Fast Data Acquisition Speed and
High Quantitative Performance in
Authors
Mark Sartain, Thomas Glauner,
Behrooz Zekavat, and Anabel Fandino
Agilent Technologies, Inc.
Santa Clara, CA, USA
the Simultaneous Determination
of Mycotoxins, Illegal Dyes, and
Pesticides in Spices
Application Note
Food
Introduction
Spices have been used since antiquity for the flavoring and coloring of food. There
have been many alerts from European countries on the safety of spices with regards to
different groups of contaminants. This raised the need for the simultaneous screening
and quantitation of pesticides, mycotoxins, and dyes using a single multiresidue method.
Major challenges are the complex matrixes, the large number of analytes, the diversity
of compound classes, and the concentration range that must be covered in the analysis.
This Application Note evaluates an ion-funnel triple quadrupole mass spectrometer
operated with dynamic MRM and fast polarity switching for the accurate quantitation of
an extended suite of analytes at trace-levels. We further evaluated a modified software
and firmware to optimize inter-MRM delay times, enabling high quality data at dwell
times as low as 0.5 ms.
The excellent sensitivity achieved using the Agilent 6495B Triple Quadrupole LC/MS
enables precise and accurate quantitation of pesticides, mycotoxins, and dyes with high
degrees of sample dilution, allowing reduction in matrix effects and improved method
robustness.
Experimental Instrument conditions
Agilent 1290 Infinity II UHPLC System
Sample preparation
Column Agilent ZORBAX RRHD Eclipse Plus C18, 2.1 × 150 mm, 1.8 µm
Ground black pepper and paprika (p/n 959759-902)
were obtained from a local grocery
Column temperature 40 °C
store, and extracted according to the
Injection volume 2 μL
EN 15662 QuEChERS protocol using
Agilent BondElut QuEChERS kits Mobile phase A) 5 mM ammonium formate + 0.1 % formic acid
(p/n 5982‑6650). The extracts were B) 5 mM ammonium formate + 0.1 % formic acid in methanol
further cleaned with the QuEChERS dSPE Flow rate 0.4 mL/min
Enhanced Matrix Removal—Lipid kit Gradient 95 %A hold for 0.5 minutes, to 40 %B in 3.5 minutes,
(p/n 5982‑1010), followed by a polishing to 98 %B in 17 minutes, hold for 3 minutes,
step (p/n 5982‑0102). Final extracts down to 5 %B in 0.1 minutes
were spiked at 2 ppb (10 µg/kg) with the Hold for 0.9 minutes, stop time 21.00 minutes
Post time 2 minutes
Agilent comprehensive pesticide mixture
Agilent 6495B Triple Quadrupole Mass Spectrometer
(p/n 5190-0551), mycotoxins, and dye
standards (Sigma). Spiked extracts were Ion source Agilent Jet Stream ESI
diluted 1:5, 1:10, 1:20, 1:50, and 1:100 with Polarity Positive and negative switching
acetonitrile. Gas temperature 120 °C
Drying gas (nitrogen) 17 L/min
Method design Nebulizer gas (nitrogen) 30 psi
Agilent MassHunter Optimizer software Sheath gas (nitrogen) 300 °C
was used to optimize MRM transitions Sheath flow 12 L/min
for five mycotoxins and nine dyes. At Capillary voltage 3,500/–3,500V
least two MRM transitions per compound Nozzle voltage 300/–500 V
and the corresponding MS conditions
iFunnel High/low pressure RF 150/60V
were also taken from the Agilent
Pesticide Triggered MRM (tMRM) LC/MS Scan type Dynamic MRM (dMRM)
Application Kit. Analysis was carried out Q1/Q2 resolution Unit (0.7 amu)
with positive and negative electrospray Delta EMV 200 V
ionization in dynamic MRM (dMRM) or Cell acceleration voltage 3–7 V
tMRM in a single analytical run. Major Total number of MRMs 542 (positive: 530, negative: 12)
UHPLC and dMRM MS parameters are
shown in the Instrument conditions table.
×10 6

Results and Discussion Tribenuron-methyl

2.5 396.0 & 155.1
Evaluation of optimized inter-MRM Peak area at 0.5 ms
= 0.80
delay times 2.0
Metobromuron Peak area at 5.0 ms
Counts

An MRM method was developed 1.5

259.0 & 179.0
targeting 100 transitions representing Peak area at 0.5 ms
= 0.94
Peak area at 5.0 ms
53 pesticides to assess a modified 1.0 ─ 5.0 ms
software and firmware that optimizes ─ 2.0 ms
─ 1.0 ms
inter-MRM delay times. Figure 1 shows 0.5
─ 0.5 ms
the overlaid MRM chromatograms of two
0
pesticides at four different dwell times, 8.6 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8
demonstrating the importance of using Acquisition time (min)
appropriate inter‑MRM delay times for
Figure 1. Overlaid MRM chromatograms of metobromuron and tribenuron-methyl (100 ppb) at dwell times
maintaining ion signal at short dwell
of 5.0, 2.0, 1.0, and 0.5 ms using optimized inter-MRM delay times.
times.

2
 Method development ×10 2
and performance A
0.8 MRM
An MRM method was first developed 5 Mycotoxins
with new transitions optimized for

Counts (%)
0.6 9 Dyes
mycotoxin and dye compounds of
0.4
interest. Next, the new compound
transition and observed RT data were 0.2
used to update a dMRM method targeting
> 250 pesticides. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Acquisition time (min)
The combination of UHPLC with an ×10 5 B Final dMRM method
optimized 6495B Triple Quadrupole Mass 5 Mycotoxins
Spectrometer allowed the quantitation 9 Dyes
6 253 Pesticides
of the majority of analytes at a lower
limit of quantitation (LLOQ) of 10 % of
Counts

4
their allowed maximum residue levels
(MRLs). The precision and accuracy of 2
measurements were evaluated at 13
standard concentrations ranging from an 0
LLOQ as low as 2 ppt, to the upper limit of 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
quantitation (ULOQ) of 100 ppb, and were Acquisition time (min)

calculated from five replicate injections Figure 2. Overlaid MRM chromatograms of mycotoxins and dyes (A). Overlaid MRM chromatograms from
at each level. Excellent assay precision a 10 ppb mixture of standards acquired with the final dMRM method targeting 267 total compounds (B).
(RSD% < 20 % at the LLOQ and <15 % at
the rest of the levels) as well as average
accuracy (80–125 % at the LLOQ and
85–115 % at the rest of the levels) were
obtained. Correlation coefficients (R2) for
calibration curves were higher than 0.99.

×10 7 A
LLOQ IDL
Mycotoxin Aflatoxin G2 Mycotoxin (ppt) (ppt)
0.1 5 ppt–100 ppb
Type: Linear, Origin:Ignore, Weight: 1/x Aflatoxin B1 10 3.7
Response

0.01 Aflatoxin B2 20 2.2

Aflatoxin G1 5 2.3
0.001 y = 38.853317*x + 76.262541
R2 = 0.99779842 Aflatoxin G2 5 2.6
0.0001 Ochratoxin A 500 274.5

10 100 1,000 10,000 100,000 LLOQ IDL

Concentration (pg/mL) Dye (ppt) (ppt)
×10 7
B Dimethyl yellow 50 7.9
Pesticide Monocrotophos Orange II 1,000 733.5
0.1 2 ppt–100 ppb
Type: Linear, Origin:Ignore, Weight: 1/x Rhodamine B 500 294.1
Response

0.01 Sudan I 50 24.0

Sudan II 50 8.8
0.001 y = 103.153461*x + 23.614258
R2 = 0.99814145 Sudan III 500 158.8
0.0001 Sudan orange G 500 190.5
Sudan red 7B 50 11.6
10 100 1,000 10,000 100,000 Sudan red G 100 24.1
Concentration (pg/mL)

Figure 3. Examples of the dynamic range and linearity achieved with the final dMRM method.
The tables provide LLOQs and instrument-detection-limits (IDLs) for the mycotoxins and dyes.

3
Minimizing matrix effects by Table 1. Recoveries for selected compounds calculated for different dilution ratios based on a solvent
dilution of extracts calibration. Cells shaded in green comply with requirements of SANCO/12571/ 2013.

The ability to dilute complex sample No 1:5 1:10 1:20 1:50 1:100
extracts to minimize matrix effects Analyte dilution Dilution Dilution Dilution Dilution Dilution
enables more accurate quantification, Acetamiprid 47.1 ± 3.7 88.5 ± 2.3 94.6 ± 3 99.2 ± 6.8 104.5 ± 6.4 116.7 ± 5.9
while also reducing contamination of Aflatoxin B1 31.1 ± 0.9 80 ± 1.3 90 ± 3.8 92.1 ± 7.7 110.2 ± 8.8 112 ± 3.4
the LC/MS system, thereby increasing Buprofezin 4.1 ± 0.3 16.6 ± 1.1 32.1 ± 1.2 47.3 ± 5.8 84.6 ± 9 109.8 ± 9.9
robustness. Table 1 shows the beneficial Chlorantraniliprole 8.2 ± 0.7 31.1 ± 1.1 50.9 ± 5.2 65.5 ± 10.9 94.2 ± 10.2 101.9 ± 20.4
effect of dilution, where compound
Lenacil 16.9 ± 1.8 57.7 ± 5.9 75.7 ± 5.4 82.4 ± 3.6 97.5 ± 9.2 106.9 ± 11.5
recoveries in black pepper improve as the
Methomyl 24.9 ± 1.9 51.3 ± 3.5 65.3 ± 6.5 77.1 ± 7.5 107.3 ± 10.7 113.3 ± 14.6
sample is further diluted.
Profenofos 2.6 ± 0.3 12.5 ± 0.6 20.4 ± 2.1 34.2 ± 1.7 59 ± 8.3 88.5 ± 15.5
Spices are very challenging food matrices, Propamocarb 105.9 ± 8 102.4 ± 3.4 101 ± 3 100.2 ± 2.8 114.5 ± 3.4 111.5 ± 6.8
as their complex nature results in Proquinazid 11.4 ± 0.6 42.8 ± 0.9 57.9 ± 1 68.3 ± 3.9 96 ± 11.6 111.3 ± 6.8
significant ion suppression effects. The Sudan red 7B 19.8 ± 2.5 57.4 ± 4.2 61 ± 6.9 70.5 ± 2.4 84 ± 5 81.5 ± 7.2
matrix effects were further enhanced
by our inability to leverage conventional
dispersive SPE cleanup to remove 120
interfering matrix compounds, due to Paprika

observed losses of dye compounds. 100

Black pepper
Therefore, the dilution of extracts was
critical to reduce suppression, and 80
Number of pesticides

the comparison shown in Figure 4

demonstrates that the degree of dilution
60
required varies considerably across types
of spices.
40
Black pepper was identified as the most
challenging spice matrix we analyzed. 20
Figure 5 shows that a 1:100 dilution of
the extract in acetonitrile was required to 0
achieve acceptable recoveries for >90 % No dilution 1 to 5 1 to 10 1 to 20 1 to 50 1 to 100
of the compounds. The innate sensitivity
Figure 4. Acceptable recoveries (70–120 %) for 194 compounds were compared for paprika and
of the 6495B LC/MS system enabled
black pepper extract dilutions.
analysis of the desired 1:100 black pepper
dilution level while still maintaining
the ability to detect the majority of the 200
compounds with an area RSD < 20 %. No Dilution
180 1 to 5 Passes
1 to 10 SANCO
160 Specifications
1 to 20
140
1 to 50
Number of pesticides

120 1 to 100

100

80

60

40

20

0
<10 % 10–30 % 30–70 % 70–120 % >120 %

Figure 5. Histogram of recoveries for pesticides, mycotoxins, and dyes spiked into black pepper at 2 ppb
(10 µg/kg), and diluted with acetonitrile. Two-hundred five compounds showed strong ion suppression,
and significantly better recoveries were observed after dilution.

4
Confirmation with triggered based on the presence of a single
MRM (tMRM) primary transition. The full compound
spectrum was compared against a
While evaluating matrix effects with
spectral library created from the pure
the dMRM method, we found > 20
standards. These same data were also
pesticides and mycotoxins present in the
used for quantitation. In the examples
paprika blank extract (without spike-in
shown in Figure 6, Ochratoxin A and
of standards). For further confirmation
Aflatoxin B1 were calculated at 28 µg/kg
of these compounds in this control
and 8.1 µg/kg in the 1:20 diluted paprika
sample, we applied a tMRM method
extract.
where up to six transitions were acquired

×10 4 ×10 3
Ochratoxin A Lib Match Score = 100.0
Ratio = 48.9 (101.5 %)
3.4 A 1.0 239.2
11.70 2.6
3.2
3.0 2.4 0.8
2.8 2.2 0.6
2.6 2.0
2.4 0.4
1.8
2.2 0.2
2.0
1.6 136.9 192.9

Counts
1.4 404.1
1.8 0
1.6 1.2
-0.2 192.9
1.4 1.0
1.2 0.8 -0.4
1.0 0.6 -0.6
0.8 0.4 -0.8
0.6 0.2
0.4 -1.0
0 239.2
0.2
-0.2 -1.2
0
11.65 11.7 11.75 11.8 11.85 11.2 11.4 11.6 11.8 12.0 150 200 250 300 350 400

Aflatoxin B1 ×10 4
×10 3
Lib Match Score = 98.7
285.0
7.64 1.0
B 4.0 Ratio = 87.1 (98.6 %) 241.2
4.5
0.8
4.0 3.5
0.6
3.5 3.0
0.4
3.0 2.5 0.2
184.9 213.0 269.9
Counts

313.1
2.5 2.0 0

2.0 1.5 -0.2 184.9 269.9

213.0
-0.4
1.5 1.0
-0.6
1.0 0.5
-0.8
0.5 0 241.2
-1.0
285.0
0 -0.5
7.60 7.65 7.70 7.75 7.80 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 180 200 220 240 260 280 300

Figure 6. Triggered MRM data for Ochratoxin A (A) and Aflatoxin B1 (B) in paprika extract, displaying compound chromatograms, qualifier details, and reference
library matching (left to right).

5
Conclusions
• Optimized inter-MRM delay times
allowed the use of MRM dwell
times as short as 0.5 ms with
minimum analyte signal losses.

• Mycotoxin and dye compounds

were incorporated into a
UHPLC/MS/MS pesticide method
for the determination of more than
260 compounds.

• The enhanced sensitivity

achieved using the Agilent 6495B
LC/MS system enabled precise
and accurate quantitation of
compounds with very high degrees
of spice extract dilution, provided
reduced matrix effects, and
improved method robustness.

• The value of tMRM acquisition

for confirmation of mycotoxin
compounds in spices was
demonstrated.

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© Agilent Technologies, Inc., 2016

Published in the USA, December 15, 2016
5991-7300EN