quantum
Pesticides, Mycotoxins, Plant toxins

High Resolution and Precise Mass Accuracy: A Perfect Combination for Food and Feed Analysis in Complex Matrices
Index-2
Index BY MATRIX
Pharmaceuticals
The Thermo Scientific Exactive Benchtop LC/MS Orbitrap Mass Spectrometer
Pharmaceuticals, Personal Care Products, and Pesticides

Detection of Pharmaceuticals, Personal Care Products, and Pesticides in Water Resources by APCI-LC-MS/MS
Phenolic Pollutants
Toxins
Analysis of Microcystins from Blue-green Algae Using the TSQ Quantum Ultra LC-MS/MS System
BY MATRIX
Catfish
Cattle forages
Analysis of Mycotoxins in Various Cattle Forages and Food Matrices with the TSQ Quantum Discovery MAX High Resolution and Precise Mass Accuracy: A Perfect Combination for Food and Feed Analysis in Complex Matrices
Crawfish
Determination of Trace Level Nitrofuran Metabolites in Crawfish Meat by Electrospray LC-MS/MS on the TSQ Quantum Discovery MAX
Crop extracts
Multi-residue Analysis of Pesticides in Food using GC/MS/MS with the TSQ Quantum GC Determination of Different Classes of Pesticide Residues in Processed Fruits and Vegetables by LC-MS Using the TSQ Quantum Ultra According to EU Directive 91/414 EEC Utility of H-SRM to Reduce Matrix Interference in Food Residue Analysis of Pesticides by LC/MS/MS using the TSQ Quantum Discovery
Eel
LC-MS/MS Determination of Malachite Green and Leucomalachite Green in Fish Products
Index-3
Index BY MATRIX
Honey
Analysis of (Fluoro)quinolones in Honey with Online Sample Extraction and LC-MS/MS Multi-class Antibiotic Screening of Honey Using Online Extraction with LCMS/MS Highly Selective Detection and Identification of Nitrofurans Metabolites in Honey using LC-MS/MS
Milk
Simple and Rapid Analysis of Chloramphenicol in Milk by LC-MS/MS
Potato chips; breakfast cereals

Red wine
Direct Analysis of Red Wine Using Ultra-Fast Chromatography and High Resolution Mass Spectrometry Testing LC-MS System Robustness with Automated Sample Cleanup Using Red Wine as a Matrix
Shrimp, fish, pork

LC-MS/MS Analysis of Malachite Green, Leucomalachite Green, Ciprofloxacin, and Tetracycline in Food Samples using a TurboFlow Method
Tea
Water (drinking)
Analysis of Haloacetic Acids in Drinking Water by IC-MS/MS Analysis of Regulated Pesticides in Drinking Water Using Accela and EQuan Analysis of Triazine Pesticides in Drinking Water Using LC-MS/MS (EPA Method 536.0) LC-MS/MS Analysis of Herbicides in Drinking Water at Femtogram Levels Using 20 mL EQuan Direct Injection Techniques Quantitation-Enhanced Data-Dependent (QED) Scanning of Drinking Water Samples Using EQuan for Pesticide Analysis on a Triple Stage Quadrupole
Index-4
Index BY PRODUCT
Water (environmental)
Analysis of Microcystins from Blue-green Algae Using the TSQ Quantum Ultra LC-MS/MS System Analysis of Sulfonamides in River Water using EQuan, an Online Concentration Analysis System Analyzing Phenolic Pollutants in Water Using U-HPLC Detection of Pharmaceuticals, Personal Care Products, and Pesticides in Water Resources by APCI-LC-MS/MS On-line Enrichment HTLC/MS/MS Assay for Multiple Classes of Antibiotics in Environmental Water Sources UHPLC Separation of Triazine Herbicides at Elevated Temperature
Water (lab)
Simultaneous Detection of 88 Pesticides on the TSQ Quantum Discovery using a Novel LC/MS/MS Method The Thermo Scientific Exactive Benchtop LC/MS Orbitrap Mass Spectrometer Zero Cross-talk on the TSQ Quantum
Water (sewage)
Determination of Sulfonamide Antibiotics in Wastewater by Liquid Chromatography Tandem Mass Spectrometry LC/MS/MS Analysis of Anti-Infectives In Raw and Treated Sewage
BY PRODUCT
Thermo Scientific Accela U-HPLC system
Analysis of Regulated Pesticides in Drinking Water Using Accela and EQuan Analysis of Triazine Pesticides in Drinking Water Using LC-MS/MS (EPA Method 536.0) Analyzing Phenolic Pollutants in Water Using U-HPLC Direct Analysis of Red Wine Using Ultra-Fast Chromatography and High Resolution Mass Spectrometry Metabolomic Analysis of Green and Black Tea Extracts Using an LTQ Orbitrap XL Hybrid Linear Ion Trap Mass Spectrometer Simple and Rapid Analysis of Chloramphenicol in Milk by LC-MS/MS UHPLC Separation of Triazine Herbicides at Elevated Temperature
Index-5
Index BY PRODUCT
Thermo Scientific Aria TLX-1

Analysis of (Fluoro)quinolones in Honey with Online Sample Extraction and LC-MS/MS LC-MS/MS Analysis of Malachite Green, Leucomalachite Green, Ciprofloxacin, and Tetracycline in Food Samples using a TurboFlow Method Multi-class Antibiotic Screening of Honey Using Online Extraction with LCMS/MS
Thermo Scientific Exactive

Direct Analysis of Red Wine Using Ultra-Fast Chromatography and High Resolution Mass Spectrometry High Resolution and Precise Mass Accuracy: A Perfect Combination for Food and Feed Analysis in Complex Matrices The Thermo Scientific Exactive Benchtop LC/MS Orbitrap Mass Spectrometer
Thermo Scientific LTQ Orbitrap XL

Thermo Scientific SEIVE

Thermo Scientific Surveyor HPLC System

Analysis of Mycotoxins in Various Cattle Forages and Food Matrices with the TSQ Quantum Discovery MAX Detection of Pharmaceuticals, Personal Care Products, and Pesticides in Water Resources by APCI-LC-MS/MS Determination of Different Classes of Pesticide Residues in Processed Fruits and Vegetables by LC-MS Using the TSQ Quantum Ultra According to EU Directive 91/414 EEC Determination of Sulfonamide Antibiotics in Wastewater by Liquid Chromatography Tandem Mass Spectrometry Determination of Trace Level Nitrofuran Metabolites in Crawfish Meat by Electrospray LC-MS/MS on the TSQ Quantum Discovery MAX LC/MS/MS Analysis of Anti-Infectives In Raw and Treated Sewage LC-MS/MS Determination of Malachite Green and Leucomalachite Green in Fish Products
Index-6
Index BY PRODUCT
Utility of H-SRM to Reduce Matrix Interference in Food Residue Analysis of Pesticides by LC/MS/MS using the TSQ Quantum Discovery Zero Cross-talk on the TSQ Quantum
Thermo Scientific Surveyor Plus HPLC system

Thermo Scientific TSQ Quantum

Analysis of Regulated Pesticides in Drinking Water Using Accela and EQuan Analysis of Sulfonamides in River Water using EQuan, an Online Concentration Analysis System Quantitation-Enhanced Data-Dependent (QED) Scanning of Drinking Water Samples Using EQuan for Pesticide Analysis on a Triple Stage Quadrupole Zero Cross-talk on the TSQ Quantum
Thermo Scientific TSQ Quantum Access

Analysis of Haloacetic Acids in Drinking Water by IC-MS/MS Analysis of Triazine Pesticides in Drinking Water Using LC-MS/MS (EPA Method 536.0) LC-MS/MS Analysis of Herbicides in Drinking Water at Femtogram Levels Using 20 mL EQuan Direct Injection Techniques LC-MS/MS Analysis of Malachite Green, Leucomalachite Green, Ciprofloxacin, and Tetracycline in Food Samples using a TurboFlow Method Simple and Rapid Analysis of Chloramphenicol in Milk by LC-MS/MS
Thermo Scientific TSQ Quantum Discovery

Highly Selective Detection and Identification of Nitrofurans Metabolites in Honey using LC-MS/MS Quantitation of Acrylamide in Food Samples on the TSQ Quantum Discovery by LC/APCI-MS/MS Simultaneous Detection of 88 Pesticides on the TSQ Quantum Discovery using a Novel LC/MS/MS Method Utility of H-SRM to Reduce Matrix Interference in Food Residue Analysis of Pesticides by LC/MS/MS using the TSQ Quantum Discovery
Thermo Scientific TSQ Quantum Discovery MAX

Index-7
Index BY PRODUCT
Determination of Trace Level Nitrofuran Metabolites in Crawfish Meat by Electrospray LC-MS/MS on the TSQ Quantum Discovery MAX LC-MS/MS Determination of Malachite Green and Leucomalachite Green in Fish Products
Thermo Scientific TSQ Quantum GC

Multi-residue Analysis of Pesticides in Food using GC/MS/MS with the TSQ Quantum GC
Thermo Scientific TSQ Quantum Ultra

Analysis of (Fluoro)quinolones in Honey with Online Sample Extraction and LC-MS/MS Analysis of Melamine and Cyanuric Acid in Food Matrices by LC-MS/MS Analysis of Microcystins from Blue-green Algae Using the TSQ Quantum Ultra LC-MS/MS System Detection of Pharmaceuticals, Personal Care Products, and Pesticides in Water Resources by APCI-LC-MS/MS Determination of Different Classes of Pesticide Residues in Processed Fruits and Vegetables by LC-MS Using the TSQ Quantum Ultra According to EU Directive 91/414 EEC Determination of Sulfonamide Antibiotics in Wastewater by Liquid ChromatographyTandem Mass Spectrometry LC/MS/MS Analysis of Anti-Infectives In Raw and Treated Sewage Multi-class Antibiotic Screening of Honey Using Online Extraction with LCMS/MS On-line Enrichment HTLC/MS/MS Assay for Multiple Classes of Antibiotics in Environmental Water Sources Testing LC-MS System Robustness with Automated Sample Cleanup Using Red Wine as a Matrix
Index-8

EFS Applications

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

EFS Applications

Uploaded by

Copyright:

Available Formats

Thermo Scientific LC-MS Applications for Food, Beverage, and Water Testing

Includes sample preparation, chromatography, and mass spectrometry conditions

Contaminants Drug Residues Natural Compounds Pesticides

Part of Thermo Fisher Scientic

Click the Search button to search for any word or phrase.

Click on a compound class at right to go to related notes.

Application Note: 319

Quantitation of Acrylamide in Food Samples on the TSQ Quantum Discovery by LC/APCI-MS/MS

Search | Contents | Index

1900 1800 1700 1600 1500

d3-Acrylamide 75 58 Area = 1507019

Figure 1: SRM chromatograms for 50 ng/mL d3-acrylamide

5500 5000 4500 4000 3500

Acrylamide 75 55 Area = 26998 S/N = 17

d3-Acrylamide 75 58 Area = 5523570

Results and Discussion

100000 1.5 2.0 2.5 3.0 3.5 4.0

0.15 0.10 0.05 0.00

Search | Contents | Index

% CV 12.1 6.7 4.6 0.9 0.7 0.5 0.8 0.6 0.6

Table 1: Statistical data for the calibration curve of acrylamide

RT: 2.97 250000 200000

Acrylamide 72 55 Area = 3268462

150000 100000 50000

RT: 2.93 400000 350000 300000

250000 200000 150000 100000 50000 0.0

d3-Acrylamide 75 58 Area = 5400939

Figure 4: SRM chromatograms of the Potato Chip 2 sample aqueous extract

20000 18000 16000

Acrylamide 75 58 Area = 115877

14000 12000 10000 8000 6000 450000 400000 350000

300000 250000 200000 150000 100000 50000 0.0

d3-Acrylamide 75 58 Area = 5541493

Table 2: Results of acrylamide assay from food samples

Search | Contents | Index

View additional Thermo Scientic LC/MS application notes at: www.thermo.com/appnotes

Part of Thermo Fisher Scientic

Search | Contents | Index

Contaminants Melamine & Cyanuric Acid

Application Note: 424

Analysis of Melamine and Cyanuric Acid in Food Matrices by LC-MS/MS

QED-MS/MS Conditions: Collision Energy: 30 eV Reverse Energy Ramp (RER): 50 eV

Search | Contents | Index

MS Conditions Cyanuric Acid

Cyanuric Acid 13C3

2.86 3 Time (min)

Melamine Y = 0.0118128*X R^2 = 0.9998 W: Equal

1.0 0.8 0.6 0.4 0.2 0.0 20 40 60 ng/mL 80 100

Search | Contents | Index

Cyanuric_acid Y = 0.00790388*X R^2 = 1.0000 W: Equal 8 7 6

5 4 3 2 1 0 0 200 400 600 ng/mL 800 1000

0 100 50 0 100 50 0 100 50 0 0

Cyanuric Acid Standard

2.69 3 Time (min) 4

Search | Contents | Index

55 50 45 40 35 30 25 20 15 10 5 0 30 40 50 60 70 80 m/z 90 100 110 120 130

Search | Contents | Index

Search | Contents | Index

Laboratory Solutions Backed by Worldwide Service and Support

View additional Thermo Scientic LC/MS application notes at: www.thermo.com/appnotes

Thermo Fisher Scientic, San Jose, CA USA is ISO Certied.

Part of Thermo Fisher Scientic

Search | Contents | Index