Determinación de Acrilamida en Dieferentes Materiales

Food T Est ing
A p p l i c at ion Not e boo k

[ TAble of Contents ]
INTRODUCTION
Solid-Phase Extraction Strategies......................................................................................................................................................... 5

Sep-Pak and Oasis Cartridges for Rapid Sample Preparation............................................................................................................. 7
Sample Preparation Solutions............................................................................................................................................................... 9
Integrated Solutions............................................................................................................................................................................... 12
Separation Solutions.............................................................................................................................................................................. 13
Veterinary Drugs in Food.....................................................................................................16
β2-Agonists in Pork and Pig Liver Tissues............................................................................................................................................ 17
Chloramphenicol in Honey..................................................................................................................................................................... 19
Dexamethasone in Pork.......................................................................................................................................................................... 20
Enrofloxacin (Baytril®) in Chicken......................................................................................................................................................... 21
Nitrofurans in Honey.............................................................................................................................................................................. 23
Nitrofurans in Tissues............................................................................................................................................................................ 25
Penicillin G in Pork................................................................................................................................................................................. 27
Penicillins, Tetracyclines, and Sulfonamides in Milk........................................................................................................................... 28
Spiramycin in Pork................................................................................................................................................................................. 29
Streptomycin in Honey.......................................................................................................................................................................... 31
Sulfonamide Antibacterials in Milk....................................................................................................................................................... 32
Tetracyclines and Sulfonamides in Milk................................................................................................................................................ 34
Tetracyclines in Animal Tissues............................................................................................................................................................ 35
Tetracyclines in Honey........................................................................................................................................................................... 36
Pesticides and Contaminants................................................................................................38
Acrylamide in Fried Potato Products...................................................................................................................................................... 39
Aflatoxins in Produce Samples............................................................................................................................................................... 40
Carbamates in Fruits and Vegetables..................................................................................................................................................... 41
Malachite Green in Fish (HPLC/UV )........................................................................................................................................................ 42
Malachite Green in Fish (UPLC/MS/MS)................................................................................................................................................. 43
Melamine and Cyanuric Acid in Infant Formula using HPLC................................................................................................................. 45
Melamine and Cyanuric Acid in Infant Formula using UPLC................................................................................................................. 47
Melamine and Cyanuric Acid in Infant Formula using LC/MS/MS......................................................................................................... 49
Microcystins in Natural Waters............................................................................................................................................................... 51
[3]
[ TAble of Contents ]
Multi-Residue Analysis of Pesticides in Grain and Beans...................................................................................................................... 52
Multi-Residue Analysis of Pesticides in Vegetables and Fruits............................................................................................................. 53
Multi-Residue LC/MS/MS Determination of 52 Non-Gas Chromatography-Amenable

Pesticides and Metabolites in Fruits and Vegetables............................................................................................................................. 54
Multi-Residue Analysis of Pesticides by QuEChERS in:
Avacado by GC/MS........................................................................................................................................................................... 55
Avocado by UPLC/MS/MS ............................................................................................................................................................... 56
Avocados and Grapes by GC/MS...................................................................................................................................................... 57
Baby Food by UPLC/MS/MS............................................................................................................................................................ 58
Flour by GC/MS................................................................................................................................................................................ 60
Flour by UPLC/MS/MS..................................................................................................................................................................... 61
Grapes by GC/MS............................................................................................................................................................................. 62
Grapes by UPLC/MS/MS.................................................................................................................................................................. 63
Oranges by GC/MS........................................................................................................................................................................... 64
Oranges by UPLC/MS/MS................................................................................................................................................................ 65
Rolled Oats by GC/MS..................................................................................................................................................................... 66
Rolled Oats by UPLC/MS/MS........................................................................................................................................................... 67
Teas by UPLC/MS/MS....................................................................................................................................................................... 68
Vegetables and fruits by UPLC/MS/MS........................................................................................................................................... 69
Paraquat and Other Quaternary Ammonium Compounds in Water....................................................................................................... 70
Patulin in Apple Juice............................................................................................................................................................................. 71
PFOS and Related Compounds in Water and Tissue............................................................................................................................... 72
Propham in Potatoes by GC/MS.............................................................................................................................................................. 74
Propham in Potatoes by LC/MS............................................................................................................................................................... 76
Sudan Dyes in Chilli Oil and Powder...................................................................................................................................................... 77
Sudan Dyes in Fresh Chillis..................................................................................................................................................................... 78
FOOD TESTING AND QUALITY CONTROL (QC)...............................................................................79
Amino Acids in Animal Feed Hydrolysates........................................................................................................................................... 80
Amino Acids in Tea................................................................................................................................................................................. 82
Food Sugars in Bran with Raisin Cereal.................................................................................................................................................. 83
Food Sugars in Milk................................................................................................................................................................................ 84
Ginsenoside Rb1 in Ginseng Root Powder Extract Nitrofurans in Tissues.............................................................................................. 85
Compound Index.....................................................................................................................86
[4]
So l id - P ha s e E x t r ac t ion S t r at egi e s
Retention-Cleanup-Elution Strategy SPE Procedure Steps
As the sample is loaded onto the cartridge, the analytes of interest The following section describes the steps involved in a complete
are retained by the sorbent. If needed, an optimized series of solid-phase extraction procedure:
washes are used to remove matrix interference from the cartridge.
A strong solvent is used to elute the analytes from the cartridge.
Sample enrichment results when the final elution volume is smaller 1. Pretreatment
than the load volume.
Solid samples (soil, tissue, etc.)
Load Sample Step Step Step  Shake, sonicate, or use soxhlet extraction.
(Black) Elute 1 Elute 2 Elute 3
- Extract sample with polar organic solvent (methanol,
acetonitrile) for polar analytes.
- Extract sample with organic solvent and drying agent
(dichloromethane, acetone) for non-polar analytes
and multi-residue extraction.
Stationary
NOTE: Different
Phase
strength solvents Non-aqueous liquid
Particles
can be used to
separate the dyes.
 If the sample is soluble in water, dilute it with water for
reversed-phase SPE.
One cartridge can separate all three dyes  If the sample is soluble in hexane, dilute it with hexane for SPE.
 Alternatively, evaporate the solvent and exchange to hexane.
Pass-Through Cleanup Strategy Wastewater
Pass-through cleanup methods optimize matrix retention while the  Filter or centrifuge as necessary.
analytes of interest pass-through the cartridge unretained. No sample
enrichment occurs during the solid-phase extraction (SPE) step. 2. Condition
1. Sample is passed through For reversed-phase sorbents, preconditioning of the sorbent

sorbent and collected. with an organic solvent, such as methanol, acetonitrile,
 No sample enrichment. isopropanol, or tetrahydrofuran, is usually necessary to obtain
reproducible results. Without this step, a highly aqueous solvent
2. Matrix interferences are cannot penetrate the hydrophobic surface and wet the sorbent.
retained on sorbent. Thus, only a small fraction of the sorbent surface area would be
available for interaction with the analyte. For the same reason,
it is important not to let silica-based SPE cartridges dry out
between the solvation step and the addition of the sample.
A complete preconditioning of a reversed-phase cartridge includes
the solvation step and an equilibration with a low-strength solvent,
such as water or buffer.
Pass-Through
[5]
So l id - P ha s e E x t r ac t ion S t r at egi e s
3. Load 4. Wash
When the analytes of interest are not retained by the sorbent, The wash steps are designed to remove unwanted matrix
this is called analyte breakthrough. For some methods, such as components that remain from the loading step. The ideal wash
pass-through cleanup, analyte breakthrough is desirable and solvent removes only the matrix while keeping the analytes
is maximized for those specific methods. However, in all other bound to the sorbent. For complex samples this is impossible,
cases, analyte breakthrough is unwanted and contributes to poor so the wash steps are optimized using pH, solvent strength, and
recovery and method reproducibility. Breakthrough occurs when: solvent polarity to remove as much matrix as possible while
 T here is too high an organic concentration in the load maintaining acceptable analyte recovery.
solution for very polar analytes. Dilute sample at least 1:1
with water or buffer prior to loading.
5. Elute
 The analytes are bound to proteins, they may pass through
the sorbent. Ensure that analytes are not bound to proteins Once the interferences are washed off the cartridge, a strong
by acidifying or basifying the sample. solvent is introduced to elute the analytes of interest. T he
 Sorbent is overloaded by the matrix component. Therefore, it volume and flow rate of the eluting solvents should be precisely
is important to choose the correct sorbent mass (see Tables 1 controlled as in the load step to ensure reproducible results.
and 2). Refer to Table 3 for guidelines on various types of separation
 The flow rate of the load step is too fast. There is not mechanisms and recommended solvents.
enough contact time between the analytes and the sorbent.
Table 2. Choice of Sep-Pak® Cartridges Based on Sample Size
Look at the drops and adjust the vacuum so that you see
Sample Size Sep-Pak Cartridge
discrete droplets, not a stream of liquid.
10–100 mL 3 cc/200 mg or 6 cc/500 mg
Table 1. Choice of Oasis® Cartridges Based on Sample Size 100–500 mL 3 cc/200 mg or 6 cc/500 mg
Sample Size Oasis Cartridge 500–1000 mL 6 cc/500 mg (LP) or 6 cc/1 g
1–10 mL 1 cc/30 mg or 3 cc/60 mg

10–100 mL 3 cc/60 mg or 6 cc/200 mg
100–500 mL 6 cc/200 mg or 6 cc/500 mg (LP*)
500–1000 mL 6 cc/500 mg (LP) or 12 cc/1 g (LP)
* LP=large particules (60 µm)
Table 3. Guidelines on the Various Types of Separation Mechanisms
Reversed Phase Normal Phase Ion Exchange

Low to moderate polarity/
Analyte Moderate to highly polar/uncharged Charged or ionized
hydrophobic
Matrix Aqueous Non-polar organic solvent Aqueous/low ionic strength
1. Solvate polar organic

Condition/Equilibrate Non-polar organic Low ionic strength buffer
2. Water
Wash Aqueous/buffer Non-polar Low ionic strength buffer
Increase polar organic content Increase moderate to high polarity organic Stronger ionic strength buffers or pH to
Elute
in steps content in steps neutralize the charge
[6]
S e p- pa k an d OAsis C a rt ridg e s fo r r a p id sam p l e p r e pa r at ion
Sorbent P rop e rties and T y pic a l Applic ations
Separation Mode
Sorbent Properties & Applications

Reversed Phase
Hydrophobic, silica-based bonded phase used to adsorb analytes of even weak hydrophobicity from aqueous
solutions; typical applications include drugs and their metabolites in serum, plasma or urine, desalting of
C18
peptides, trace organics in environmental water samples, organic acids in beverages; similar to reversed-
phase HPLC columns in elution behavior.
Strongly hydrophobic silica-based bonded phase; trifunctional bonding chemistry gives it increased hydrolytic
tC18
stability over C18; applications similar to those of C18.
Moderately hydrophobic, silica-based bonded phase; use for methods requiring less retention than C18. Typical
C8
applications include drugs and their metabolites in serum, plasma or urine, peptides in serum and plasma.
Silica-based bonded phase with low hydrophobicity; use for methods requiring less retention than C8;
tC 2
applications are similar to C18 and C8.
Strongly hydrophobic, water-wettable polymer with unique hydrophilic-lipophilic balance. Maintains high
retention and capacity even it it runs dry after conditioning. Stable in organic solvents. Typical applications
Oasis HLB include drugs and metabolites in biofluids, isolation of peptides and oligonucleotides, high-throughput
biopolymer desalting, trace organics, priority pollutants, endocrine disruptors, and PMHLW official food
methods for antibiotics and pesticides.
Normal or Reversed Phase

Silica-based, moderately polar, bonded phase with weakly basic surface; can be used as a polar sorbent, like
silica, with different selectivity for acidic/basic analytes or as weak anion exchanger in aqueous medium
Aminopropyl [NH2]
below pH 8; applications include phenols and phenolic pigments, petroleum fractionation, saccharides, drugs,
and drug metabolites.
Silica-based bonded phase of low hydrophobicity; can be used as less polar alternative to silica in normal-phase
Cyanopropyl [CN] applications or as less hydrophobic alternative to C18 or C8 in reversed-phase applications; typical applications
include drugs, drug metabolites, and pesticides.
Silica-based, moderately polar, bonded phase with neutral surface; can be used as an alternative to silica in
normal-phase applications, where the acidic character of silica is undesirable or as very weakly interacting
Diol
hydrophobic phase in aqueous media; applications include antibiotics from cosmetics; isolation of proteins or
peptides by hydrophobic-interaction chromatography [HIC].
Normal Phase
Polar sorbent, used primarily to adsorb analytes from non-polar solvents like hydrocarbons, chloro- or
fluoro-substituted hydrocarbons or less polar esters and ethers; elution with more polar solvents like
Silica polar esters, ethers, alcohols, acetonitrile, or water; the binding mechanism can be hydrogen bonding or
dipole-dipole interaction; silica can also be used in aqueous medium as a cation exchanger of intermediate
strength, or as a support for liquid-liquid partition separations with a polar stationary phase.
Similar in use to silica; available in acidic [A], neutral [N], and basic [B] grades; highly active, polar surface;
alumina also exhibits specific interactions with the π-electrons of aromatic hydrocarbons, making it useful
Alumina A, N, B
for applications like crude oil fractionation; acidic and basic grades can also be used as low-capacity ion
exchangers, which, unlike polymer-based exchangers, are unaffected by high energy, radioactive materials.
Highly active, polar sorbent with a slightly basic surface for adsorption of low to moderate polarity species
from nonaqueous solutions; specifically designed for the adsorption of pesticides using official Association
Florisil ®
of Analytical Communities (AOAC) and Environmental Protection Agency (EPA) methods; other applications
include polychlorinated biphenyls in transformer oil.
[7]
S e p- pa k an d OAsis C a rt ridg e s fo r r a p id sam p l e p r e pa r at ion
Ion Exchange
Silica-based, hydrophilic, strong anion exchanger with large pore size; extraction of anionic analytes in
aqueous and non-aqueous solutions; due to the large pore size, it is excellent for the isolation of anionic
Accell™ Plus QMA
proteins, eg., immunoglobulins, enzymes; other applications include the removal of acidic pigments from
wines, fruit juices and food extracts, isolation of phenolic ccompounds, and peptide pool fractionations.
Silica-based, hydrophilic, weak cation exchanger with large pore-size; extraction of cationic analytes in
Accell Plus CM aqueous and non-aqueous solutions; due to the large pore-size, it is excellent for the isolation of cationic
proteins; other applications include pesticides, herbicides, and steroids.
Waters patented mixed-mode, reversed-phase/strong cation-exchange, water-wettable polymer, highly selective
for bases, used to isolate basic, neutral and acidic compounds with high recoveries. Highly cross-linked polymer
Oasis MCX
is stable in organic solvents. Typical applications include basic drugs from biofluids and tissue extracts; drug
monitoring: screening, identification, confirmation, quantitation and pesticides and herbicides.
Waters patented mixed-mode, reversed-phase/weak cation-exchange, water-wettable polymer used to retain
and release strong bases [e.g., quaternary amines]. Highly cross-linked polymer is stable in organic solvents.
Oasis WCX Typical applications include strongly basic compounds in biofluids and tissue extracts, drug monitoring
(screening, identification, confirmation, quantitation), and Japan Ministry of Health, Labor and Welfare
(JPMHLW) official method for streptomycin and dihydrostreptomycin in vegetable crops.
Waters patented mixed-mode, reversed-phase/strong anion-exchange, water-wettable polymer, highly selective
for acids, used to isolate acidic, neutral and basic compounds with high recoveries. Highly cross-linked polymer
Oasis MAX is stable in organic solvents. Typical applications include acidic compounds and metabolites in biofluids and
tissue extracts, drug monitoring: screening, identification, confirmation, quantitation, food additives, and
contaminants [e.g., Sudan Red].
Waters patented mixed-mode, reversed-phase/weak anion-exchange polymer used to retain and release
strong acids [e.g., sulfonic acids]. Highly cross-linked polymer is stable in organic solvents. Typical applica-
Oasis WAX
tions include strongly acidic compounds and metabolites in biofluids and tissue extracts, drug monitoring
(screening, identification, confirmation and quantitation), and emerging contaminants [e.g., perfluoroacids].
Silica-based phase containing primary and secondary amines with similar selectivity to Aminopropyl, but with
PSA
higher pKa’s and increased ion exchange capacity. Strong affinity for fatty acids, polar pigments, and sugars.
Product for Specific Applications

Sorbent Properties & Applications
Two-layer sorbent bed used for pesticide cleanup in food matrices prior to gas chromatography (GC) and liquid
Carbon Black/Aminopropyl chromatography (LC) analysis. Typical applications include JPMHLW official methods for pesticides in food and
JPMHLW official method for propham.
Very hydrophobic copolymer designed for multi-residue pesticide analysis in water samples. Typical
Sep-Pak PS2 applications include JPMHLW official methods for pesticides in water and JPMHLW official methods for
pesticides in food.
Highly hydrophobic, low ash content, activated carbon used to remove or enrich very polar organic molecules
Sep-Pak AC2 from water. Typical applications include JPMHLW official method for 1,4-dioxane analysis in water and pesticides,
herbicides, especially highly polar small molecules.
Sep-Pak Dry Anhydrous sodium sulfate, a high capacity desiccant used to remove residual water from extracts.
Two layered sorbent bed used for pesticide clean-up in food matrices prior to GC analysis. PSA provides
Carbon Black/PSA
an alternative selectivity compared to Aminopropyl.
[8]
Sam p l e p r e pa r at ion so lu t ions
 Traditional SPE phases

 Many product formats
 Many literature references and validated methods available
 Ultra low extractables from Certified Sep-Pak cartridges
 Reduced interferences and increased sensitivity using
Certified Sep-Pak cartridges
The convenient format and features of Sep-Pak cartridges overcome many of the procedural difficulties of traditional column
liquid-solid extraction and allow the enormous benefits of solid-phase extraction to be realized. Adsorbent and packed bed quality,
reproducibility, versatility, and ease-of-use are assured through intelligent design, production control, and quality testing.
S e p- Pak C a rtridg e S e pa ration Guid elin es

Chromatographic Mode Normal Phase Reversed Phase Ion Exchange
Silica, Florisil, Alumina, C18, tC18, C8, Diol, Accell Plus QMA,
Separation Characteristic
Diol, NH2, CN PoraPak® RDX, NH2, CN Accell Plus CM, NH2
Packing Surface Polarity High Low High
Typical Solvent Polarity Range Low to medium High to medium High
Typical Sample Loading Solvent Hexane, toluene, dichloromethane Water with low ionic strength Water, buffers
Ethyl acetate, acetone, Methanol, acetonitrile, Buffers, salt solutions with high ionic
Typical Elution Solvent
acetonitrile dichloromethane strength
Least polar sample Most polar sample Most weakly ionized sample
Sample Elution Order
components first components first components first
Increase ionic strength
Solvent Change Required to Elute
Increase solvent polarity Decrease solvent polarity or increase pH (anion exchange)
Retained Compounds
or decrease pH (cation exchange)
Certified Sep-Pak Cartridges
One key to success in developing a rugged solid-phase extraction method is the reproducibility of the
solid-phase sorbent. Waters takes significant steps to ensure that the solid-phase chemistry does not
change between batches. Each batch of Certified Sep-Pak cartridge stationary phase undergoes a variety
of stringent analytical checks and functional chromatographic testing to ensure that each application will
perform identically on every batch of sorbent. From start to finish, the production of Certified Sep-Pak
cartridges is done in an ISO 13485 and ISO 9002-certified facility under strict GLP and cGMP guidelines.
[9]
SAM P LE P RE PARATION SOLUTIONS
 Waters premium brand for SPE
 Cartridges, plates, and µElution technology
 Co-polymer, water wettable, reproducible
 Outperforms C18 for polar bases
Oasis 2x4 Method—The fastest, simplest, and cleanest approach to SPE method development
 Characterize your analyte (acid, base, pKa)
 Choose 1 of 5 Oasis sorbents
HLB: Hydrophilic-Lipophilic-Balanced reversed-phase sorbent for acids, bases and neutrals

MCX: Mixed-mode Cation eXchange sorbent for bases
MAX: Mixed-mode Anion eXchange sorbent for acids
WCX: Mixed-mode Weak Cation eXchange sorbent for strong bases and quaternary amines
WAX: Mixed-mode Weak Anion eXchange sorbent for strong acids
 Apply designated protocol (1 or 2)  Analyze chromatograms and SPE recoveries
[ 10 ]
SAM P LE P RE PARATION SOLUTIONS
 Easy and straightforward method to implement, requiring little training
 Conforms to the AOAC and CEN official methods for determining

pesticide residues in fruits in vegetables
 Cost effective
 Reliable, high quality product in a simple kit format
DisQu E DIS P E RSIV E SAM P L E P R E PA RAT ION K IT
Dispersive sample preparation, commonly referred to as “QuEChERS”, is a simple and straightforward sample preparation technique
suitable for multi-residue pesticide analysis in a wide variety of food and agricultural products. Waters DisQuE ™ Dispersive Sample
Preparation Kit contains conveniently packaged centrifuge tubes with pre-weighed sorbents and buffers designed for use with AOAC
and European Committee for Standardization (CEN) official methods. DisQuE dispersive sample preparation is a well proven, high
throughput sample preparation method for a wide array of pesticide in produce samples.*
*Reference: Lehotay, J.AOAC Int. 90(2) 2007, 485-520.
FILT E RS
Filtration provides immediate protection for analytical system components and

minimizes downtime. In partnership with Pall Life Sciences, Waters offers filtration
products that are Certified for Compliance, which means they have been designed and
developed to comply with regulatory and quality objectives.
C E RT IFI ED V IA L S
Sample vials are a critical part to sample preparation. Ensure that the vials you use do not introduce
unwanted contaminants and interferences. Waters provides a wide selection of certified vials tested to
maximize sensitivity and improve detection limits for LC/UV/MS and LC/MS analysis. Do not compromise
your test results; avoid ghost peaks, dislodged septa, and damaged needles.
[ [1111] ]
Int e rg r at e d so lu t ions
The UPLC® amino acid analysis solution consists of:
 Waters ACQUITY UPLC® system and tunable UV detector
 Full system and application level support documentation
 Application-specific performance qualification
 Connections INSIGHT® remote, intelligent services
 Empower™ 2 software’s pre-configured projects, methods, and report formats
 AccQ•Tag™ Ultra derivatization c hemistry including column, reagents, and eluents
AMINO AC ID ANA LYSIS
Amino acid composition is a critical component of the nutritional value of foods and feeds. Qualitative and quantitative amino acid
analysis is used to determine the concentration and identity of a protein, or to confirm the origin of natural products based on the free
amino acid content of a particular commodity. When used for food safety testing, amino acid analysis can determine protein deficiencies
in processed food and to detect food adulteration that masks true protein content.
C A RBAMAT E ANA LYSIS K IT
Containing a Waters Carbamate column, Oasis HLB cartridges, vials, and Reference
Standards, this kit is optimized to simplify your analysis while increasing your
confidence in the results.
SOF T D RINK ANA LYSIS K IT
The soft drink mobile phase and soft drink standards separate caffeine, aspartame, benzoic acid, and sorbic acid.
MEL AMIN E ANA LYSIS PAC KAGES
Based on United States Food and Drug Administration (US FDA) Laboratory Information
Bulletin No. 4422, these packages offer a comprehensive solution for screening Melamine
and Melamine-related compounds in foods, including infant formula and dairy products.
Available in both HPLC and UPLC formats.
[ [1212] ]
S E PARATION SOLUTIONS
Column S el ection Guid e
Waters is committed to material sciences and, with our ongoing research into HPLC and UPLC column chemistries, we continue to develop
ground-breaking column technologies. As scientific challenges evolve, Waters meets these changing needs with
new column innovations.
XSelect™
Selectivity Features: General purpose reversed-phase column that offers excellent pH stability and rapid mobile-phase
re-equilibration for method development. Charged Surface Hybrid (CSH) technology enables superior peak shape and
C18 increased loading capacity for basic compounds.
Bonding: Trifunctional C18 ligand, fully end-capped, bonded to a CSH particle substrate.
Selectivity Features: General purpose alternative selectivity ligand that provides pi-pi interactions with polyaromatic
CSH compounds, while maintianing excellent reproducibility at pH extremes. CSH technology enables superior peak shape and
Phenyl-Hexyl increased loading capacity for basic compounds..
Bonding: Trifunctional C6 Phenyl ligand, fully end-capped, bonded to a CSH particle substrate.
Selectivity Features: General purpose column that provides a very high degree of analyte selectivity, especially
CSH when using low-pH mobile phases. CSH technology enables superior peak shape and increased loading capacity for
Fluoro-Phenyl basic compounds.
Bonding: Trifunctional propyl fluorophenyl ligand, non-endcapped, bonded to a CSH particle substrate.
Selectivity Features: High performance C18 chemistry, increased retention, superior peak shape, resists acid hydrolysis at
HSS C18 low pH. Designed for UPLC separations where silica-based C18 selectivities are desired.
Bonding: High coverage trifunctional C18, fully endcapped, bonded to High Strength Silica (HSS) HPLC particle substrate.
Selectivity Features: Unique, non-endcapped C18 chemistry designed specifically for method development scientists.
Offers unique Selectivity for Bases (SB) when operating under low pH conditions and transferability between UPLC and
HSS C18 SB
HPLC separations.
Bonding: Intermediate coverage trifunctionally bonded C18, no endcapping, bonded to HSS HPLC particle substrate.
Selectivity Features: Aqueous mobile-phase compatible HPLC column designed for extreme retention.
HSS T3 Combines polar compound retention with transferability between UPLC and HPLC separations.
Bonding: T3 (C18) bonding and endcapping, bonded to HSS HPLC particle substrate.
XBridge™
Selectivity Features: General purpose column ideally suited for method development due to extreme pH stability and
C18 applicability to the broadest range of compound classes.
Bonding: Trifunctional C18, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate.
Selectivity Features: Alternate selectivity as compared to straight chain C18, particularly with phenolic analytes.
Shield RP18 Compatible with 100% aqueous-phase composition.
Bonding: Monofunctional embedded polar C18, fully endcapped, bonded to substrate.
C8 applicability to the broadest range of compounds classes.
Bonding: Trifunctional C 8, fully endcapped, bonded to BEH substrate.
Selectivity Features: Excellent method development column for alternate selectivity, particularly for polyaromatic compounds.
Phenyl Unique level of pH stability for a phenyl-bonded phase.
Bonding: Trifunctional C 6 phenyl, fully endcapped, bonded to BEH substrate.
Selectivity Features: Excellent for retention of very polar, basic, water-soluble analytes. Specifically designed and
HILIC tested for HILIC separations using mobile phases containing high concentrations of organic solvent.
Bonding: Unbonded BEH substrate.
[ [1313] ]
XBridge™ continued
Selectivity Features: Rugged HILIC stationary phase designed to separate a wide range of very polar compounds.
Especially good at separating carbohydrates (saccharides) using high concentrations of organic modifier, elevated tem-
Amide
perature and high pH. Compatible with all modern detectors including MS, ELSD, UV and Fluorescence.
Bonding: Trifuncional amide bonded to BEH substrate.
Atlantis®
Selectivity Features: Retention of polar compounds, compatible with 100% aqueous mobile phases, superior stability
T3 under low pH conditions. Specifically designed for enhanced retention of polar analytes.
Bonding: T3 (C18) bonding and endcapping, bonded to high purity silica substrate.
HILIC tested for HILIC separations using mobile phases containing high concentrations of organic solvent.
Bonding: Unbonded high purity silica substrate.
Selectivity Features: Retention of polar compounds. Designed for compatibility with 100% aqueous mobile phases.
C18
Bonding: Difunctional C18 bonding, fully endcapped, bonded to high purity silica substrate.
SunFire™
Selectivity Features: General purpose method development column. Very high loading capacity, particularly for basic
C18 analytes in low pH mobile phases. Ideally suited for purification and impurity profile assays.
Bonding: Difunctional C18, fully endcapped, bonded to high purity silica substrate.
Selectivity Features: General purpose method development column. Very high loading capacity, particularly for basic
C8 analytes in low pH mobile phases. Less hydrophobic, therefore, less retentive than C18 for most analytes.
Bonding: Difunctional C 8, fully endcapped, bonded to high purity silica substrate.
ACQUITY UPLC®
Selectivity Features: General purpose reversed-phase column that offers excellent pH stability and rapid mobile-phase
re-equilibration for method development. Charged Surface Technology (CSH) technology enables superior peak shape and
CSH C18
increased loading capacity for basic compounds.
Bonding: Trifunctional C18 ligand, fully end-capped, bonded to a CSH particle substrate.
Selectivity Features: General purpose alternative selectivity ligand that provides pi-pi interactions with polyaromatic
CSH compounds, while maintaining excellent reproducibility at pH extremes. CSH technology enables superior peak shape and
Phenyl-Hexyl increased loading capacity for basic compounds.
Bonding: Trifunctional C 6 phenyl ligand, fully end-capped, bonded to a CSH particle substrate.
Selectivity Features: General purpose column that provides a very high degree of analyte selectivity, especially when
CSH using low-pH mobile phases. CSH technology enables superior peak shape and increased
Fluoro-Phenyl loading capacity for basic compounds.
Bonding: Trifunctional propyl fluorophenyl ligand, non-endcapped, bonded to a CSH particle substrate.
BEH C18 applicability to the broadest range of compound classes.
Bonding: Trifunctional C18, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate.
Selectivity Features: Alternate selectivity as compared to straight chain C18, particularly for phenolic analytes.
BEH Shield Compatible with 100% aqueous-phase composition.
RP18
Bonding: Monofunctional embedded polar C18, fully endcapped, bonded to BEH substrate.
[ 14 ]
ACQUITY UPLC® continued

BEH C 8 applicability to the broadest range of compounds classes.
Bonding: Trifunctional C 8, fully endcapped, bonded to BEH substrate.
Selectivity Features: Excellent method development column for alternate selectivity, particularly in regard to polyaro-
BEH Phenyl matic compounds. Unique level of pH stability for a phenyl-bonded phase.
Bonding: Trifunctional C 6 phenyl, fully endcapped, bonded to BEH substrate.
BEH HILIC tested for HILIC separations using mobile phases containing high concentrations of organic solvent.
Bonding: Unbonded BEH substrate.
Selectivity Features: Ultra performance C18 chemistry, increased retention, superior peak shape, resists acid hydrolysis
BEH HSS C18 at low pH. Designed for UPLC separations where silica-based C18 selectivities are desired.
Bonding: High coverage trifunctional C18, fully endcapped, bonded to High Strength Silica (HSS) UPLC particle substrate.
Selectivity Features: Rugged HILIC stationary phase designed to separate a wide range of very polar compounds.
Especially good at separating carbohydrates (saccharides) using high concentrations of organic modifier, elevated
BEH Amide
temperature and high pH. Compatible with all modern detectors including MS, ELSD, UV and Fluorescence.
Bonding: Trifunctional amide bonded to BEH substrate.
Selectivity Features: Ultra performance C18 chemistry, increased retention, superior peak shape, resists acid hydrolysis
HSS C18 at low pH. Designed for UPLC separations where silica-based C18 selectivities are desired.
Bonding: High coverage trifunctional CC18, fully endcapped, bonded to HSS UPLC particle substrate.
Selectivity Features: Unique, non-endcapped C18 chemistry designed specifically for method development scientists.
HSS C18 SB Offers unique Selectivity for Bases (SB) when operating under low pH conditions.
Bonding: Intermediate coverage tri-functionally bonded C18, no endcapping, bonded to HSS UPLC particle substrate.
Selectivity Features: Aqueous mobile-phase compatible UPLC column designed for extreme retention. Combines polar
HSS T3 compound retention with UPLC efficiencies and performance.
Bonding: T3 (C18) bonding and endcapping, bonded to HSS UPLC particle substrate.
FOOD TESTING SPECIALITY COLUMNS
In addition to a complete selection of UPLC and HPLC column chemistries, Waters also provides
columns optimized for specific food testing analysis. These columns are ideal for fermentation
analysis, organic acids, alcohols, and carbohydrates, triglycerides and cholesterol analysis, and fatty
acid analysis.
GUARD COLUMNS
VanGuard™ Pre-columns, Sentry™ guard columns, and Guard-Pak™ inserts prolong column lifetime by
removing contaminants from the sample, giving you enhanced reproducibility and performance. They are
packed with the same high performance stationary phases used in Waters analytical columns.
[ 15 ]
Veterinary Drugs in Food
Antibiotics are given to animals to prevent or treat diseases. When trace amounts of antibiotics show up
in meat, milk, and other foods, there is concern of developing antibiotic-resistant strains of diseases.
Some of the applications in this section cover antibiotics in certain foods that are banned outright.
For example, the presence of chloramphenicol residue in honey would indicate the improper use the
antibiotic in the bee keeping industry.
Other applications in this section are for monitoring antibiotic residues for possible future regulation of
their proper use.
β2 -Agonis t s in p o r k an d p ig l iv e r t issu e s
INTRODUCTION SPE Procedure
β2-Agonists, veterinary drugs such as albuterol, are used to Oasis® MCX, 3 cc/60 mg
force pigs to mature faster with a higher amount of lean meat.
Trace levels of β2-Agonists can cause palpitation, headaches, Condition/Equilibrate:
A. 2 mL methanol
and even death in heart patients. β2-Agonists have been B. 2 mL water
banned as growth promoters in pork production.
Load:
5 mL sample
Pretreatment
1. Add 8 mL 0.2 M sodium acetate (pH 5.2) to 2 g of sample. Wash:

A. 2 mL water
Homogenize and take out supernatant. Add 50 μL B. 2 mL (2:98, v/v) formic acid in water
β-Glucuronidase/arylsulfatase and hydrolyze at 37 °C overnight.
Dry cartridge by vacuum
2. Shake the hydrolysate for 15 minutes. Centrifuge at 5000 rpm
for 10 minutes and take out 4 mL supernatant.
Elute:
3. Add 100 μL of 10 ng/mL standards (clenbuterol-D9, 2 mL 5% ammonia solution in methanol
salbutamol-D3) and mix.

Evaporate to dryness at 40 °C under nitrogen gas
4. Add 5 mL 0.1 M perchloric acid and adjust pH to 1 ± 0.3.
5. Centrifuge at 5000 rpm for 10 minutes. Add 200 μL 0.1% formic acid in methanol (5:95, v/v), ultrasonicate
6. Collect supernatant and add 10 M sodium hydroxide to

Centrifuge at 15000 rpm for 10 minutes
adjust pH to 11.
7. Add 10 mL saturated sodium chloride and 10 mL LC Conditions

isopropanol-ethyl acetate (60:40, v/v). Instrument: Waters Alliance ® HPLC 2695 System
8. After centrifugation, take organic layer and evaporate to Column: Atlantis ® dC18, 2.1 x 150 mm, 5 μm
dryness at 40 °C under nitrogen gas. Guard Column: Atlantis dC18, 2.1 x 10 mm, 5 μm
Flow Rate: 0.2 mL/min
9. Dissolve residue in 5 mL 0.2 M sodium acetate (pH 5.2).
Mobile Phase: A. 0.1% formic acid
B. 0.1% formic acid in acetonitrile
Gradient: Time (min) A% B%
0.00 96 4
2.00 96 4
8.00 77 23
21.00 77 23
22.00 5 95
25.00 5 95
25.50 96 4
Injection Volume: 20 μL
Column Temperature: 35 °C
MS Conditions
Instrument: Waters Quattro Premier™
Ionization Mode: Positive electrospray (ESI +)
Multiple reaction monitoring
[ 17 ]
β2 -Agonis t s in p o r k an d p ig l iv e r t issu e s
MRM for MRM for A

Analyte
Quantification Confirmation
Salbutamol 240→148 240→222
Terbutaline 226→152 226→125 B
Cimaterol 202→160 202→143
Cimbuterol 234→160 234→143
C
Ractompamine 302→164 302→284
Clenbuterol 277→203 277→259
Bromclenbuterol 323→249 323→168 D
Bromobuterol 367→293 367→349
Isoxsuprine 302→150 302→284
E
Mabuterol 311→237 311→293
Mapenterol 325→237 325→217
Clenbuterol-D9 (IS) 286→204 286→204
F
Salbutamol-D3 (IS) 243→151 243→151
MRM method parameters.

G
Results
7 β2-agonists by multiple reaction monitoring (MRM) scan mode
A (A) salbutamol-d3 (B) salbutamol (C) terbutaline (D) cimaterol
(E) cimbuterol (F) ractompamine (G) clenbuterol-D 9.
Pig liver tissues were spiked with 11 β2-agonists standard mixture

B
of concentrations 0.5 ng/g, 1 ng/g and 2 ng/g respectively. The
SPE recoveries are between 89.4% and 110.5%, RSD are between
1% and 2.8%.
C
Ordering Information
F Description Part Number

Oasis MCX, 3 cc/60 mg, 30 μm, 100/box 186000254
Atlantis dC18, 2.1 x 150 mm, 5 μm 186001301
6 β2-agonists by multiple reaction monitoring (MRM) scan mode Atlantis dC18, 2.1 x 10 mm, 5 μm 186001379
(A) clenbuterol (B) bromclenbuterol (C) bromobuterol (D) isoxsuprine Sentry 2.1 mm Guard Holder
™
WAT097958
(E) mabuterol (F) mapenterol.
Qsert Vials, LCGC Certified Combination Packs
™
186001126C
Ref: T he determination of β2-agonists by LC/MS-MS JIN Yu-E1, GUO De-Hua

2, ZHENG Ye 3,WANG Guo-Quan1 (Shanghai Municipal Center for Disease
Control and Prevention; Shanghai Entry-Exit Inspection and Quarantine Bureau;
Shanghai University)
©2011 Waters Corporation. Waters, Oasis, Atlantis, Sentry, Alliance and Quattro Premier are trademarks of Waters
Corporation. All other trademarks are the property of their respective owners.
[ 18 ]
C h lo r am p h enico l in Hon e y
INTRODUCTION MS Conditions
Instrument: Waters Quattro micro™ API
Bee keeping and honey production is a world-wide industry.
Ionization Mode: Negative electrospray (ESI -)
Trace levels of the antibiotic chloramphenicol have been
detected in honey. The antibiotic can be detected when bee
Analyte MRM Transition
keepers apply the antibiotic on hives to control bacteria that
321→152
affect bee larvae. Chloramphenicol is banned in food products. Chloramphenicol (CAP)
321→257
354→185
Pretreatment Thiamphenicol (TAP)
354→290
1. Dissolve 5 g of honey (spiked with D5-CAP) in 5 mL water. 356→336
Florfenicol (FP)
356→185
2. Extract with 15 mL ethyl acetate and centrifuge.
Internal Standard D5-CAP 327→157
3. Transfer the supernatant to a clean tube and evaporate to
dryness under nitrogen at 50 °C.
Results
4. Reconstitute the residue in 1 mL methanol and dilute with
20 mL water. Chloramphenicol
SPE Procedure
Oasis® HLB, 6 cc/200 mg Florfenicol
Condition/Equilibrate: Thiamphenicol
A. 5 mL methanol
B. 5 mL water
Load:
20 mL of sample at 2 drops/second
Wash:
5 mL water
Overlays of chloramphenicol, thiamphenicol, florfenicol at 2 μg/kg for
Elute: each MRM transition.
2 x 2.5 mL of methanol
Evaporate to dryness under nitrogen at 50 °C Analyte Mean Recovery (%) RSD (%)
Chloramphenicol 91.1 2.2
Reconstitute residue with 9:1 water/methanol (500 µL)
Thiamphenicol 91.9 5.9
LC Conditions Florfenicol 104.6 1.7
Recovery data for CAP, TAP and FP at 0.3 µg/kg (n = 5).
Instrument: Waters Alliance ® HPLC 2695 System
Column: Symmetry ® C 8, 2.1 x 50 mm, 3.5 µm
Guard Column: Symmetry Sentry™ C 8, 2.1 x 10 mm, 3.5 µm Ordering Information
Mobile Phase: A. water Description Part Number
B. methanol
Oasis HLB, 6 cc/200 mg, 30 µm, 30/box WAT106202
Gradient: Time (min) A% B% Symmetry C8, 2.1 x 50 mm, 3.5 µm WAT200624
0.00 90 10
Symmetry Sentry Guard C8, 2.1 x 10 mm WAT106128
8.00 10 90
10.00 10 90 Sentry 2.1 mm Guard Holder WAT097958
10.10 90 10 Total Recovery Vials 186000750CV
15.00 90 10
Ref: Waters Application Note 720001015EN
Injection Volume: 20 µL ©2011 Waters Corporation. Waters, Oasis, Symmetry, Sentry, Alliance, and Quattro micro are trademarks of
Waters Corporation.
[ 19 ]
D e x am e t hason e in P o r k
INTRODUCTION Gradient: Time (min) A% B%

0.00 70 30
3.00 10 90
The European Union (EU) considers dexamethasone residues a
3.10 70 30
high priority in food animals as they are synergistic with illegal 4.00 70 30
growth promoters, such as beta-agonists or anabolic steroids.
MS Conditions
Pretreatment
Instrument: Waters Quattro Premier™ XE
1. Add 5 g of the ground pork into 30 mL of 95:5 MRM Transitions: 1. 393.00 → 373.00
acetonitrile:water (v/v). 2. 393.00 → 393.00
2. Shake for 20 minutes, homogenize, and shake for 10 minutes. Multiple reaction monitoring
3. Centrifuge for 5 minutes.
Results
4. Repeat extraction. 100
5. Combined both extractions and load onto Sep-Pak ® Vac 3 cc Blank
(500 mg), Florisil cartridges and elute with 30 mL of 95%

® %
acetonitrile and bring up to 100 mL with acetonitrile. 0

0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3
6. Take out 20 mL of extract add 10 mL of hexane. Shake for 100
3 minutes and leave to stand. 80 ppb spiked

%
7. Take acetonitrile layer and evaporate to dryness.

0
8. Reconstitute with 4 mL of 0.2 M sodium phosphate buffer 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 min
50 ppb dexamethasone spiked in pork muscle.

(pH 5) to a final volume of 6 mL with water.
Compound Name: Dexamethasone 393 393 RT Area
SPE Procedure 50 ppb-spiked 1.64 333.30
50 ppb-spiked 1.63 322.59
Sep-Pak Plus C18, 360 mg 50 ppb-spiked 1.63 548.12
50 ppb-spiked 1.63 506.48
Condition: 50 ppb-spiked 1.63 386.79
A. 10 mL methanol RSD (%) 24.43 -
B. 10 mL water
Recovery (%) 90.09 -
C. 2 mL sodium phosphate buffer (pH 5)
Compound Name: Dexamethasone 393 393 RT Area
LOAD 50 ppb-spiked 1.64 270.38
50 ppb-spiked 1.64 250.18
Wash: 50 ppb-spiked 1.63 503.14
A. 5 mL sodium phosphate buffer (pH 5) 50 ppb-spiked 1.63 404.46
B. 10 mL 25% methanol in water 50 ppb-spiked 1.63 327.81
RSD (%) 29.59 -
Elute: Recovery (%) 89.00 -
10 mL 60% acetonitrile 40% water (60:40, v/v) Recovery data for 50 ppb dexamethasone spiked in pork muscle.
Reconstitute in 200 μL in 10% acetonitrile and 90% water (10:90, v/v) O rd e ring Information
Description Part Number

LC Conditions
Sep-Pak Vac, 3cc/500mg, Florisil Cartridges WAT020815
Instrument: Waters ACQUITY UPLC® System Sep-Pak Plus C18, 360 mg WAT020515
Column: ACQUITY UPLC BEH C18, 2.1 x 100 mm, 1.7 µm ACQUITY UPLC BEH C18, 2.1 x 100 mm, 1.7 µm 186002353
Flow Rate: 400 µL/min Qsert Vials, LCGC Certified Combination Packs
™
186001126C
Mobile Phase: A. 0.1% formic acid in water
©2011 Waters Corporation. Waters, Sep-Pak, ACQUITY UPLC, and Quattro Premier. All other trademarks are the property of
B. 0.1% formic acid in acetonitrile their respective owners.
[ 20 ]
En ro f lox ac in (Bay t ril ®) in C hic k en
INTRODUCTION Cartridge II: Sep-Pak® Accell™ QMA, 3 cc/500 mg
The United States Food and Drug Administration (US FDA) CONDITION/EQUILIBRATE:
3 mL 5% ammonia in methanol
banned the use of enrofloxacin for growth enhancement in
poultry production. Ciprofloxacin is a degradant of enrofloxacin, Attach Sep-Pak Accell QMA cartridge to outlet of MCX cartridge.
Elute from MCX cartridge into Sep-Pak Accell QMA cartridge.
therefore, both compounds need to be screened in the assay. (6 cc cartridge on top)
Any detectable residue can be evidence of inappropriate
Elute*:
poultry farming practice. 3 mL (5:95, v/v) ammonia in methanol
Remove Oasis MCX cartridge

Pretreatment
WASH:
1. Extract 1.5 g homogenized sample with 30 mL ethanol/ 3 mL ethanol
acetic acid (99:1, v/v) and centrifuge at 4000 rpm for 5 minutes. ELUTE:
3 mL methanol in formic acid (98:2, v/v)
2. Take 10 mL aliquot of the supernatant for SPE
enrichment and cleanup. Evaporate solvent and reconstitute in 150 µL of
acetonitrile in water (15:85, v/v)
3. For muscle samples, dilute 10 mL of supernatant with 5 mL * Note: As the analytes are eluted from cartridge I, they are subsequently
retained by anion exchange on cartridge II. Therefore, the eluate from
water prior to SPE; liver samples are not diluted.
cartridge I becomes the load for cartridge II.
SPE Procedure UPLC Conditions

Cartridge I: Oasis® MCX, 6 cc/150 mg Instrument: Waters ACQUITY UPLC® System
Column: ACQUITY UPLC BEH C18, 1 x 50 mm, 1.7 μm

Condition/Equilibrate:
A. 3 mL methanol Flow Rate: 0.12 mL/min
B. 3 mL water Mobile Phase: A. 1% formic acid in water
C. 3 mL ethanol B. acetonitrile
Load:
10 mL sample 0.00 95 50
3.00 50 50
6.50 50 50
Wash:
10.50 95 50
A. 3 mL 1% acetic acid/ethanol
B. 3 mL water 15.50 95 50
C. 3 mL methanol Injection Volume: 10 μL
MS Conditions

360→342
Enrofloxacin
360→316
332→314
Ciprofloxacin
332→288
[ 21 ]
En ro f lox ac in (Bay t ril ®) in C hic k en
Results
Ciprofloxacin
Enrofloxacin
Typical UPLC®/MS/MS chromatogram of chicken muscle spiked (2 ng/kg).
Recovery averaged 75% measured by comparison of results

from chicken samples spiked before and after sample preparation.
Precision for six replicate samples spiked at 2 μg/kg was 12%.
Oasis MCX
6 cc/150 mg SPE cartridge
Cation-exchange retention of bases.

Neutrals or acids, are poorly retained and washed
off before elution.
Sep-Pak Accell QMA

3cc/500 mg SPE cartridge
Anion-exchange retention of acids.

Neutrals or bases are poorly retained and are
washed off before elution.
Tandem SPE cartridges setup.
O rd e ring Information

Sep-Pak Accell Plus QMA, 3 cc/500 mg, 50/box WAT020850
ACQUITY UPLC BEH C18, 1 x 50 mm, 1.7 μm 186002344
ACQUITY UPLC BEH C18, 1 x 50 mm, 1.7 μm, 3/pk 176000861

Qsert™ Vials 186001126
Ref: Waters application WA43206

©2011 Waters Corporation. Waters, Oasis, Sep-Pak, ACQUITY UPLC, UPLC, Quattro micro, and Accell are
trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
[ 22 ]
Nit ro f u r ans in Hon e y
INTRODUCTION SPE Step II: Oasis HLB, 3 cc/60 mg
Nitrofurans are a class of antibiotic drugs, used to treat bacterial

infections, which may induce carcinogenic residues in animal tissues. A. 1 mL methanol
B. 1 mL water
Due to public health concerns, nitrofurans have been banned for use
in food producing animals in many countries.
Load:
Prepared honey sample pH 7
Pretreatment
Wash:
1. Dilute 2 g honey sample with 5 mL of 0.12 M hydrochloric acid. A. 2 mL water
B. 2 mL 30% methanol in water
SPE Procedure
DRY:
SPE Step I: Oasis® HLB, 3 cc/60 mg 20 minutes
Elute:
Condition/Equilibrate: 3 mL formic acid methyl-t-butyl/methanol/formic acid (79:9:2, v/v/v)
A. 2 mL methanol
B. 2 mL water
Evaporate and reconstitute in 200 µL mobile phase
Pass-through:
2 g prepared honey in 5 mL of 0.1 N hydrochloric acid
LC Conditions
Wash: Instrument: Waters Alliance ® HPLC 2695 System
2 mL water
Column: XTerra ® MS C18, 2.1 x 100 mm, 3.5 µm
Combine the solutions
Mobile Phase: Isocratic 70% 20 mM ammonium formate
pH 4.0, 30% acetonitrile
1. Collect quantitatively the eluent from the pass-through and
Injection Volume: 20 µL
wash steps into a 15 mL capped sample tube.
2. Add 300 μL of 50 mM 2-nitrobenzaldehyde in dimethyl-
sulfoxide. Hydrolyse and derivatize for 18 hours at 37 ˚C.
MS Conditions
3. Cool the sample to room temperature and adjust to pH 7 by
adding 6 mL of 0.1 M dipotassium hydrogen phosphate.
4. Put sample through SPE Step II. Multiple reaction monitoring

AOZ 236→134
AMOZ 335→291
Semicarbizide (SC) 209→192
1-Aminohydantion (AH) 178→249
[ 23 ]
Nit ro f u r ans in Hon e y
Results
AOZ
AMOZ
SC
AH
Spiked honey (400 ng/kg) metabolites as 2-nitrobenzaldehyde derivatives.
RSD (%)
Analytes
Raw Wild Honey Buckwheat Honey
Semicarbizide 9.8 9.7
AOZ 13.9 9.6
AMOZ 3.8 2.9
AH 14 3.8
Relative standard deviation obtained from two different lots of honey spiked at
500 ng/kg (ppt). Metabolite recovery was greater than 85% post-derivatization
for each analyte. The samples used for spiking tested negative before the study.

XTerra MS C18, 2.1 x 100 mm, 3.5 µm 186000404
LCMS Certified Vials 600000751CV

©2011 Waters Corporation. Waters, Oasis, XTerra, Alliance, and Quattro micro are trademarks of Waters Corporation.
[ 24 ]
Nit ro f u r ans in T issu e s
INTRODUCTION LC Conditions
The United States Food and Drug Administration (US FDA) banned
Column: XTerra ® MS C18, 2.1 x 100 mm, 3.5 µm
Nitrofuran drugs are banned in food producing animals because
they pose a public health risk. The rule went into effect as a result
Mobile Phase: Isocratic 70% 20 mM ammonium formate
of evidence that the drugs may induce carcinogenic residues in
pH 4, 30% acetonitrile
animal tissues.
Pretreatment
1. Homogenize 10 g of sample in 100 mL of 0.12 M MS Conditions

hydrochloric acid.
2. Take 1 mL aliquot and treat with 400 µL of 50 mM Ionization Mode: Positive electrospray (ESI +)
2-nitrobenzaldehyde in dimethylsulfoxide. Multiple reaction monitoring
3. Hydrolyze/derivatize the sample for 16 hours at 37 °C. Analyte MRM Transition

AOZ 236→134
4. Adjust the sample to pH 7.4 with potassium hydrogen phosphate.
AMOZ 335→291
5. Centrifuge sample for 5 minutes at 8000 rpm.
SC 209→192
AH 249→178
SPE Procedure
Oasis® HLB 3 cc/60 mg
Results
A. 1 mL methanol
B. 1 mL water AOZ
Load:
Approximatively 100 mL of sample
Wash: AMOZ
A. 2 mL water
B. 2 mL 30% methanol in water
DRY: SC
20 minutes
Elute:
3 mL methyl-t-butyl/methanol/formic acid (89:9:2, v/v/v)
AH
Evaporate and reconstitute in 200 μL mobile phase
Spiked chicken muscle (1 ng/g) metabolites as 2-nitrobenzaldehyde derivatives.
[ 25 ]
Nit ro f u r ans in T issu e s
STRUCTURES
OHC NO 2
O O
NO 2
N
N
AOZ O N
NH2
O
H + ,H 2 O, 37 o
C, 18 hr
H 2N NO 2 N
N N N O
O
O
AMOZ O
N
O O
O NO 2
NH 2
1-Aminohydantion (AH) HN
N
O
N N
NH
O
O
NO 2 H
O N NH 2
N
Semicarbizide (SC) H 2N
N NH 2
O
H
Chemical structures of 2-nitrobenzaldehyde derivatives.

XTerra MS C18, 2.1 x 100 mm, 3.5 µm 186000404
Qsert™ Vials, LCGC Certified
186001126C
Combination Packs
©2011 Waters Corporation. Waters, Oasis, Alliance, XTerra, and Quattro micro are trademarks of Waters Corporation. All
other trademarks are the property of their respective owners
[ 26 ]
P enic il l in G in P o r k
INTRODUCTION
100
Blank Sample
World organizations are concerned about the overuse of %
antibiotics and antibacterials levels in foods due to possible

bacteria resistances and health concerns. 0
100
Spiked Sample
Pretreatment
%
1. Homogenize 3 g of sample with 3 mL of 5% sodium tungstate,

3 mL of 0.17 M sulfuric acid and 30 mL of water. 0
0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 min
2. Centrifuge 3100 rpm for 10 minutes.

5 ppb penicillin spiked in pork muscle.
3. Filter with glass fiber filter.
Compound Name: penicillin MRM
RT Area
SPE Procedure 335→176
Pork blank 1 1.83 4.50
Sep-Pak® Plus Short C18, 360 mg Pork blank 2 1.83 3.23
5 ppb spiked pork 1 1.83 23.30
Load
Wash:
3 mL water 5 ppb spiked pork 4 1.83 15.45
Elute: RSD (%) 15.56 -
4 mL 20% acetonitrile water
Recovery (%) 58.26 -
Concentrate to 2.5 mL and adjust the volume to 6 mL water
Compound Name: penicillin MRM
RT Area
335→160
LC Conditions Pork blank 1 1.82 5.81
Pork blank 2 1.83 5.02
Instrument: Waters ACQUITY UPLC® System
Column: ACQUITY UPLC BEH C18, 2.1 x 50 mm, 1.7 µm 5 ppb spiked pork 2 1.83 26.59
Flow Rate: 600 µL/min 5 ppb spiked pork 3 1.83 21.89
Mobile Phase: A. 0.1% formic acid in water 5 ppb spiked pork 4 1.82 25.22
RSD (%) 29.75 -
0.00 90 10
5.00 10 90 Recovery (%) 78.30 -
5.50 90 10 Recovery results for 5 ppb penicillin spiked in pork muscle.
6.00 90 10
MS Conditions
Instrument: Waters Quattro Premier™ XE O rd e ring Information
Multiple reaction monitoring Description Part Number
Sep-Pak Plus Short C18 WAT020515
MRM Transitions: 1: 335160
2: 335176 ACQUITY UPLC BEH C18, 2.1 x 50 mm, 1.7 µm 186002350
©2011 Waters Corporation. Waters, ACQUITY UPLC, Sep-Pak, and Quattro Premier are trademarks of Waters Corporation.
[ 27 ]
P enic il l ins, T e t r ac yc l in e s, an d su l fonamid e s in mil k
INTRODUCTION Results
1
100
World organizations are concerned about the overuse of anti-

2
biotics and antibacterials. This method can be used to monitor
antibacterials in milk.
3
Pretreatment
1. Penicillin
Milk (1 mL) is first extracted with 3 mL acetonitrile (1 minute % 2. Oxacillin
shake). The sample is centrifuged and the supernatant is collected. 3. Cloxacillin
The acetonitrile extract (extract 1) is evaporated to just under 1 mL

using a gentle nitrogen stream and a water bath at 45 °C (this step
extracts the penicillins and partially extracts the sulfonamides).
The milk solids pellet is then extracted with 3 mL of pH 5 suc-

cinate/EDTA buffer. The sample is centrifuged and the supernatant 0
2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 min
is collected (extract 2, this step extracts the tetracyclines and any 100 ppb spiked in milk.
remaining sulfonamides).
Compound MRM Transition Recovery
Extract 2 is combined with the evaporated extract 1 and the Oxytetracydine 461→426 78
volume is made up to 10 mL with water. The combined extracts Tetracycline 445→410 69
are then processed using an Oasis HLB cartridge.
®
Chlortetracycline 479→444 76
SPE Procedure Sulfadiazine 251→92 83

Sulfathiazole 256→92 87
Oasis HLB 1cc/30mg
Sulfapyridine 250→92 66
Condition/Equilibrate: Sulfamerazine 265→92 95
1 mL MeOH, 1 mL water
Sulfamethazine 279→92 90
Load SAMPLE: Sulfamethoxypyridazine 281→92 70
From pretreatment Sulfachloropyridazine 85
285→92
Wash: Sulfamethoxazole 254→92 97
0.5 mL 5% methanol/water Sulfadimethoxine 311→92 83
Elute:
60:40 methanol/water Penicillin G 335→160 93
60 mM ammonium acetate
Oxacillin 402→160 83
LC Conditions Cloxacillin 436→160 96
Column: Waters ACQUITY UPLC® BEH Shield RP18, MRM method parameters and recovery data.
2.1 x 100 mm, 1.7 µm
Flow Rate: 4 mL/min
Mobile Phase: A: 0.1% Formic acid in water O rd e ring Information
B: Acetonitrile
Gradient: Time (min) A% B% Description Part Number
Initial 85 15 Oasis HLB, 1 cc/30 mg, 100/box WAT094225
2.00 60 40 ACQUITY UPLC BEH Shield RP18,
2.50 40 60 186002854
2.1 x 100 mm, 1.7 µm
3.00 10 90
4.50 10 90
4.60 85 15
5.50 85 15 ©2011 Waters Corporation. Waters, Oasis, and ACQUITY UPLC are trademarks of Waters Corporation.
[ 28 ]
S p i r am yc in in P o r k
Instrument Waters ACQUITY UPLC® System
World health organizations are concerned about the antibiotics
Column: ACQUITY UPLC BEH C18, 2.1 x 50 mm, 1.7 µm
and antibacterials levels in food. This method can be used to
Flow Rate: 400 µL/min
monitor Spiramycin, a macrolide antibiotic, in pork.
Pretreatment
0.00 90 10
1. Weigh 5 g of sample into 25 mL of water. Homogenize.
2.00 60 40
2. Centrifuge at 3000 rpm for 10 minutes. 2.10 90 10
3.00 90 10
3. Collect supernatant.
4. Re-extract pellet with 15 mL 1.2% metaphosphoric acid/ MS Conditions

methanol (50:50, v/v).
5. Vortex. MRM transitions: 1: 422.50→101.00
2: 422.50→174.10
6. Centrifuge at 3000 rpm for 10 minutes. Collect supernatant.
Ionization mode: Positive electrospray (ESI +)
7. Combine both supernatants. Multiple reaction monitoring
8. Filter sample using glass fiber filter.

Results
SPE Procedure 100
Oasis® MCX, 6 cc/150 mg %

Quantification
0
Condition:
A. 3 mL phosphate buffer/methanol (1:9, v/v) 100
B. 5 mL water
%
Confirmation
Load: 0
0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 min
Sample (approximately 40 mL)
100 ppb spiramycin spiked sample in pork muscle.
Wash:
A. 3 mL 0.1 M dipotassium hydrogen phosphate (pH 3)
B. 15 mL 0.1 M potassium phosphate/methanol (1:9, v/v)
Evaporate:
Approximately to 1 mL and bring up to 2 mL with 20% methanol
SPE Buffer:
Dissolve 8.7 g dipotassium hydrogen phosphate in 1 L of deionized
water, adjust to pH 3 with phosphoric acid.
[ 29 ]
S p i r am yc in in P o r k
Compound Name: Spiramycin 174.1 RT Area

100 ppb spiked 1 1.34 902.79
100 ppb spiked 2 1.34 911.36
100 ppb spiked 3 1.34 1041.24
100 ppb spiked 4 1.34 1030.33
100 ppb spiked 5 1.34 1101.27
RSD (%) - 7.97
Recovery (%) - 73.26
Compound Name: Spiramycin 101 RT Area

100 ppb spiked 1 1.34 1063.65
100 ppb spiked 2 1.34 1267.22
100 ppb spiked 3 1.34 1368.84
100 ppb spiked 4 1.34 1227.10
100 ppb spiked 5 1.34 1379.40
RSD (%) - 5.96
Recovery data for 100 ppb spiramycin spiked sample in pork muscle.

Oasis MCX, 6 cc/150 mg, 30/box 186000256
ACQUITY UPLC BEH C18, 2.1 x 50 mm, 1.7 µm 186002350
©2011 Waters Corporation. Waters, ACQUITY UPLC, Oasis, and Quattro Premier are trademarks of Waters Corporation.
[ 30 ]
S TRE P TOMYCIN IN HONEY
Bee keeping and honey production is a world-wide industry. Trace Instrument: Waters Quattro micro™
levels of the Streptomycin, an aminoglycoside, has been detected Multiple reaction monitoring
in honey. Streptomycin is not permitted in food products.
Pretreatment Streptomycin
582→263
582→176
1. Dissolve 20 g of honey in approximately 75 mL water. 584→263
Internal Standard
2. Bring to a total volume of 100 mL with water. 584→246
3. Filter through a fluted filter to remove suspended solids.
Results
SPE Procedure
100 Internal Standard
Sep-Pak® Vac 6 cc Accell™ Plus CM cartridge % Quantification
0
Condition:
2 x 5 mL 2% acetic acid 100 Internal Standard
% Confirmation
0
RINSE:
2 x 5 mL water*
100
Streptomycin
% Quantification
LOAD: 0
50 mL of honey solution at approximately 2 drops per second
100 Streptomycin
RINSE: % Confirmation
2 x 5 mL water 0
min
ELUTE: MRM for streptomycin and the internal standard.

2 x 5 mL (80:20, 2% acetic acid/acetonitrile)
Spiked Concentration µg/kg Mean Std. Dev. RSD (%)

Adjust to 10 mL with water 2 1.99 0.09 4.5
* Do not allow the cartridge to dry out through the procedure 20 20.17 0.076 3.7
100 100.91 3.75 3.7
LC Conditions Method accuracy and precision over three days.
Instrument: Waters Alliance ® 2795 HPLC System

Column: Atlantis ® HILIC Silica, 2.1 x 50 mm, 3 μm
Guard Column: Atlantis HILIC Silica, 2.1 x 10 mm, 3 μm
Mobile Phase: A. 200 mM ammonium formate in 100 mM formic acid O rd e ring Information
B. 100mM Formic Acid in acetonitrile
Column Temperature: 30 °C Description Part Number
Injection Volume: 20 μL Sep-Pak Vac, 6 cc Accell Plus CM cartridge WAT054545
Atlantis HILIC Silica, 2.1 x 10 mm, 3 μm 186002005
0.00 10 90 Atlantis HILIC Silica, 2.1 x 50 mm, 3 µm 186002011
6.00 60 40 LCMS Certified Vials 600000751CV
10.00 60 40
10.10 10 90 Ref: Waters Application Note 720000981EN
16.00 10 90 ©2011 Waters Corporation. Waters,Sep-Pak, Quattro micro, Alliance, Accell and Atlantis are trademarks of Waters
Corporation.
[ 31 ]
Su l fonamid e Ant ibac t e ria l s in Mil k
Sulfonamides are widely used for therapeutic and prophylactic Instrument: Waters Quattro Premier™ XE
purposes in animals. When sulfonamides are retained in food Ionization Mode: Positive electrospray (ESI +)
stuff, this may result in allergic or toxic reactions in sensitive
consumers. This method can be used to monitor the presence of
Compounds Precusor (m/z)
sulfonamides in milk. 249.80→91.90
Sulfapyridine
249.80→155.80
SPE Procedure 253.70→91.90
Sulfamethoxazole
253.70→155.80
Oasis® MCX, 3 cc/60 mg
250.80→91.90
Sulfadiazine
250.80→155.90
Condition:
255.70→91.90
A. 2 mL methanol Sulfathiazole
B. 2 ml water 255.70→155.90
264.80→92.00
Sulfamerazine
264.80→155.80
Load:
5 mL whole homogenized milk 270.90→91.70
Sulfamethizole
270.90→155.80
Wash 1: 278.80→91.90
Sulfamethazine
2 mL 5% methanol in water 278.80→155.80
280.80→91.90
Sulfamethoxy-pyridazine
WASH 2: 280.80→155.80
1 mL 0.5 M aqueous hydrochloric acid 284.70→92.00
Sulfachloropyridazine
284.70→155.90
WASH 3: 310.80→91.90
Sulfadimethoxine
2 mL 20% methanol in water 310.80→155.90
WASH 4:
1 mL methanol
ELUTE:
2.5 mL 90:10 methanol/water (200 mM ammonium bicarbonate pH 8.5)
Reconstitute in 0.5 mL mobile phase
LC Conditions
Mobile Phase: A. 0.05% formic acid/water
B. 0.05% formic acid/methanol
0.00 90 10
3.25 80 20
3.26 90 10
Detector: ACQUITY UPLC PDA Detector
[ 32 ]
Su l fonamid e Ant ibac t e ria l s in Mil k
Results
10
7
1- Sulfadiazine
3 6
2- Sulfathiazole
9
2 3- Sulfapyridine
4
4- Sulfamerazine
5- Sulfamethizole
6- Sulfamethazine
7- Sulfamethoxypyridine
8- Sulfachloropyridazine
9- Sulfamethoxazole
1
8 10-Sulfadimethoxine
%
0
1 1.5 2 2.5 3 3.5 4 min
Whole milk (5 ng/mL) typical LC/MS/MS analysis (MRM).
Compound Recovery (%) RSD (%)

1-Sulfadiazine 79 9.2
2-Sulfathiazole 75 6.5
3-Sulfapyridine 68 10.2
4-Sulfamerazine 77 9.1
5-Sulfamethizole 73 10.1
6-Sulfamethazine 67 7.0
7-Sulfamethoxypyridine 79 10.0
8-Sulfachloropyridazine 60 7.0
9-Sulfamethoxazole 71 9.1
10-Sulfadimethoxine 80 9.2
Recovery data for whole milk (5 ng/mL).

ACQUITY UPLC BEH C18, 2.1 x 100 mm, 1.7 μm 186002352
©2011 Waters Corporation. Waters , Oasis, ACQUITY UPLC, and Quattro Premier are trademarks of Waters Corporation. All
other trademarks are the property of their respective owners.
[ 33 ]
T e t r ac yc l in e s an d Su l fonamid e s in Mil k
Tetracyclines and sulfonamides are widely used for therapeutic Instrument: Waters Quattro Premier™ XE
and prophylactic purposes in animal diseases. When these classes Ionization Mode: Positive electrospray (ESI +)
of veterinary drugs are retained in food stuff, this may result in
allergic or toxic reactions in sensitive consumers. Results
Pretreatment 100
4
1. Mix 1.5 mL milk with 6 mL pH 4 McIivaine buffer. 1
2 1. Oxytetracycline
2. Centrifuge. 2. Tetracycline
3. Chlortetracycline
3. Take supernatant/adjust to pH 10 with 0.75 mL 1 M NaOH. % 4. Doxycycline
SPE Procedure 3
Oasis® MAX, 1 cc/300 mg

0
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 min
2 mL MeOH, 2 mL water 100 ppb spiked sample.
Load SAMPLE:
From pretreatment
Wash 1:
0.5 mL 5% NH2OH/water
WaSH 2:
0.5 mL methanol
ELUTE:
0.5 mL 45.55 acetonitrile/75 mM oxalic acid.
Dilute 1:3 with water for LC
LC Conditions
Column: Waters ACQUITY UPLC® BEH C18, 2.1 x 50 mm, 1.7 µm
Flow Rate: 4 mL/min
Mobile Phase: A: 0.1% Formic acid/water
B: Acetonitrile
Initial 85 15
2.50 50 50
3.50 30 70 O rd e ring Information
3.60 85 15
4.00 85 15 Description Part Number
Oasis MAX, 1 cc/30 mg, 100/box 186000366
©2011 Waters Corporation. Waters, Oasis, and ACQUITY UPLC are trademarks of Waters Corporation. All other trademarks are the
property of their respective owners.
[ 34 ]
T e t r ac yc l in e s in Anima l T issu e
Tetracyclines (TCs) are broad-spectrum antibiotics used in
human and veterinary medicine. The use of TCs is increasing, Column: Nova-Pak ® C 8, 3.9 x 150 mm, 4 μm
as they are used not only in treatment, but also prevention Flow Rate: 0.8 mL/min
of illnesses. Moreover, TCs are given to animals destined for Mobile Phase: Acetonitrile: 50 mM oxalic acid (1:4)
human consumption to promote growth and may potentially Injection Volume: 60 μL
result in the presence of residues in edible animal tissues, Detector: 2487 Dual Wavelength Detector
which can be toxic and dangerous for human health. UV Wavelength: 365 nm
Pretreatment 1. Oxytetracycline (Spike level 0.5 µg/g)

2. Tetracycline
1 3. Chlortetracycline
1. Add 20 mL McIlvaines buffer-EDTA to 5 g homogenized
sample in a 50 mL centrifuge tube and mix thoroughly. 2
2. Centrifuge at 3000 rpm for 10 minutes.

3
spiked pork
3. Collect supernatant.
nonspiked sample
4. Repeat steps 1 and 2 with the residue. Collect supernatant.
5. Combine both supernatant portions for SPE analysis.

5
0.05 µg/g sample.
SPE Procedure
Oasis® HLB, 6 cc/200 mg

Solutions
A. 3 mL methanol
B. 2 mL water McIlvaines Buffer
1. Thoroughly mix 1000 mL 0.1 M citric acid with 625 mL

Load: 0.1 M disodium hydrogen phosphate dihydrate.
40 mL sample (4 mL/min)
2. Adjust with sodium hydroxide or hydrochloric acid to
Wash: pH 4 ± 0.05, if necessary.
2 mL 5% methanol in water
EDTA-McIlvaines Buffer
Elute:
3 mL methanol 1. Add 60.5 g disodium EDTA to 1625 mL McIlvaines
Buffer and mix thoroughly.
Evaporate to 0.2 mL
Reconstitute with 1 mL 50 mM oxalic acid

Nova-Pak C8, 3.9 x 150 mm, 4 μm WAT035876
©2011 Waters Corporation. Waters, Oasis, Alliance, and Nova-Pak are trademarks of Waters Corporation.
[ 35 ]
T e t r ac yc l in e s in Hon e y
INTRODUCTION
Solutions
Tetracyclines (TCs), an antibiotic, is not permitted in apiculture.
This method can monitors the presence of TC’s in honey. McIlvaines Buffer
1. Thoroughly mix 1000 mL 0.1 M citric acid with

Pretreatment 625 mL 0.1 ML disodium hydrogen phosphate
dihydrate.
1. Add 30 mL EDTA-McIlvaines buffer to 6 g sample, mix
thoroughly for 1 minute. 2. Adjust with sodium hydroxide or hydrochloric acid to
pH 4 ± 0.05, if necessary.
2. Centrifuge at 3000 rpm for 5 minutes and collect supernatant
for SPE. EDTA-McIlvaines Buffer
1. Add 60.5 g disodium EDTA to 1625 mL McIlvaines

SPE Procedure Buffer and mix thoroughly.
Cartridge I: Oasis HLB, 6 cc/500 mg
®
LC Conditions
A. 5 mL methanol
B. 10 mL water Column: SunFire™ C 8, 2.1 x 150 mm, 3.5 µm
Mobile Phase: Acetonitrile:methanol: 0.4% fomic acid (18:4:78)
Load: Flow Rate: 0.2 mL/min

Honey sample (3 mL/min) Injection Volume: 20 μL
Column Temperature: 25 ˚C
Wash:
5 mL methanol/water (5:95, v/v)
MS Conditions
DRY:
By vacuum for 20 minutes Instrument: Waters Quadrupole MS
Elute: Multiple reaction monitoring
15 mL ethyl acetate
MRM for MRM for

Solution I Analyte
Quantification Quantification
461→426
Cartridge II: Sep-Pak® Accell™ CM, 3 cc/500 mg Oxytetracycline 461→443 461→426
461→381
Condition/Equilibrate: 445→410
5 mL ethyl acetate Tetracycline 445→154 445→410
445→428
Load: 479→444
Solution I (3 mL/min) Chlortetracycline 479→154 479→444
479→462
Wash: 445→428
5 mL methanol Doxycycline 445→410 445→428
445→154
DRY:
By cartridge by vacuum for 5 minutes MRM method parameters.
Elute:
4 mL mobile phase
[ 36 ]
T e t r ac yc l in e s in Hon e y
Results
B
A. Oxytetracycline, B. Tetracycline, C. Chlortetracycline and D. Doxycycline

standards.
Concentration
Analyte Average Recovery
(mg/kg)
0.002 88.0
0.010 95.3
A. Oxytetracycline
0.100 95.8
0.050 93.6
0.002 81.9
0.010 82.6
B. Tetracycline
0.050 84.5
0.100 89.3
0.002 87.2
0.010 86.0
C. Chlortetracycline
0.050 86.6
0.100 90.8
0.002 85.2
0.010 85.3
D. Doxycycline
0.050 86.8
0.100 87.9
Recovery data for tetracyclines in honey.

Oasis HLB, 6 cc/500 mg, 60 µm 30/box 186000115
Sep-Pak Accell CM, 3 cc/500 mg, 50/box WAT020855
SunFire C8, 2.1 x 150 mm, 3.5 μm 186002712
Ref: China GB/T 18932.23 - 2003

©2011 Waters Corporation. Waters, Sep-Pak, Accell, Oasis, and SunFire are trademarks of Waters Corporation.
[ 37 ]
Pesticides and Contaminants
The presence of contaminants in food, such as pesticides, herbicides, illegally-added dyes, mycotoxins, and melamine,
are a concern to regulatory bodies, public health agencies, and the public at large. Methods in this section cover:
 Sample pretreatment
 Sample preparation
 Instrumentation methods and results
These methods meet, or exceed, the level of detection and quantitation required by government agencies.
This section of the notebook also includes applications of multi-residue pesticides analysis by QuEChERS and other official
methods. There are QuEChERS* application briefs on a variety of different commodities to show examples requiring:
 Different pretreatment requirements, i.e. soaking of dry commodities before the extraction procedure
 Alternative d-SPE sorbent selection, i.e. for removing fats
* For further details on this method, see Waters QuEChERS White Paper on our website (literature number 720003643en).
Ac ryl amid e in F ri e d P otat o P ro du c t s
INTRODUCTION Mobile Phase: 0.1% formic acid in water

Acrylamide, a chemical contaminant, is produced during the Column Temperature: 30 ˚C
cooking of french fries, potato chips, and other processed foods.
Acrylamide is considered to be a possible cancer causing agent.
MS Conditions
Pretreatment Instrument: Waters ZMD Mass Detector

Ionization Mode: Positive Electrospray (ESI +)
1. 1 g crushed potato product was weighed into a centrifuge tube. Selected-Ion Recording (SIR)
Compound: Mass Cone Voltage (V)
2. 15 mL of 2 M sodium chloride and 10 μL of internal standard
Acrylamide 72 20
(acrylamide-D3) solution was added to the tube. Shake
55 40
contents vigorously for 30 minutes. Acrylamide-D3 75 20
58 40
3. Centrifuge at 10000 x g for 12 minutes.
4. Take a 1.5 mL aliquot of the supernatant from the centrifuge

tube for SPE extraction and cleanup. Results
Acrylamide
SPE Procedure 710 µg/kg
Cartridge I: Oasis® HLB, 6 cc/200 mg

abundance, m/z 72
Condition:
A. 2mL methanol
B. 2mL 2 M sodium chloride
Load:
1.5 mL potato extract
Wash: 2 3 4 5 min
0.8 mL water LC/MS determination of acrylamide in potato chips.
Elute: Fortification Level (µg/kg) Amount Found (μg/kg) RSD (%)*

3 mL 1% formic acid in methanol
100 96 12
200 211 8.7
Cartridge II: Oasis MCX, 3 cc/60 mg 500 488 5.8
1000 1010 8.0
Condition:
2 mL methanol 2000 2000 6.5
*Five replicate samples analyzed per level.
A. Pass eluent from Part A.
B. Rinse vial in 0.5 mL methanol, combine with passed eluent
C. Collect in total
Evaporate and Reconstitute:
0.4 mL water Description Part Number
Atlantis dC18, 2.1 x 150 mm, 5 μm 186001301
LC Conditions Oasis HLB, 6 cc/200 mg WAT106202

Oasis MCX, 3 cc/60 mg 186000254
Qsert™ Vials, LCGC Certified Combination Packs 186001126C
Column: Atlantis ® dC18, 2.1 x 150 mm, 5 μm
Ref: Waters Application Note, 720000688EN
Flow Rate: 0.2 mL/min ©2011 Waters Corporation. Waters, Oasis, Alliance, and Atlantis are trademarks of Waters Corporation. All other
trademarks are the property of their respective owners
[ 39 ]
A f l at ox ins in P ro du c e SAm p l e s
Aflatoxins are naturally occurring mycotoxins. Aflatoxins often

cause disease even when ingested in minute amounts and are most
commonly known for causing acute, or chronic liver disease, and
liver cancer.
Pretreatment
1. Weigh 50 g ground sample with 5 g sodium chloride and

place in blender jar.
2. Add 100 mL 80:20 methanol: water (v/v) to jar.
3. Blend at high speed for 1 minute.
4. Filter extract with fluted filter paper. Collect filtrate in a

clean vessel.
5. Pipette or pour 65 mL filtered extract into a clean vessel.
6. Dilute extract with 60 mL of phosphate buffer saline. Mix well.

Aflatoxins in red pepper extract. Recovery: 76% at 20 ppb (7B1:1B2:3G1:1G2
aflatoxin mix).
7. Filter extract through glass microfiber filter into a clean vessel.
Action Levels for Aflatoxins
SPE Procedure United States (FDA) action levels (B1, B2, G2, G2, M1)
AflaTest® Affinity Column
Food Stuff Level Regulation
Pass 4 mL of filtered diluted extract (4 mL = 0.2 g sample equivalent) All products, except milk, designated Policy Guides 7120.26,
20 ng/g
completely through AflaTest affinity column at a rate of about for humans 7106.10, 7126.33
1–2 drops/seconds until air comes through column Policy Guides 7120.26,
Milk 0.5 ng/g
7106.10, 7126.33
Pass 10 mL of 20:80 methanol:water through the column Corn for immature animals and Policy Guides 7120.26,
20 ng/g
at a rate of about 2 drops/second dairy cattle 7106.10, 7126.33
Corn for breeding beef cattle, swine Policy Guides 7120.26,
100 ng/g
Repeat previous step once more until air comes through column and mature poultry 7106.10, 7126.33
Policy Guides 7120.26,
Corn for finishing swine 200 ng/g
Place glass cuvette under AflaTest column and add 1 mL HPLC grade 7106.10, 7126.33
methanol into glass syringe barrel Policy Guides 7120.26,
Corn for finishing beef cattle 300 ng/g
7106.10, 7126.33
Elute AflaTest column at a rate of 1 drop/second by passing Policy Guides 7120.26,
methanol through the column and collecting all the sample eluate Cottonseed meal (as feed ingredient) 300 ng/g
7106.10, 7126.33
(1 mL) in a glass cuvette
Policy Guides 7120.26,
All feedstuff other than corn 200 ng/g
Add 1 mL of purified water to eluate. Inject 20-100 μL onto HPLC 7106.10, 7126.33
Aflatoxin regulatory action limits.
LC Conditions O rd e ring Information
Instrument: Waters Alliance HPLC System
®
Column: XBridge™ C18, 4.6 x 250 mm, 5 μm Description Part Number

Flow Rate: 1 mL/min VICAM aflaOchra HPLC Columns, 25/box
™
G1017
Mobile Phase: Acetonitrile/water/methanol (17:54:29, v/v/v) VICAM Glass Cuvette 34000
Injection: 100 µL XBridge C18, 4.6 x 250 mm, 5 μm 186003117
Detector: 2475 Multi Wavelength Fluorescence LCMS Certified Combination Packs 600000751CV
Detection: Excitation Wavelength: 333 nm ©2011 Waters Corporation. Waters, Alliance, AflaTest, aflaOchra HPLC, Viacam and XBridge are trademarks of
Emmission Wavelength: 460 nm Waters Corporation.
[ 40 ]
C a r bamat e s in F ruit s an d V eg e ta bl e s
INTRODUCTION Gradient: Time (min) A% B% C%

0.00 88 12 0
Carbamates have been identified as a health risk. They affect 5.30 88 12 0
the nervous system by reducing the ability of cholinesterase, an 5.40 68 16 16
enzyme, to function properly in regulating the neurotransmitter 14.00 68 16 16
16.10 50 25 25
acetylcholine.
20.00 50 25 25
22.00 88 12 0
Pretreatment 30.00 88 12 0
Sample: 10 ng of each analyte on column
1. Add 50 mL of acetonitrile to 25 g of sample. Homogenize
for 2 minutes and filter.
Post Column Addition: OPA*/NaOH @ 0.5 mL/min
2. Collect 40-50 mL of filtrate into a flask containing 5–7 g
Detector: 2475 Multi Wavelength Fluorescence Detector
sodium chloride.
Excitation Wavelength: 339 nm
3. Shake vigorously for 1 minute. Leave to stand at room Emission Wavelength: 445 nm
temperature. *OPA: Orthophthaldehyde
4. Take 10 mL aliquot from the acetonitrile layer and evaporate 5

6
sample to dryness (80 °C under nitrogen or air).
7 9 11
1 8
5. Reconstitute with 2 mL methanol/dichloromethane (1:99, v/v). 234 12
10
SPE Procedure
Sep-Pak® Aminopropyl, 6 cc/500 mg

Aldicarb standards.
4 mL methanol/dichloromethane (1:99, v/v) Peak Analyte 400 µL
1 Aldicarb Sulfoxide 3.77
PASS-THROUGH: 2 Aldicarb Sulfone 4.66
2 mL sample
3 Oxamyl 5.17
Collect the solution 4 Methomyl 6.03
5 3-Hydroxycarbofuran 9.83
Elute: 6 Aldicarb 11.46
2 mL methanol/dichloromethane (1:99, v/v) x 2
7 Propoxur 14.35
8 Carbofuran 14.94
Combine the solutions
9 Carbaryl 17.37
10 1-Naphthol 18.99
Evaporate to dryness under nitrogen at 50 °C
11 Methiocarb 22.02
12 BDMC 22.56
Reconstitute in 2.5 mL methanol
Expected retention times for aldicarb standards.
LC Conditions
Column: Carbamate Analysis Column, 3.9 x 150 mm Description Part Number
Sep-Pak Aminopropyl, 6 cc/500 mg, 30/box WAT054560
Carbamate Analysis Column, 3.9 x 150 mm WAT035577
Mobile Phase: A. water
B. methanol LC Certified Vials 186000272C
C. acetonitrile
Ref: Ministry of Agriculture, China (NY/T 761.1 – 2004 and NY/T761.3 – 2004)
©2011 Waters Corporation. Waters, and Sep-Pak and Alliance are trademarks of Waters. Corporation.
[ 41 ]
Ma l ac hit e G r e en in F ish (H P LC / UV )
INTRODUCTION
Solutions
Malachite Green (MG) is an effective and inexpensive fungicide
used in aquaculture. During metabolism MG reduces to McIlvaines Buffer (pH 2.6) :
Leucomalachite Green (LMG), which has been shown to accumulate 1. 0.1 M citric acid monohydrate (A) - Dissolve citric acid
in fatty fish tissues. Both MG and LMG have demonstrated putative monohydrate (10.5 g) in water (500 mL).
carcinogenic activity, and thus, have been banned for use in aqua-
2. 0.2 M disodium hydrogen phosphate dihydrate (B) -
culture by both the United States Food and Drug Administration
Dissolve disodium hydrogen phosphate dihydrate
(US FDA) and European Union (EU).
(14.2 g) in water (500 mL).
Pretreatment 3. Mix A (445.5 mL) and B (54.5 mL).
1. Weigh 1 g sample into a 30 mL centrifuge tube. McIlvaines Buffer (pH 2.6): methanol (50:50 v/v):
2. Add 50 μL TMPD* solution (1 mg/mL). Mix McIlvaines Buffer (pH 2.6) (250 mL) with methanol (250 mL).
3. Add standard solutions (MG, LMG, 0.1 µg/mL) and internal
standard, leave to stand for 10 minutes.
LC Conditions
4. Add 10 mL McIlvaines Buffer (pH 2.6) /methanol
(50:50, v/v) solution; homogenize for 45 seconds.
Column: SunFire™ C18, 4.6 x 150 mm, 5 μm
5. Centrifuge at 5000 rpm for 20 minutes, transfer supernatant Self-packed PbO2 oxidising column: 4.6 x 20 mm,
into centrifuge tube. (PbO2 : diatomaceous earth = 3:1); Both ends are
fitted with 2 μm stainless steel frits.This column
6. Repeat steps 4 and 5, combine the two portions of supernatant. is connected between the analytical columns and
A 20 mL aliquot will be used for SPE. the detector.
*TMPD= N, N, N’, M’- Tetramethyl-1,4-phenylenediamine dihydrochloride Flow Rate: 2 mL/min
Mobile Phase: A. 125 mM ammonium acetate, adjust to pH 4.5
SPE Procedure with formic acid
B. acetonitrile
Oasis® MCX 6cc/150 mg Gradient: Time (min) A% B%
0.00 45 55
A. 5 mL methanol 7.00 10 90
B. 5 mL water 10.00 10 90
C. 5 mL McIlvaines Buffer (pH 2.6) Injection Volume: 50 µL
Detector: 2487 Dual Wavelength UV Detector
Load:
20 mL of sample UV Wavelength: 619 nm
Wash:
A. 5 mL 0.1 N hydrochloric acid C. 4 mL 50% methanol/water
B. 2 x 5 mL water D. 6 mL hexane, vacuum dry
elute: Ordering Information

10 mL
50% ethyl acetate: 45% methanol: 5% ammonium hydroxide (v/v/v) Description Part Number
Dry eluant at 50 °C under nitrogen SunFire C18, 4.6 x 150 mm, 5 μm 18600255
Reconstitute with 50% acetonitrile (100 µL)
Ref: Jointly developed by Waters China applications team and Beijing CIQ
©2011 Waters Corporation. Waters, Oasis, SunFire, and Alliance are trademarks of Waters Corporation. All other trademarks are
the property of their respective owners.
[ 42 ]
Ma l ac hit e G r e en in F ish (U P LC / MS / MS)
INTRODUCTION
Solutions
Malachite Green (MG) is an effective and inexpensive fungicide
used in aquaculture. During metabolism MG reduces to McIlvaines Buffer (pH 2.6) :
Leucomalachite Green (LMG), which has been shown to accumlate 1. 0.1 M citric acid monohydrate (A) - Dissolve citric acid
in fatty fish tissues. Both MG and LMG have demonstrated monohydrate (10.5 g) in water (500 mL).
putative carcinogenic activity, and thus, have been banned for
2. 0.2 M disodium hydrogen phosphate dihydrate (B) -
use in aquaculture by both the United States Food and Drug
Dissolve disodium hydrogen phosphate dihydrate
Administration (US FDA) and European Union (EU).
(14.2 g) in water (500 mL).
Pretreatment 3. Mix A (445.5 mL) and B (54.5 mL).
1. Weigh 1 g sample into a 30 mL centrifuge tube. McIlvaines Buffer (pH 2.6): methanol (50:50 v/v):
2. Add 50 μL TMPD* solution (1 mg/mL). Mix McIlvaines Buffer (pH 2.6) (250 mL) with methanol (250 mL).
3. Add standard solutions MG, LMG, 0.1 µg/mL and internal

standard, leave to stand for 10 minutes.
LC Conditions
4. Add 10 mL McIlvaines Buffer (pH 2.6) /methanol (50:50 v/v)
solution; homogenize for 45 seconds. Instrument: Waters ACQUITY UPLC® System
5. Centrifuge at 5000 rpm for 20 minutes, transfer supernatant
into centrifuge tube.
6. Repeat steps 4 and 5, combine the two portions of supernatant. B. 0.1% formic acid in acetonitrile
A 20 mL aliquot will be used for SPE. Gradient: Time (min) A% B%
0.00 40 60
*TMPD= N, N, N’, M’- Tetramethyl-1,4-phenylenediamine dihydrochloride 3.00 5 95
5.00 40 60
SPE Procedure

MS Conditions
Condition/Equilibrate: Instrument: Waters Quattro Premier™

A. 2 mL methanol Ionization Mode: Positive electrospray (ESI +)
B. 2 mL water
C. 2 mL McIlvaines Buffer (pH 2.6)
Load: Analyte MRM Transition

20 mL of sample 331.2→239.1
LMG
331.2→316.2
Wash:
A. 2 mL 0.1N HCl B. 2 x 2.5 mL water 329.2→208.1
MG
C. 2 mL 50% methanol/water D. 3 mL hexane, vacuum dry 329.2→313.1

elute:
5 mL
50% ethyl acetate:45% methanol:5% ammonium hydroxide (v/v/v)
Dry eluant at 50 °C under nitrogen
Reconstitute with 50% acetonitrile (100 µL)
[ 43 ]
Ma l ac hit e G r e en in F ish (U P LC / MS / MS)
Results
LMG
MG
The LOD of LMG and MG are 0.02 ppb and 0.01 ppb, respectively. The recoveries
of LMG and MG in fish is between 50-80%.

ACQUITY UPLC BEH C18, 2.1 x 50 mm, 1.7 μm, 3/pk 176000863
©2011 Waters Corporation. Waters, Oasis, ACQUITY UPLC, and Quattro Premier are trademarks of Waters Corporation. All other
trademarks are the property of their respective owners.
[ 44 ]
M e l amin e an d C yanu ric ac id in in fant fo rmu l a using H P LC
INTRODUCTION Cyanuric Acid SPE Cleanup

Oasis MAX cartridges, 6 cc/150 mg
Responding to recent worldwide concern related to melamine
in food, the United States Food and Drug Administration Condition:
(US FDA) issued an interim method for the determination of A. 5 mL 0.1 M HCl in acetonitrile
B. 5 mL 0.1 M NaOH in acetonitrile
residual melamine and cyanuric acid in foods using LC/MS/MS.
Equilibrate:
Pretreatment A. 5 mL acetonitrile
B. 5 mL 5% NH 4OH in water
1. Weigh 5 g of liquid infant formula, or 1 g of dry infant

Load:
formula, and add 4 mL of water.
A. 3 mL 5% NH 4OH in water
B. Add 2 mL of sample supernatant
2. Add 500 ng (500 μL of 1 μg/mL stock) of isotopically-
labeled melamine.
WASH:
3. Add 2500 ng (250 μL of 10 μg/mL stock) of isotopically- 5 mL acetonitrile
labeled cyanuric acid. ELUTE:

2 mL 4% formic acid in acetonitrile
4. Add 20 mL of 50:50 acetonitrile:water.
5. Shake 10-20 minutes. Filter eluent into vial using 0.2 μm PTFE syringe filters and syringes
6. Centrifuge for 10 minutes at 3400 rpm. DILUTE:

Cyanuric acid calibration standards accordingly
SPE Procedure LC Conditions

Melamine SPE Cleanup Instrument: Waters ACQUITY UPLC® System
Oasis MCX, 6 cc/150 mg
®
Column: Atlantis ® HILIC Silica 2.1 x 150 mm, 3 μm
Mobile Phase A: 10 mM Ammonium acetate in 50/50 Acetonitrile/H2O
Condition:
A. 5 mL 0.1 M NaOH in acetonitrile Mobile Phase B: 10 mM Ammonium acetate in 95/5 Acetonitrile/H2O
B. 5 mL 0.1M HCl in acetonitrile Gradient Table: Time Flow Rate %A %B Curve
(min) (mL/min)
Equilibrate: Initial 0.5 0 100 -
A. 5 mL acetonitrile 2.00 0.5 0 100 6
B. 5 mL 4% formic acid in water 3.50 0.5 60 40 6
5.00 0.5 60 40 6
5.20 0.8 0 100 6
Load:
11.00 0.8 0 100 6
A. 3 mL of 4% formic acid in water
B. Add 2 mL of sample supernatant 11.10 0.5 0 100 6
14.00 0.5 0 100 6
Wash:
A. 5 mL acetonitrile
B. 5 mL 0.2% diethylamine in acetonitrile
ELUTE:
4 mL 2% diethylamine in acetonitrile
Filter eluent into vials using 0.2 μm PTFE syringe filters and syringes
[ 45 ]
M e l amin e an d C yanu ric ac id in in fant fo rmu l a using H P LC
MS Conditions 100
Liquid Infant Formula Fortified Cyanuric Acid at 500 µg/kg
MS System: Waters ACQUITY TQD ®
Software: MassLynx™ v.4.1

Ionization Mode: ESI Positive (melamine and 13C 315N3 melamine) ESI
Negative (cyanuric acid and 13C 315N3 cyanuric acid)
%
MRM Cone Collision

Compound Ionization
Transitions Voltage(V) Energy (eV) Liquid Infant Formula Fortified Cyanuric Acid at 100 µg/kg
0.59
127→85 40 17
Liquid Infant Formula Blank
Melamine Positive
127→68 40 25
0
C 315N3
13 133→89 40 17 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 min
Positive Cyanuric Acid in Liquid Infant Formula at 100 ppb and 500 ppb using
Melamine 133→45 40 26
Atlantis HILIC Silica Column.
128→42 30 13
Cyanuric Acid Negative
128→85 30 11 Single Day Results Multi-Day Results
Spiking
134→44 30 13 Type Average Spike % Average Spike %
C 3 N3
13 15 Concentration
Negative Recovery ± % RSD (n) Recovery ± % RSD (n)
Cyanuric Acid
134→89 30 11 500 µg/kg Dry 113.3 ± 8.8 (n = 5) 109.2.0 ± 7.7 (n = 11)
MRM Conditions for Melamine, 13C315N3 Melamine, Cyanuric Acid, and 13C315N3 2500 µg/kg Dry 108.5 ± 5.5 (n = 5) -
Cyanuric Acid. 10 µg/kg Liquid 110.2 ± 13.2 (n = 5) 104.7 ± 8.2 (n = 8)
100 µg/kg Liquid 113.8 ± 12.7 (n = 5) 116.4 ± 10.6 (n = 8)
Results
Melamine Spike % Recovery in Dry and Liquid Infant Formula using Atlantis
1.81
100 HILIC Silica Column.
Dry Infant Formula Fortified Melamine at 2500 µg/kg
Single Day Results Multi-Day Results

Spiking
Type Average Spike % Average Spike %
Concentration
Recovery ± % RSD (n) Recovery ± % RSD (n)
500 µg/kg Dry 115.7 ± 1.8 (n = 5) 113.9 ± 2.7 (n = 8)
% 2500 µg/kg Dry 104.7 ± 3.9 (n = 5) 104.7 ± 3.1 (n = 8)
100 µg/kg Liquid 109.2 ± 2.9 (n = 5) 108.4 ± 3.1(n = 8)
500 µg/kg Liquid 103.9 ± 2.1 (n = 5) 103.4 ± 3.3 (n = 8)
Dry Infant Formula Blank MRM method parameters.
0
1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 min
Melamine in Dry Infant Formula at 500 ppb and 2500 ppb using Atlantis Ordering Information
HILIC Silica Column.
HPLC Melamine Analysis Package 176001773
Atlantis HILIC Silica, 2.1 x 150 mm, 3 µm 186002015

©2011 Waters Corporation. Waters, Oasis, ACQUITY UPLC, ACQUITY, Atlantis, and MassLynx are trademarks of
Waters Corporation.
[ 46 ]
M e l amin e an d C yanu ric ac id in in fant fo rmu l a using U P LC
INTRODUCTION Cyanuric Acid SPE Cleanup

Oasis MAX cartridges, 6 cc/150 mg
in food, the United States Food and Drug Administration Condition:
(US FDA) issued an interim method for the determination of A. 5 mL 0.1 M HCl in acetonitrile
B. 5 mL 0.1 M NaOH in acetonitrile
Equilibrate:
Pretreatment A. 5 mL acetonitrile
B. 5 mL 5% NH 4OH in water
1. Weigh 5 g of liquid infant formula, or 1 g of dry infant
formula, and add 4 mL of water. Load:
3 mL 5% NH 4OH in water
2. Add 500 ng (500 μL of 1 μg/mL stock) of isotopically- Add 2 mL of sample supernatant
labelled melamine.
WASH:
3. Add 2500 ng (250 μL of 10 μg/mL stock) of isotopically- 5 mL acetonitrile
labelled cyanuric acid.
ELUTE:
4. Add 20 mL of 50:50 acetonitrile:water. 2 mL 4% formic acid in acetonitrile
5. Shake 10-20 minutes.

Filter eluent into vial using 0.2 μm PTFE syringe filters and syringes
6. Centrifuge for 10 minutes at 3400 rpm.
DILUTE:
Cyanuric acid calibration standards accordingly
SPE Procedure
LC Conditions
Melamine SPE Cleanup
Oasis® MCX, 6 cc/150 mg Instrument: Waters ACQUITY UPLC® System
Column: ACQUITY UPLC BEH HILIC, 2.1 x 100 mm, 1.7 μm
Condition: Mobile Phase A: 10 mM Ammonium acetate
A. 5 mL 0.1 M NaOH in acetonitrile
Mobile Phase B: 10 mM Ammonium acetate in 95/5 Acetonitrile/H2O
B. 5 mL 0.1M HCl in acetonitrile
Gradient Table: Time Flow Rate %A %B Curve
Equilibrate: (min) (mL/min)
A. 5 mL acetonitrile Initial 0.6 0 100 -
B. 5 mL 4% formic acid in water 0.80 0.6 0 100 6
2.30 0.6 22 78 6
2.80 0.6 22 78 6
Load: 2.90 0.6 0 100 6
A. 3 mL of 4% formic acid in water
4.00 0.6 0 100 6
B. Add 2 mL of sample supernatant
Wash:
A. 5 mL acetonitrile
B. 5 mL 0.2% diethylamine in acetonitrile
ELUTE:
Filter eluent into vials using 0.2 μm PTFE syringe filters and syringes
[ 47 ]
M e l amin e an d C yanu ric ac id in in fant fo rmu l a using U P LC
MS Conditions 100

MS System: Waters ACQUITY® TQD
Software: MassLynx™ v.4.1
Ionization Mode: ESI Positive (melamine and 13C 315N3 melamine) ESI
Negative (cyanuric acid and 13C 315N3 cyanuric acid)
%
MRM Cone Collision

Compound Ionization
Transitions Voltage (V) Energy (eV) 0.59
127→85 40 17
Melamine Positive
127→68 40 25 Liquid Infant Formula Blank
133→89 40 17
C 3 N3
13 15
Positive 0
Melamine 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 min
133→45 40 26
128→42 30 13 Cyanuric Acid in Liquid Infant Formula at 100 ppb and 500 ppb using
Cyanuric Acid Negative Atlantis HILIC Silica Column.
128→85 30 11
134→44 30 13 Single Day Results Multi-Day Results

C 315N3
13
Spiking
Negative Type Average Spike % Average Spike %
Cyanuric Acid 134→89 30 11 Concentration
MRM method parameters. 500 µg/kg Dry 115.0 ± 4.7 (n = 5) 110.7 ± 6.9 (n = 11)
2500 µg/kg Dry 109.6 ± 3.1 (n = 5) -
Results
10 µg/kg Liquid 103.9 ± 10.5 (n = 5) 104.7 ± 8.2 (n = 8)
1.81
100
100 µg/kg Liquid 105.7 ± 3.2 (n = 5) 105.1 ± 4.5 (n = 8)
Melamine spike % recovery in dry and liquid infant formula using BEH HILIC
column.
Single Day Results Multi-Day Results

Spiking
Type Average Spike % Average Spike %
% Concentration
500 µg/kg Dry 114.9 ± 3.9 (n = 5) 116.1 ± 4.8 (n = 8)
2500 µg/kg Dry 109.6 ± 3.1 (n = 5) 104.9 ± 4.8 (n = 8)

Dry Infant Formula Blank 100 µg/kg Liquid 117.7 ± 4.0 (n = 5) 115.0 ± 5.0 (n = 8)
500 µg/kg Liquid 103.8 ± 5.9 (n = 5) 103.1 ± 2.9 (n = 8)

0
1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 min Cyanuric Acid Spike % Recovery in Dry and Liquid Infant Formula using BEH
HILIC Column.
tMelamine in Dry Infant Formula at 500 ppb and 2500 ppb using Atlantis
HILIC Silica Column.

UPLC® Melamine Analysis Package 176001791
ACQUITY UPLC BEH HILIC, 2.1 x 100 mm, 1.7 µm 186003461
©2011 Waters Corporation. Waters, Oasis, ACQUITY UPLC, ACQUITY, UPLC, Atlantis, and MassLynx are trademarks of
Waters Corporation.
[ 48 ]
M e l amin e an d C yanu ric ac id in in fant fo rmu l a using lc /ms / ms
Column: ACQUITY UPLC® BEH HILIC 2.1 x 150 mm, 1.7 µm
in food, the United States Food and Drug Administration
Mobile Phase A: 10 mM ammonium acetate at pH 3.9 in 50/50
(US FDA) issued an interim method for the determination of
water/acetonitrile
Mobile Phase B: 10 mM ammonium acetate at pH 3.9 in 11.5/88.5
water/acetonitrile
Sample Extraction Gradient Table: Time Flow Rate %A %B Curve
(min) (mL/min)
1. Weigh 1 g infant formula. Initial 0.45 0.0 100.0 -
5.00 0.45 0.0 100.0 6
2. Add 9 mL of sample extraction buffer into a 50 mL 6.00 0.45 0.0 100.0 11
centrifuge tube. Injection Volume: 10 µL
3. Shake or vortex the solution for 1 minute.

MS Conditions
4. Centrifuge for 15 minutes at 3500 rpm
MS System: Waters ACQUITY® TQD
Melamine SPE Cleanup
Ionization Mode: ESI Positive (melamine, ammelide and ammeline)
ESI Negative (cyanuric acid)
Condition:
A.5 mL 0.1 M NaOH in acetonitrile
B.5 mL 0.1M HCl in acetonitrile
Cone
MRM Dwell Collision
Compounds Voltage
CONDITION: Transitions (sec) Energy (eV)
(V)
A. 5 mL methanol
B. 5 mL 0.5N HCl 127→85 0.07 30 18
Melamine
127→68 0.07 30 22
LOAD:
A. 3 mL of 0.5N HCl 129→87 0.07 30 14
B. 2 mL of sample supernatant Ammelide
129→43 0.07 30 18
WASH: 128→86 0.08 30 15
A. 5 mL water Ammeline
B. 2 mL acetonitrile 128→43 0.08 30 20
128→42 0.055 25 12
Cyanuric Acid
ELUTE: 128→85 0.055 25 6
4 mL 5% ammonium hydroxide in acetonitrile
Cyanuric Acid SPE Cleanup

Sep-Pak Aminopropyl (NH2), 6 cc/500 mg
®
Condition:
LOAD:
A. 2 mL 5% NH 4OH in water
B. 3 mL of sample supernatant
ELUTE:
5 mL 10:10:80 water:formic acid:acetonitrile
[ 49 ]
M e l amin e an d C yanu ric ac id in in fant fo rmu l a using lc /ms / ms
2.24
100
%
Ammeline
0
1.87
100
%
Melamine
0
1.58
100
Ammelide
%
1.41 1.75
0
1.10
100
%
Cyanuric Acid
0
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 min
Melamine, cyanuric acid, ammelide, and ammeline of infant formula

fortified at 1.0 µg/g.
Recovery Overall Recovery

Compound Average Spike % Average % Overall
Recovery ± % RSD (n) Recovery (n)
Cyanuric Acid 102.8 ± 12.8 (n = 5) 74.4 (n = 5)
Ammelide 97.4 ± 2.5 (n = 5) 60.2 (n = 5)
Melamine 106.8 ± 2.7 (n = 5) 58.5 (n = 5)
Ammeline 94.9 ± 3.5 (n = 5) 46.2 (n = 5)
Melamine, ammelide, and cyanuric acid spike % recovery in infant

formula.

Oasis MCX, 6 cc/150 mg 186000256
Sep-Pak Aminopropyl (NH2), 6 cc/500 mg WAT054560
ACQUITY UPLC BEH HILIC 2.1 x 150 mm, 1.7 µm 186003462
Ref: Waters Application Note, 720000688EN

©2011 Waters Corporation. Waters, Oasis, Sep-Pak, ACQUITY UPLC, and ACQUITY are trademarks of Waters Corporation.
[ 50 ]
Mic ro c ys t ins in Nat u r a l Wat e rs
Microcystin-LR is a potent mammalian toxin which is known to have Instrument: Waters Quattro Ultima Pt™
been responsible for the deaths of domesticated animals, livestock Ionization Mode: Positive electrospray (ESI +)
loss, and the potential presence in potable water supplies.
Characteristic
Pretreatment Analyte MRM MW [M+H]+ [M+H]2+
Ion Fragment
278.0
1. Filter water sample through 0.45 μm membrane filter. Enkephalin 556.1→278.0 555.6 556.1 N.D
397.1
2. Add 100 µL of enkephalin (concentration 10 µg/L) to 10 mL 135.0
MCYST-LR 519.9→135.0 994.5 995.7 498.4
filtered water sample and mix thoroughly. 861.5
135.0
MCYST-RR 498.4→135.0 1037.6 1038.4 519.9
SPE Procedure 620.0
897.1
MCYST-LW 1025.8→891.7 1024.5 1025.8 N.D
Oasis HLB, 3 cc/60 mg
®
583.2
852.5
Condition/Equilibrate: MCYST-LF 986.8→852.5 985.5 986.8 N.D
A. 3 mL methanol 544.0
B. 6 mL water MRM method parameters.
Load: Results
10 mL sample (1 mL/min)
Average
Concentration RSD
Wash: Analyte Recovery
(μg/L) (%)
A. 3 mL water (%)
B. 5 mL 20% methanol
0.10 100.0 6.45
MCYST-RR 0.20 95.2 4.02
Dry cartridge by vacuum for 1 minute
0.40 90.0 4.35
0.02 105.0 5.40
elute:
5 mL methanol MCYST-LR 0.05 96.0 4.53
0.08 93.8 4.22
Evaporate to dryness at 50 °C under nitrogen stream 0.40 103.8 5.30
MCYST-LW 1.00 102.7 5.87
Reconstitute residue with 1 mL 50% methanol 1.60 93.8 5.67
0.20 103.0 7.03
LC Conditions
MCYST-LF 0.50 109.8 5.69
Instrument: Waters Alliance ® HPLC 2695 System 0.80 102.3 4.57
Column: Symmetry300 C18, 4.6 x 75 mm, 3.5 µm
™ Recovery data for spiked samples at various concentrations.
Flow Rate: 0.2 mL/min Ordering Information

B. 0.2% formic acid in methanol Description Part Number
Gradient: Time (min) A% B% Oasis HLB, 3 cc/60 mg, 30 μm, 100/box WAT094226
0.00 45 55 Symmetry300 C18, 4.6 x 75 mm, 3.5 μm 186000189
12.00 10 90
Nylon Filter 0.45 μm WAT200524
12.50 0 100
15.00 0 100 LCMS Certified Vials 600000751CV
15.10 45 55 Ref: Determination of Microcystins in Natural Water by Liquid Chromatography
25.00 45 55 Tandem Mass Spectrometry, Chen Qi, Huang Baifen, Zhang Jing, Ren Yiping;
Injection Volume: 10 µL Zhejiang Provincial Center for Disease Prevention and Control
©2011 Waters Corporation. Waters, Oasis, Symmetry300, Alliance, and Quattro Ultima Pt are trademarks of
Column Temperature: 30 °C Waters Corporation.
[ 51 ]
Mu lt i - r e sidu e ana lysis o f p e s t ic id e s in G r ain an d B eans
INTRODUCTION Sep-Pak Vac Carbon Black/Aminopropyl, 6 cc/500 mg/500 mg
This application brief shows the step wise procedure of the Japan CONDITION:
Ministry of Health, Labor and Welfare (JPMHLW) Official Method for 10 mL of toluene:acetonitrile (1:3, v/v)
multi-residue analysis of pesticides for grain and bean samples.

Load the sample extract
Pretreatment
ELUTE:
20 mL of toluene:acetonitrile (1:3, v/v)
1. Extract 10 g sample with 20 mL of water. Add 50 mL
of acetonitrile.
Collect eluate
2. Homogenize sample. Centrifuge and collect supernatant.
3. Repeat extraction with 20 mL of acetonitrile. Evaporate to dryness
4. Collect acetonitrile layer and dilute to 100 mL with acetonitrile.

Dissolve the residue with 1 mL of acetone:n-hexane (1:1, v/v)
5. Sample 20 mL of diluted acetonitrile extract, add 10 g
sodium chloride and 20 mL of 0.5 M of phosphate buffer.
Shake for 10 minutes.
6. Collect acetonitrile layer for SPE treatment.
SPE Procedure
Sep-Pak® Vac C18, 1 g/6 cc
CONDITION:
10 mL of acetonitrile
Load the sample extract
ELUTE:
2 mL of acetonitrile
Collect eluate
Dry over anhydrous sodium sulfate
Filter
Evaporate to dryness
Dissolve the residue with 2 mL of toluene:acetonitrile (1:3, v/v)
Sep-Pak Vac C18, 1 g/6 cc WAT036905
Sep-Pak Vac Carbon Black/Aminopropyl,
186003369
6 cc/500 mg/500 mg
©2011 Waters Corporation. Waters, and Sep-Pak are trademarks of Waters Corporation.
[ 52 ]
Mu lt i - r e sidu e ana lysis o f p e s t ic id e s in v eg e ta bl e s an d f ruit s
INTRODUCTION
Pesticides* Spike Conc./μg/g Recovery (%)
Abamectin 0.1 102.0
This application brief shows the Japan Ministry of Health, Labor and
Anibfos 0.1 111.7
Welfare (JPMHLW) Method for multi-residue analysis of pesticides
Azinphos-methyl 0.1 107.6
in vegetables and fruit. This sample preparation method calls for Benzofenap 0.1 139.5
an extract from the commodity, followed by a SPE extract from a Butafenacil 0.1 104.5
Sep-Pak Vac Carbon Black/Aminopropyl column. Chloridazon 0.1 106.0
Chromafenozide 0.1 108.2
SPE Procedure Clomeprop 0.1 104.4
Cloquintocet-mexyl 0.1 108.7
Sep-Pak® Vac Carbon Black/Aminopropyl, Clothianidin 0.1 101.5
6 cc/500 mg/500 mg Cyazofamid 0.1 108.3
Cyflufenamid 0.1 110.1
CONDITION:
Dimethirimol 0.1 101.0
Toluene/acetonitrile 10 mL (1:3 v/v). Do not allow to dry
Fenoxycarb 0.1 108.7
Ferimzone 0.1 112.6
LOAD:
Formetanate hydrochloride 0.1 86.7
Rinse vials with toluene/acetonitrile (1:3 v/v) solution and load
2 mL of extracted sample Furathiocarb 0.1 100.5
Imidacloprid 0.1 111.8
ELUTE : Indoxacarb 0.1 121.2
20 mL toluene/acetonitrile (1:3 v/v). Flow: 2 mL/min Iprovalicarb 0.1 106.2
Isoxaflutole 0.1 99.5
LC Conditions Lactofen 0.1 106.8
Methoxyfenozide 0.1 103.3
Mibemectin A3 0.1 114.5
Column XTerra ® MS C18, 2.1 x 150 mm, 3.5 μm
Mibemectin A4 0.1 101.2
Flow Rate: 0.2 mL/min Naproanilide 0.1 115.9
Mobile Phase: A. water Oryzalin 0.1 103.8
B. methanol Oxycarboxin 0.1 85.1
C. 100 mM ammonium acetate in water Oxydemeton-methyl 0.1 108.0
Gradient: Time (min) A% B% C% Phenmedipham 0.1 102.2
0.00 80 15 5 Pyrazolynate 0.1 72.7
1.00 55 40 5
Quizalofop-P-tefuryl 0.1 145.3
3.50 55 40 5
Simeconazole 0.1 106.0
6.00 45 50 5
8.00 40 55 5 Thiacloprid 0.1 109.2
17.50 00 95 5 Thiamethoxam 0.1 108.3
30.00 80 15 5 Tridemorph 0.1 94.6
47.00 80 15 5 Etriticonazole 0.1 113.3
Injection Volume: 5 µL *Five replicate samples analyzed per level.
Temperature: 40 ˚C
MS Conditions Ordering Information

Multiple reaction monitoring 186003369
6 cc/500 mg/500 mg
XTerra MS C18, 2.1 x 150 mm, 3.5 μm 186000506
LCGC Certified Vials 186000272C
©2011 Waters Corporation. Waters, Alliance, XTerra, Sep-Pak and Quattro Premier are trademarks of Waters Corporation.
[ 53 ]
Multi-residue LC /MS/MS det ermination of 52 non-gas
chromatography-amenable p esticides and metabolit es in F ruit s and V egetables
This multi-residue pesticide sample preparation shows the steps
Column: Atlantis ® dC18, 2.1 x 100 mm, 5 μm
used to process fruit and vegetable samples, extract and concen-
trate the extract by an Oasis® HLB SPE method.
B. 0.01% formic acid in methanol
Pretreatment
0.00 95 50
1. Samples (lemon, raisin, tomato and avocado) were cut into
1.00 95 50
small pieces. 8.50 50 50
25.00 10 90
2. A 20 g portion of homogenized sample was mixed with 60 mL 28.00 10 90
0.1% formic acid in methanol:water (80:20, v/v). 29.00 95 50
3. Extraction for 2 minutes with Ultra-Turrax at 8000 rpm.
4. Filtration and dilution with methanol:water 0.1% formic acid

MS Conditions
(80:20, v/v) to a final volume of 100 mL.
Instrument: Waters Quattro micro™
5. 2.5 mL aliquot diluted to 20 ml with 0.1% formic acid in water.
Load 5 mL of the diluted extract onto the SPE cartridge.
Results
SPE Procedure
Methamidophos Omethoate
Oasis HLB, 6cc/200mg
Condition:
A. 5 mL methanol
B. 5 mL methanol: MTBE* (10:90) 0.1% formic acid
C. 5 mL methanol 0.1% formic acid
Ethiofencarbsulfone Ethiofencarbsulfoxide
Equilibrate:
5 mL water 0.1% formic acid
LOAD:
5 mL diluted extract
LC/MS/MS data for 4 representative pesticides.
DRY:
By passing air for through cartridge 1 hour
ELUTE:
5 mL methanol:MTBE (10:90, v/v) 0.1% formic acid, by means of gravity
Sample post-treatment:
0.5 mL water added to the extract. Evaporate with nitrogen at 40 ˚C until O rd e ring Information
0.5 mL. Adjust final volume to 1 mL with methanol:water (10:90, v/v).
*MBTE: methyl-t-buthyl ether Oasis HLB, 6 cc/200 mg, 30/box WAT106202
Atlantis dC18, 2.1 x 100, 5 µm 186001297
LCMS Certified Combination Packs 600000751CV
Ref: Journal of Chromatography A, 1109 (2006) 242-252

©2011 Waters Corporation. Waters, Oasis, Alliance, Atlantis, and Quattro micro are trademarks of Waters Corporation.
[ 54 ]
Mu lt i - r e sidu e Ana lysis o f p e s t ic id e s by Qu EC h E RS in:
Avo c a do by gc /ms
INTRODUCTION 180
160
140
Avocado is a fruit that contains fat; therefore, the recommended 120
% Recovery
100
selection of the dispersive solid-phase extraction (SPE) tube 80
60
should include C18 to remove the fats. 40
20
E x traction P roc edur e 0
in
e
nid
yl
hio
s
yl
in
zin
yl
ini
ifo
ole
ate
ral
ar
eth
ob
th
lua
Et
od
ra
r
rb
flu
az
ulf
me
py
str
-m
At
pr
Ca
lyf
Tri
on
ns
lor
y
s-
1. Add 15 mL 1% acetic acid in acetonitrile into the 50 mL
Cy
im
To
ox
uc
ifo
lfa
Ch
ox
Az
b
r
su
Te
es
py
do
Kr
lor
En
DisQuE ™ extraction tube containing 1.5 g of sodium acetate
Ch
Pesticide
and 6 g of magnesium sulfate. Pesticides in Avocados by GC/MS.
2. Add 15 g of homogenized sample into the 50 mL tube.
3. Add any internal standards and standard mixture.
4. Shake vigorously for 1 minute and centrifuge > 1500 rcf

for 5 minutes.
5. Transfer 1 mL of the acetonitrile extract into the 2 mL

clean-up tube containing 50 mg PSA, 150 mg MgSO 4, and
50 mg C18.
6. Shake for 30 seconds and centrifuge >1500 rcf for

1 minute.
7. Transfer 0.5 mL extract into a tube.
8. Add any post-extraction internal standards.
9. Add 0.25 mL toluene.
10. Evaporate at 50 °C with N2 to < 0.1 mL.
11. Bring volume up to 0.2 mL with toluene.
12. Transfer to vial with insert for analysis.
T EST CONDIT IONS

GC Conditions
Instrument: Agilent ® 6890N GC
Column: RTX-5MS, 30 x 0.25 mm, (0.25 µm film)
Carrier Gas: Helium
O RD E RING INFO RMAT ION*
Temperature Program: Initial 100 °C, hold 1 min, then 10 °C/min to Description Part Number
320 °C, hold for 7 minute DisQuE 50 mL Tube-AOAC/Acetate 186004571
Injection Volume: 2 µL splitless DisQuE 2 mL Tube-AOAC/C18 186004830
MS Conditions
Insert 300 µL with Poly Spring WAT094170
Instrument: Waters Quattro micro™ GC-MS
* For all AOAC and CEN QuEChERS methods, refer to the DisQuE Dispersive
Ionization: Electron Impact (70 eV)
Sample Preparation Brochure (lit code: 720003048EN)
Acquisition: Single Ion Recording (SIR) Mode
© 2011 Waters Corporation. Waters, DisQuE and Quattro micro are trademarks of Waters Corporation. All other trademarks
are the property of their respective owners.
[ 55 ]
Avo c a do by lc / ms / ms
LC System: Waters ACQUITY UPLC® System
Avocado is a fruit that contains fat; therefore, the recommended
Column: ACQUITY UPLC BEH C18, 2.1 x 100 mm, 1.7 μm
selection of the dispersive solid-phase extraction (SPE) tube should
include C18 to remove the fats.
Sample Temperature: 4 °C
Extraction Procedure Flow Rate: 0.3 mL/min.

Mobile Phase A: Water + 0.1% formic acid
Mobile Phase B: Methanol + 0.1% formic acid
DisQuE ™ extraction tube.
Gradient: Time Flow Rate A% B%
2. Add 15 g of homogenized sample into the 50 mL tube. 0.00 0.3 75 25
0.25 0.3 75 25
3. Add any internal standards and standard mixture. 7.75 0.3 5 100
8.50 0.3 0 100
8.51 0.5 75 25
for 5 minute. 10.50 0.5 75 25
11.0 0.3 75 25
Injection Volume: 15 μL, Partial loop injection
clean-up tube containing 50 mg PSA, 150 mg MgSO 4,
and 50 mg C18.
MS Conditions
6. Shake for 30 seconds and centrifuge >1500 rcf for 1 minute.
Instrument: Waters ACQUITY® TQ Detector
7. Transfer 100 μL of final extract into a 1.5 mL centrifuge tube.
Ionization: Positive electrospray (ESI+)
8. Add any post-extraction internal standards. Acquisition: Multiple reaction monitoring (MRM)
9. Dilute as needed with an appropriate buffer or solvent.
10. Centrifuge > 16000 rcf for 5 minutes.
11. Transfer to autosampler vial.
TEST CONDITIONS
160
140
120
100
% Recovery
80
60
40
20
O RD E RING INFO RMAT ION*

on
l
yl
l
e
nid
s
ali
yl
ini
id
os
zin
vo
bin
le
ar
ur
om
pr
zin
az
od
zo
lua
ph
rb
lor
ra
Lin
tro
clo
Im
th
pr
da
tro
Ca
ido
At
lyf
ch
ys
Me
ida
Cy
en
me
Di
am
To
ox
iab
Im
Py
Az
th
Th
Me
Pesticides
DisQuE 50 mL Tube-AOAC/Acetate 186004571
Pesticides in Avocados by UPLC®/MS/MS
DisQuE 2 mL Tube-AOAC/C18 186004830
© 2011 Waters Corporation. Waters, The Science of What’s Possible, DisQuE, ACQUITY UPLC, and ACQUITY are trademarks
of Waters Corporation.
[ 56 ]
Avo c a do an d g r a p e s by gc /ms
INTRODUCTION GC Conditions
Avocado is a fruit that contains fat; therefore, the recommended Instrument: Agilent ® 6890N GC system
selection of the dispersive SPE tube should include C18 to remove Column: Restek ® Rtx-5MS, 30 x 0.25 mm i.d., 0.25 μm df
the fats. Carrier Gas: Helium

Temperature Program: Initial temp at 80 °C, hold for 1 min, 10 °C/min to
320 °C, hold for 5 min
Extraction Procedure
1. Add 15 g of homogenized sample to the 50 mL DisQuE ™
Injection Volume: Split/splitless mode with 0.5 min purge time and
extraction tube containing 1.5 g of sodium acetate and 6 g 1 μL injection
of magnesium sulphate.
MS Conditions
2. Add 15 mL of 1% acetic acid in acetonitrile.
Instrument: Waters Quattro micro™ GC mass spectrometer
3. Add any pre-extraction internal standards.
Ionization Mode: Positive electrospray (70 eV)
4. Shake vigorously for 1 minute and centrifuge > 1500 rcf for
Selected-Ion Recording (SIR)
1 minute.
5. Transfer 1 mL of the acetonitrile extract into the 2 mL GC/MS (SIR)

DisQuE extraction tube containing 50 mg PSA and 150 mg
of magnesium sulphate. Channel Mass Mass
Phenylphenol 170 141
Atrazine 200 173
1 minute.
Chlorpyrifos methyl 286 109
7. Transfer 250 μL of final extract into an autosampler vial. DDD 235 165
Ethion 231 153
Cylohalothrin 197 181
SIE method parameters.
Chlorpyrifos Methyl Phenylphenol DDD Ethion Cyclohalothrin Thiabendazole
%Recovery (± %RSD) 97.07 % 105.19% 109.42% 103.19% 102.27% 103.58%

in Grape (± 4.98%) (± 10.31%) (± 3.01%) (± 2.10%) (± 7.33%) (± 3.99%)
%Recovery (± %RSD) 107.45% 109.64% 96.85% 99.65% 95.62% 101.46%

in Avocado (± 1.93%) (± 4.45%) (± 3.04%) (± 9.03%) (± 12.56%) (± 3.86%)
GC separation of five basic/neutral pesticides. Compounds (1) phenylphenol, (2) atrazine (IS), (3) chloropyrifos methyl, (4) DDD, (5) ethion, (6) cyclohalothrin.
O rd e ring Information*

DisQuE Dispersive Sample Preparation Kit (100/pk) 176001676
©2011 Waters Corporation. Waters, DisQuE, and Quattro micro are trademarks of Waters Corporation. All other trademarks
[ 57 ]
BAby foo d by u p lc / ms / ms
The application brief uses QuEChERS extraction procedure plus

UPLC®/MS/MS to analyze pesticides in fruit- and meat-based
baby food extracts. This method exceeds both current European
and worldwide legislation requirements.
Sample Temperature: 4 ˚C
1. Add 15 g of homogenized sample to the 50 mL DisQuE ™ Mobile Phase B: Methanol + 0.1% formic acid
extraction tube containing 1.5 g of sodium acetate and Gradient: Time (min) A% B%
0.00 99 1
6 g of magnesium sulphate.
5.00 1 99
2. Add 15 mL of 1% acetic acid in acetonitrile. 6.00 1 99
6.10 99 1
3. Add any pre-extraction internal standards. 8.00 99 1
Total Run Time: 8 min
Injection volume: 50 μL, full loop injection
for 1 minute.
5. Transfer 1 mL of the acetonitrile extract into the 2 mL MS Conditions

MS System: Waters Xevo™ TQ MS
6. Shake for 30 seconds and centrifuge >1500 rcf for Multiple reaction monitoring
1 minute.
100
7. Transfer 250 μL of final extract into an autosampler vial.

1.35
17
1
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 min
17 pesticide residues in one injection at 1 ng/mL in water.
[ 58 ]
BAby foo d by u p lc / ms / ms
MRM
Peak Pesticide RT Dwell Time (s) Cone Voltage (V) Collision Energy (eV)
Transitions
214→183 12
1 Omethoate 0.97 0.08 16
214→155 15
247→169 14
2 Oxydemeton-S-methyl 1.35 0.04 18
247→109 18
263→169 16
3 Demeton-S-methyl sulfone 1.39 0.04 20
263→121 16
230→125 20
4 Dimethoate 1.79 0.10 12
230→171 14
293→237 18
5 Fensulfothion-oxon 2.32 0.04 22
293→265 13
309→253 15
6 Fensulfothion-oxon-sulfone 2.39 0.04 19
309→175 25
231→89 12
7 Demeton-S-methyl 2.63 0.10 12
231→61 22
291→185 13
8 Disulfoton sulfoxide 2.93 0.04 15
291→97 32
307→97 28
9 Disulfoton sulfone 2.98 0.02 16
307→115 23
309→281 14
10 Fensulfothion 3.10 0.02 25
309→157 24
325→269 15
11 Fensulfothion sulfone 3.17 0.02 19
325→297 11
321→171 11
12 Terbufos sulfone 3.30 0.03 19
321→115 28
305→187 11
13 Terbufos sulfoxide 3.32 0.03 10
305→131 27
243→131 19
14 Ethoprophos 3.68 0.10 18
243→173 14
275→89 10
15 Disulfoton 4.03 0.08 14
275→61 32
271→159 14
16 Cadusafos 4.09 0.02 16
271→131 22
289→103 9
17 Terbufos 4.28 0.06 12
289→233 5
Xevo TQ MS MRM method parameters.

ACQUITY UPLC BEH C18, 2.1 x 50, 1.7 µm 186002350
©2011 Waters Corporation. Waters, DisQuE, ACQUITY UPLC, and Xevo are trademarks of Waters Corporation.
[ 59 ]
f lou r by gc / ms
Flour is low water content commodity that requires the addition of
water and soak time as a pretreatment step before extraction.
Carrier Gas: Helium
Sample Pretreatment:
Temperature Program: Initial 100 °C, hold 1 min, then 10 °C/min to
1. Add 5 g of flour and 10 mL of water in a tube and soak for 320 °C, hold for 7 minute
10 minutes. Injection Volume: 2 µL splitless
Extraction Procedure MS Conditions

DisQuE ™ extraction tube 1 containing 1.5 g of sodium
acetate and 6 g of magnesium sulfate.
160
2. Add soaked flours sample into the 50 mL tube. 140
120
3. Add any internal standards and standard mixture. 100
% Recovery
80
4. Shake vigorously for 1 minute and centrifuge > 1500 rcf for 60
5 minute. 40
20
0
in
Kr ben ion
lor hlo aryl
Ca in
n s nil
str e
Tri id
-m os
en thrin
zin
ral
Bi n
bu done
ym l
r
yl
To le
n
o
Pe yl
lfa rodi
f
th
i
ate
DisQuE clean-up tube 2, containing 50 mg PSA and 150 mg
h
ox zene
ob
lua
rif pyri
Pr hen
zo
eth
C rb
flu
eth
Et
ra
fen
o- rme
ulf
na
At
lyf
do Cyp
ylp
-m
r
y
co
ox
os
im
oc
o
Az
Ph
of magnesium sulphate. lor
Te
su
py
es
ch
xa
En
Ch
He
Pesticides
6. Shake for 30 seconds and centrifuge >1500 rcf for 1
Pesticides in Flour by GC/MS.
minute.

[ 60 ]
f lou r by u p lc / ms / ms
Flour is low water content commodity that requires the addition of
Ionization: Positive electrospray (ESI +)
water and soak time as a pretreatment step before extraction.
Acquisition: Multiple reaction monitoring (MRM)
Sample Pretreatment: 160

140
1. Add 5 g of flour and 10 mL of water in a tube and soak for 120

100
% Recovery
10 minutes. 80
60
40
Extraction Procedure 20
0
ch l
Cy yl
ida lil
am uron
nid
ine
id
me yl
ini
in
le
e
vo
yl
os
le
ar
pr
zin
zo
ob
az
od
z
lua
eth
zo
lor
ph
rb
o
ra
clo
Lin
da
tro
str
Im
th
pr
na
Ca
ido
At
lyf
-m
Me
en
y
co
Di
To
ox
im
DisQuE ™ extraction tube 1 containing 1.5 g of sodium
iab
bu
Im
Py
Az
ox
th
Te
Th
es
Me
Kr
acetate and 6 g of magnesium sulfate. Pesticides
Pesticides in Flour by UPLC/MS/MS.
2. Add soaked flours sample into the 50 mL tube.
5 minute.

LC Conditions
Flow Rate: 0.3 mL/min.
0.00 0.3 75 25
0.25 0.3 75 25
7.75 0.3 5 100
8.50 0.3 0 100
8.51 0.5 75 25 O RD E RING INFO RMAT ION*
10.50 0.5 75 25
11.0 0.3 75 25 Description Part Number
Injection Volume: 15 μL, Partial loop injection DisQuE Dispersive Sample Preparation Kit (100/pk) 176001676
© 2011 Waters Corporation. Waters, DisQuE, ACQUITY UPLC, and ACQUITY are trademarks of Waters Corporation.
[ 61 ]
G r a p e s by GC / ms
Grapes are a commodity containing an ample amount of water. This
sample uses the standard Association of Analytical Communities
(AOAC) extraction and clean-up tubes.
140
100
% Recovery
80
1. Add 15 mL 1% acetic acid in acetonitrile into the 50 mL 60
DisQuE ™ extraction tube 1 containing 1.5 g of sodium 40

20
acetate and 6 g of magnesium sulfate. 0
in
im hion
Ca n
lor hlor yl
n s inil
ys e
Tri d
-m s
ylp n
ri
bin
ym l
n
rif yrifo
bu one
ni
ral
yl
r
Pe yl
le
o
i
ate
Az razi
hr
th
en
d
lua
eth
zo
eth
rb
flu
t
tro
o
fen
o- rmet
id
E
ulf
h
do Cypr
na
p
At
lyf
-m
Bi
co
To
os
ox
en
oc
lfa
C
ox
Pr
Ph
Te
su
py
es
Kr
En
Ch
Pesticide
4. Shake vigorously for 1 minute and centrifuge > 1500 rcf Pesticides in Grapes by GC/MS.
for 5 minute.


minute.
GC Conditions
Carrier Gas: Helium
Temperature Program: Initial 100 °C, hold 1 min, then 10 °C/min to
320 °C, hold for 7 minute O rd e ring Information*
Injection Volume: 2 µL splitless
[ 62 ]
G r a p e s by u p lc / ms / ms
Grapes are a commodity containing an ample amount of water. This
sample uses the standard Association of Analytical Communities
(AOAC) extraction and clean-up tubes.
160
120
100
% Recovery
40
0
ida lil
ido n
ys e
yl
Di inil
yl
nid
s
id
bin
ro
zin
e
a
ar
vo
le
os
om
iab ole
zin
pr
az
u
od
lua
zo
rb
tro
ra
lor
ph
Lin
clo
z
th
Im
tro
pr
da
Ca
At
na
lyf
ch
Me
Cy
me
en
co
ox
To
am
Im
bu
Py
Az
th
Te
Th
Me
4. Shake vigorously for 1 minute and centrifuge > 1500 rcf Pesticides
for 5 minute. Pesticides in Grapes by UPLC /MS/MS.

7. Transfer 100 μL of final extract into an autosampler vial.
LC Conditions
0.00 0.3 75 25
0.25 0.3 75 25
7.75 0.3 5 100
8.50 0.3 0 100 O rd e ring Information*
8.51 0.5 75 25
11.0 0.3 75 25 DisQuE Dispersive Sample Preparation Kit (100/pk) 176001676
Injection Volume: 15 μL, Partial loop injection ACQUITY UPLC BEH C18, 2.1 x 100 mm, 1.7 μm 186002352
© 2011 Waters Corporation. Waters, T he Science of W hat’s Possible, DisQuE, ACQUITY UPLC, and ACQUITY are trademarks
of Waters Corporation.
[ 63 ]
O r ang e s by gc / ms
Instrument: Waters Quattro micro™ GC/MS
Oranges are a commodity containing an ample amount of
water. This sample uses the standard Association of Analytical
Communities (AOAC) extraction and clean-up tubes.
180

140
120
% Recovery
100
60
20
in
Ca n
l
ion
lor aryl
Tri d
e
-m l
ini
ylp n
ri
ym l
i
in
yl
zin
bu one
ni
ral
Pe yl
To le
on
ri
im e
th
n
th
ob
eth
od
lua
th
zo
eth
n
rb
flu
py thal
he
ra
fen
es nze
id
E
o- rme
str
pr
na
At
lyf
-m
Cy
Bi
o
y
co
e
os
ox
ob
en
oc
rif
Az
Ch
lor
ox
Ph
Pr
Te
ch
lor
Kr
xa
Ch
He
Pesticide
4. Shake vigorously for 1 minute and centrifuge > 1500 rcf Pesticides in Oranges by GC/MS.
for 5 minute.


1 minute.
GC Conditions
Carrier Gas: Helium
Temperature Program: Initial 100 °C, hold 1 min, then 10 °C/min to Ordering Information*
320 °C, hold for 7 minute
DisQuE 2 mL Tube-AOAC/C18 186004830
[ 64 ]
O r ang e s by u p lc / ms / ms
Oranges are a commodity containing an ample amount of
water. This sample uses the standard Association of Analytical
Communities (AOAC) extraction and clean-up tubes.
E x traction P roc edur e 160

140
120
100
Recovery (%)
DisQuE™ extraction tube 1. 80
60
2. Add 15 g of homogenized orange with skin into the 50 mL 40
tube. 20
0
am u ron
yl
id
e
os
yl
rid
ini
zin
bin
le
an
ar
os
om
z in
v
hy
od
lop
zo
rb
lor
ra
flu
Lin
ph
tro
et
th
pr
tro
na
Ca
At
ac
ch
ly
ido
-m
Me
ys
Cy
co
me
To
id
Di
ox
im
Im
bu
Py
Az
ox
th
Te
es
Me
Kr
for 5 minute. Pesticides
5. Transfer 1 mL of the acetonitrile extract into the 2 mL clean-up Pesticides in Oranges by UPLC /MS/MS. ®
tube containing 50 mg PSA, 150 mg MgSO4, and 50 mg C18.
7. Transfer 100 μL of final extract into a 1.5 mL centrifuge tube.
LC Conditions
0.00 0.3 75 25 O rd e ring Information*
0.25 0.3 75 25
8.50 0.3 0 100
8.51 0.5 75 25
10.50 0.5 75 25 DisQuE 2 mL Tube-AOAC/C18 186004830
11.0 0.3 75 25 ACQUITY UPLC BEH C18, 2.1 x 100 mm, 1.7 μm 186002352
Injection Volume: 15 μL, Partial loop injection LCMS Certified Vials 600000749CV
© 2011 Waters Corporation. Waters, DisQuE, ACQUITY UPLC, UPLC, and ACQUITY are trademarks of Waters Corporation.
[ 65 ]
ro l l e d Oat s by gc / ms
Instrument: Waters Quattro micro™ GC/MS
Rolled oats are a low water content commodity that requires the
addition of water and soak time as a pretreatment step before
extraction.
140
120
Sample Pretreatment: 100
% Recovery
80
60
1 Add 7.5 g of ground rolled oats and 15 mL of water in a 40
tube and soak for 10 minutes. 20

0
in
rin
n s nil
lor lorp l
-m n
ine
-m s
ry
in
ym l
Cy l
in
bu one
hio
ifo
ral
yl
le
y
ate
i
Ph ethr
th
n
eth
ob
od
z
eth
zo
yr
rb
flu
he
ra
Et
fen
id
ulf
str
pr
na
Ca
Extraction Procedure:
At
ylp
rm
Tri
Bi
y
co
os
Pe
ox
im
en
oc
Ch
lfa
rif
Az
ox
Pr
Te
su
py
es
o-
do
Kr
En
Ch
1. Add 15 mL 1% acetic acid in acetonitrile into the 50 mL Pesticide
DisQuE extraction tube 1 containing 1.5 g of sodium

™ Pesticides in Rolled Oats by GC/MS.
acetate and 6 g of magnesium sulfate.
2. Add soaked oat sample into the 50 mL tube.
5 minute.


1 minute.
GC Conditions
Instrument: Agilent ® 6890N GC O rd e ring Information*
Carrier Gas: Helium
Temperature Program: Initial 100 °C, hold 1 min, then 10 °C/min to Insert 300 µL with Poly Spring WAT094170
320 °C, hold for 7 minute
[ 66 ]
Ro l l e d oat s by u p lc / ms / ms
Rolled oats are a low water content commodity that requires the
addition of water and soak time as a pretreatment step before
extraction.
Extraction Procedure Flow Rate: 0.3 mL/min.

1. Add 15 mL 1% acetic acid in acetonitrile into the 50 mL Mobile Phase B: Methanol + 0.1% formic acid
DisQuE ™ extraction tube 1 containing 1.5 g of sodium Gradient: Time Flow Rate A% B%
acetate and 6 g of magnesium sulfate. 0.00 0.3 75 25
2. Diluted 7.5 g ground rolled oats with 15 mL water and soak 0.25 0.3 75 25
for 10 min. 7.75 0.3 5 100

8.50 0.3 0 100
3. Add sample into the 50 mL tube.
8.51 0.5 75 25
4. Add any internal standards and standard mixture. 10.50 0.5 75 25
5. Transfer 1 mL of the acetonitrile extract into the 2 mL 11.0 0.3 75 25
DisQuE clean-up tube 2, containing 50 mg PSA and 150 mg Injection Volume: 15 μL, Partial loop injection
6. Transfer 1 mL of the acetonitrile extract into the clean-up MS Conditions

tube 2. Instrument: Waters ACQUITY® TQ Detector
7. Shake for 30 seconds and centrifuge >1500 rcf for 1 Ionization: Positive electrospray (ESI+)
minute. Acquisition: Multiple reaction monitoring (MRM)

140
8. Transfer 100 μL of final extract into a 1.5 mL centrifuge 120
tube. 100
80
% Recovery
9. Add any post-extraction internal standards. 60
40
10. Dilute as needed with an appropriate buffer or solvent. 20
0

yl
ida lil
ido n
nid
e
id
me yl
ini
o
in
le
iab ine
zin
vo
os
ar
le
a
ur
pr
om
zo
az
ob
od
lua
zo
lor
rb
ph
ra
z
Lin
clo
da
Im
th
tro
str
pr
na
Ca
At
lyf
ch
Me
Cy
en
y
co

Di
To
ox
am
Im
bu
Py
Az
th
Te
Th
Me
Pesticide
Pesticides in Rolled Oats by UPLC®/MS/MS.

© 2011 Waters Corporation. Waters, DisQuE, ACQUITY UPLC, UPLC, and ACQUITY are trademarks of Waters Corporation.
[ 67 ]
t ea s by U P LC / MS / ms
Teas are a low water content commodity that requires the addition
of hot water and soak time as a pretreatment step before extraction.
Sam pl e P r e pa ration P roc edur e Sample Temperature: 4 °C
1. Tare weigh an empty beaker. Flow Rate: 0.3 mL/min.

2. Weigh out 100 g of tea leaves in the beaker.
3. Add in 600 g of hot water at 80-85 °C to the beaker. Brew Gradient: Time Flow Rate A% B%
the tea for 20 minutes. 0.00 0.3 75 25
0.25 0.3 75 25
4. Weigh the beaker with water and tea. 7.75 0.3 5 100
8.50 0.3 0 100
5. Calculate the weight of water loss due to evaporation. 8.51 0.5 75 25
10.50 0.5 75 25
Add water to the beaker to make up for the loss of water.
11.0 0.3 75 25
6. Homogenize the sample until it reaches consistent texture. Injection Volume: 15 μL, Partial loop injection
Extraction Procedure MS Conditions

1. Transfer 15 g of homogenized sample into an empty Instrument: Waters ACQUITY® TQ Detector
50 mL tube. Ionization: Positive electrospray (ESI+)

225
200

% Recovery
125
4. Transfer all the powder in the DisQuE extraction tube 1 into 100
75
the 50 mL containing sample and solvent. 50
25
5. Shake vigorously for 1 minute and centrifuge > 1500 rcf 0
n
for 5 minute.
hio
n
os
s
s
s
s
ne
l
ole
im
hio
ion
ho
zo
vo
ifo
ho
ph
Et
alo
az
on
laz
lP
lor
lat
th
op
yr
za
d
ida
os
ch
op
ina
en
ch
Ma
yc
en
Tri
Ph
xa
lor
Di
of
th
rb
Qu
Tri
He
Pr
Me
Ch
Ca
6 Transfer 1 mL of the acetonitrile extract into the 2 mL

Pesticide Green Tea Black Tea
Pesticides in Teas by UPLC /MS/MS. ®

minute.
8. Transfer 100 μL of final extract into an autosampler vial. Ordering Information*

10. Dilute as needed with an appropriate buffer or solvent. DisQuE Dispersive Sample Preparation Kit (100/pk) 176001676
© 2011 Waters Corporation. Waters, T he Science of W hat’s Possible, DisQuE, ACQUITY UPLC, UPLC, and ACQUITY are
trademarks of Waters Corporation.
[ 68 ]
v eg e ta bl e s an d f ruit s by u p lc / ms / ms
Most fruits and vegetables are commodities containing an ample
Ionization Mode: Positive electrospray (ESI+)
amount of water. These samples use the standard Association of
Analytical Communities (AOAC) extraction and clean-up tubes.
100
1. Add 15 g of homogenized sample to the 50 mL DisQuE ™

extraction tube containing 1.5 g of sodium acetate and 6 g
of magnesium sulphate. %
2. Add 15 mL of 1% acetic acid in acetonitrile.
3. Add any pre-extraction internal standards.
4. Shake vigorously for 1 minute and centrifuge >1500 rcf

0
for 1 minute. 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 min
5. Transfer 1 mL of the acetonitrile extract into the 2 mL 402 Pesticide Residues In One 10 Minute Run In Injection Solvent.

of magnesium sulphate. 120
Baby Food Mango Avocado
100
Recovery (%)
80
7. Transfer 250 μL of final extract into an autosampler vial. 60
40
8. Add any post-extraction internal standards. 20
0
9. Dilute as needed with an appropriate buffer or solvent. Atrazine Carbendazim Cyprodinil Flufenacet Imazalil Tebuconazole Triabendazole
LC Conditions Pesticide
Recovery Data for Three Types of Sample Matrices.

Mobile Phase A: 98:2 water: 0.1% formic acid in methanol
0.00 90 10
0.25 90 10 O rd e ring Information*
7.75 0 100
8.50 0 100 Description Part Number
8.51 90 10 DisQuE Dispersive Sample Preparation Kit (100/pk) 176001676
Total Run Time: 10 min ACQUITY UPLC BEH C18, 2.1 x 100, 1.7 µm 186002352
Injection Volume: 20 μL, full loop injection LCMS Certified Vials 600000751CV
©2011 Waters Corporation. Waters, DisQuE, ACQUITY UPLC, and ACQUITY are trademarks of Waters Corporation.
[ 69 ]
Pa r aquat an d ot h e r Quat e rna ry Ammonium Com p oun ds in Wat e r
INTRODUCTION Analyte MRM Transition

171→77
Paraquat is one of the most widely used herbicides in the world. Paraquat
171→155
As with many herbicides it is dangerously poisonous to humans
183→157
if swallowed. Diaquat
183→168

Pretreatment
Results
1. Adjust sample to pH 7 by adding 1 M ammonium phosphate
10
buffer dropwise to 20 mL river water.
SPE Procedure Paraquat
Oasis® WCX, 3cc/60mg
A. 1 mL methanol
B. 1 mL water
1
10
Load:
20 mL sample
Diquat
Wash:
A. 1 mL 1 M pH 7 phosphate buffer
B. 1 mL water
C. 1 mL methanol
ELUTE:
1
1.5 mL acetonitrile/water/trifuoroacetic acid (84:14:12, v/v/v)
1 2 3 4 5 6 7
Reconstitute in 0.5 mL mobile phase LC/MS separation of paraquat and diquat at 0.5 μg/L.
LC Conditions  Paraquat  Diquat

Day 1 1.08 μg/L (8.1% RSD) 1.05 μg/L (2.9% RSD)
Column: Atlantis ® HILIC, 2.1 x 150 mm, 3.5 μm
Mobile Phase: 40% acetonitrile Intraday results obtained from spiked water samples (spike level 1.0 µg/L).
60% 250 mM ammonium formate (pH 3.7)
MS Conditions
Instrument: Waters Quattro micro™
Oasis WCX, 3 cc/60 mg, 60 µm, 100/box 186002497
Multiple reaction monitoring Atlantis HILIC, 2.1 x 150 mm, 3.5 μm 186002015
750 μL Polypropylene Vials 186002635
Ref: Waters Application Note WA40524

©2011 Waters Corporation. Waters, Oasis, Atlantis, Alliance, and Quattro micro are trademarks of Waters Corporation.
[ 70 ]
Pat u l in in App l e Juic e
Patulin is a mycotoxin that is produced by certain species of
Ionization Mode: Negative electrospray (ESI -)
Penicillium, Aspergillus, and Byssochylamys molds that may grow
on a variety of foods including fruit, grains, and cheese. Patulin is a
safety concern in apple juice.
Analytes MRM Transition
153→109
SPE Procedure Patulin
153→81
5-hydroxymethylfurfural (HMF) 125→95
Oasis® HLB, 3cc/60mg
Condition:
A. 3 mL methanol Results
B. 3 mL water
HMF
3. 0e - 1
Load:
2.5 mL sample 2.5e - 1
2.0e - 1
Wash 1:
AU
1.5e - 1
3 mL 1% sodium bicarbonate (1g/100mL)
1.0e - 1 Patulin
Wash 2: 5.0e - 2
1 mL 0.1% acetic acid

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 min
DRY: Apple juice extract at 50 µg/kg containing patulin and 5-hydroxymethylfurfural

Using vacuum manifold (HMF) at 276 nm.
100
Patulin
Elute:
2 x 1.5 mL 10% ethyl acetate in methyl t-butyl ether (MTBE) %
0
Reconstitute:
500 µL water 100
7528
HMF
%
LC Conditions
0
0.6 0.8 1 1.2 1.4 1.6 1.8 min
Apple juice extract at 50 µg/kg containing patulin and 5-hydroxymethylfurfural
Column: ACQUITY UPLC BEH Shield RP18, 2.1 x 100 mm, 1.7 µm in negative electrospray mode.
Flow Rate: 600 µL/min Concentration Average Recovery (%RSD)
Mobile Phase: A. 0.1% aqueous ammonium hydroxide 5 µg/kg 86.1% (13.6)
B. 0.1% ammonium hydroxide in acetonitrile 50 µg/kg 95.4% (5.9)
Gradient: Time (min) A% B% 500 µg/kg 89.9% (17.5)
0.00 99 10
1.80 99 10 Recovery data obtained from Oasis HLB extraction of patulin in apple juice.
Four data points were measured at each level.
2.30 10 90
2.80 10 90 Ordering Information
2.81 99 10
Injection Volume: 20 µL, Full loop injection Description Part Number
Column Temperature: 40 ˚C Oasis HLB, 3cc/60mg, 100/box WAT094226
Sample Temperature: 4 ˚C ACQUITY UPLC BEH Shield RP18,

186002854
2.1 x 100 mm, 1.7 μm
Detector: ACQUITY UPLC PDA
Detection: 276 nm
Ref: Developed by Vural Gökmen, Food Engineering Department, Hacettepe
University, Ankara, Turkey and John Martin, Waters Corporation
©2011 Waters Corporation. Waters, Oasis, ACQUITY UPLC, and ACQUITY are trademarks of Waters Corporation.
[ 71 ]
P FOS an d R e l at e d Com p oun ds in Wat e r an d T issu e
INTRODUCTION Note:
 The SPE eluate is collected in polypropylene test tubes,
Perfluorinated compounds (PFCs) such as perfluorooctanesul-
diluted with 2 mL of 2% aqueous formic acid and brought to
fonic acid (PFOS) and perfluorooctanoic acid are persistent
5 mL with water.
organic pollutants (POPs). PFCs may be toxic and have
bioaccumulative properties. There is growing interest in the  Alternatively, the eluate may be evaporated and reconstituted
development of analytical methods for PFCs in food, drinking in 1 mL mobile phase prior to analysis. Polypropylene
water, tissue, plasma, and blood. labware should be used exclusively.
Pretreatment LC Conditions
Water samples
1. Adjust 100 mL of sample to pH 3 with formic acid prior to SPE.
Chicken liver samples Mobile Phase: A. 20 mM ammonium acetate in water/acetonitrile (90:10)

B. acetonitrile/methanol
1. Homogenize 1 g of sample and extract with 10 mL of 10 mM Gradient: Time (min) A% B%
potassium hydroxide in methanol. Shake for 16 hours. 0.00 85 15
8.00 5 95
2. Centrifuge the sample for 10 minutes at 8000 rpm.
Injection Volume: 10 µL (full loop injection mode)
3. Dilute 1 mL aliquot of supernatant to 20 mL with water and Column Temperature: 40 ˚C
adjust the pH to 4-5 using 2% formic acid.
MS Conditions
SPE Procedure
Oasis® WAX, 3 cc/60 mg Ionization Mode: Negative electrospray (ESI -)

PFC MRM
A. 2 mL methanol
B. 2 mL water PFBS (Perfluorobutane Sulfonate) 299→80
PFOS (Perfluorooctane Sulfonate) 499→80
Load: C3 163→119
100 mL water or 20 mL diluted tissue sample C4 213→169
C5 263→219
Wash: C6 313→269
A.1 mL 2% formic acid
B. 2 mL methanol C7 363→319
C8 413→369
Elute: C9 463→419
2 mL 1% ammonia in methanol C10 513→469
C11 563→519
[ 72 ]
P FOS an d R e l at e d Com p oun ds in Wat e r an d T issu e
UPLC®/MS/MS of 12 PFCs spiked at 10 μg/kg in chicken liver.
Recovery from Drinking Water (%)

Spike Level µg/L PFBS PFOS C3 C4 C5 C6 C7 C8 C9 C10 C11
0.10 122 109 108 119 97 184 107 83 121 101 101
0.30 110 117 95 132 105 110 119 126 137 118 94
0.70 102 98 91 107 93 118 100 78 103 126 119
1.0 113 94 128 106 98 130 100 88 100 110 117
4.0 104 86 101 99 99 102 102 92 115 99 84
10 104 100 98 101 100 87 89 82 103 99 101
Recovery data for PFCS from drinking water.
Recovery from Chicken Liver (%)

Spike Level µg/kg PFBS PFOS C3 C4 C5 C6 C7 C8 C9 C10 C11
2 LOQ – 81 LOQ – 108 132 165 100 97 –
5 98 LOQ 138 148 LOQ 97 100 133 89 73 LOQ
10 93 50 102 134 121 99 87 123 95 101 33
20 102 50 128 144 94 96 110 117 90 80 25
30 87 51 104 102 124 89 86 103 91 84 20
50 92 54 92 92 118 94 97 97 86 86 22
Recovery data for PFCs from chicken liver.

PFC Analysis Kit 176001744
PFC Column Kit 176001692
PFC QC Standard 186004597
Oasis WAX, 3 cc/60 mg, 60 µm, 100/box 186002492
ACQUITY UPLC BEH C18, 2.1 x 50 mm, 1.7 µm, 3/pk 176000863
750 μL Polypropylene Vial 186002635

©2011 Waters Corporation. Waters, Oasis, ACQUITY UPLC, UPLC, Quattro Premier are trademarks of Waters Corporation.
[ 73 ]
P ro p ham in P otat o e s by GC / MS
Instrument: Agilent ® 6890
Propham is the active substance used as herbicides and potato
GC Column: DB-5ms, 30m x 0.25mm (i.d.), 0.25 µm
sprout inhibitor. This analytical method can be used to monitor
film. Direct connection of column to
Propham residues in potatoes. injection-port liner
Transfer Line to MS: 300 °C
Pretreatment Source Temperature: 200 °C
1. Add 15 g of ground potatoes into a 50 mL centrifuge tube.
Injection Port
2. Add 15 mL 1% acetic acid in acetonitrile with 1.5 g anhydrous Temperature: 180 °C
sodium acetate and 6 g anhydrous magnesium sulfate. Initial Temperature: 80 °C
3. Shake vigorously for 1 minute. Time at Initial

Temperature: 1 min.
4. Centrifuge >1500 rcf for 1 minute. Then Program at 10 °C/ min to 200 °C
5. Take out 7.5 mL extract and dilute to 10 mL with 2.5 mL toluene. Then at 25 °C minute to 300, hold 5 minutes
SPE Procedure GC/MS Conditions

Sep-Pak® Vac Carbon Black/Aminopropyl, Instrument: Waters Quattro micro™ GC
6 cc/500 mg/500 mg Ionization Mode: Electron Impact (70 eV)
Selected-Ion Recording (SIR)
CONDITION:
10 mL 25:75 toluene: actonitrile (v/v)
HP6890 GC Flow 1
Add 200 mg anhydrous magnesium sulfate to top of
cartridge to remove water Initial Flow: 1 mL/min
Time Rate Final Flow
(min) (mL/min) (mL/min)
LOAD:
0.00 50 3
Extract (collect)
0.50 50 3
0.60 50 1
ELUTE:
10 mL 25:75 toluene: acetonitrile (collect)
GC/MS (SIR)
Combine both collected fractions and adjust volume to exactly 20 mL
by addition of toluene:acetonitrile (25:75, v/v) Channel Mass
1 (Quantification) 92.8
Take 5 mL and evaporate to just below 1 mL and bring up to 1 mL 2 (Confirmation) 119
with toluene. Inject onto GC/MS. 3 (Confirmation) 120
SIR method parameters.
[ 74 ]
P ro p ham in P otat o e s by GC / MS
Results
Blank
9.79
1 ppm spike
1
4 6 8 10 12 14 min
1 µg/g spiked potato sample.
Compound Name: propham 92.8 RT Area

1 ppm spiked 1 9.78 379.00
1 ppm spiked 2 9.82 382.00
1 ppm spiked 3 9.80 458.00
1 ppm spiked 4 9.79 399.00
1 ppm spiked 5 9.75 421.00
Mean - 407.80
RSD (%) - 8.01
Recovery data for 1 μg/g spiked potato sample.

186003369
6 cc/500 mg/500 mg
©2011 Waters Corporation. Waters, Quattro micro, and, Sep-Pak are trademarks of Waters Corporation. All other
trademarks are property of their respective owners.
[ 75 ]
P ro p ham in P otat o e s by lc /MS
Propham is the active substance used as herbicides and potato 100

Blank
sprout inhibitor. This analytical method can be used to monitor
Propham residues in potatoes.
%
Pretreatment
0
1. Add 15 g of ground potatoes to 50 mL centrifuge tube.
2.15
2. Add 15 mL 1% acetic acid in acetonitrile with 1.5 g anhydrous 100
1 ppm spiked sample
sodium acetate and 6 g anhydrous magnesium sulfate.
3. Shake vigorously for 1 minute. %
4. Centrifuge >1500 rcf for 1 minute.
SPE Procedure
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 min
Pass through 1 μg/g spiked potato sample.

Sep-Pak® Light NH2
Compound 1: propham 138.3 RT Area
Transfer 5 mL to another tube and add 0.5 mg of anhydrous
magnesium sulfate 1 ppm spiked 1 2.15 5895.34
1 ppm spiked 2 2.15 6424.93
Vortex and allow powder to settle 1 ppm spiked 3 2.15 6996.63
1 ppm spiked 4 2.15 7557.80
1 ppm spiked 5 2.15 7567.60
Pass through 2 mL of prepared sample extract
Mean - 6888.46
RSD (%) - 10.57
Take out 200 µL dilute with 800 µL water Recovery (%) - 84.43
Inject onto LC/MS (10 µL) Compound 2: propham 120.3 RT Area

1 ppm spiked 1 2.15 1849.85
LC Conditions 1 ppm spiked 2 2.15 1950.71
Instrument: Waters ACQUITY UPLC System
® 1 ppm spiked 3 2.15 2091.4
Column: ACQUITY UPLC BEH C18, 2.1 x 100 mm, 1.7 µm 1 ppm spiked 4 2.15 2276.4
Mobile Phase: A. 0.1% formic acid in water 1 ppm spiked 5 2.15 2306.56
B. 0.1% formic acid in acetonitrile Mean - 2094.98
Gradient: Time (min) A% B% RSD (%) - 9.5
0.00 80 20 Recovery (%) - 81.35
3.00 20 80
3.20 80 20 Recovery data for of 1 μg/g spiked potato sample.
5.00 80 20
MS Conditions O rd e ring Information

Instrument: Waters Quattro micro™ Description Part Number
Ionization Mode: Positive electrospray (ESI ) +
Sep-Pak Light NH2 WAT023513
ACQUITY UPLC BEH C18, 2.1 x 100 mm 186002352
MRM Transitions: 1. 180.3→120.3
©2011 Waters Corporation. Waters, ACQUITY UPLC, Quattro micro and Sep-Pak are trademarks of Waters Corporation.
2. 180.3→138.3
[ 76 ]
SUDAN DYE S IN CHILLI OIL AND P OW DER
Sudan dyes are red dyes that are used for coloring solvents, Instrument: Waters Quattro micro™ API
oils, waxes, petrol, shoe and floor polishes. Sudan dyes are not Ionization mode: Positive electrospray (ESI +)
allowed to be added to food in by the United States Food and Drug
Administration (US FDA), European Union (EU), and other countries.
249→156
Pretreatment Sudan I 249→93
249→128
For Chilli Oil 277→156
Sudan II 277→121
1. Dilute 0.1 g chilli oil in 1 mL with hexane. 277→106
353→77
For Chilli Powder Sudan III 353→120
353→196
1. Homogenize and extract 1 g chilli powder with 10 mL acetone. 381→91
Sudan IV 381→106
2. Centrifuge.
381→224
3. A 1 mL aliquot is evaporated to complete dryness and the
Results
residue is taken up in 1 mL hexane.
SPE Procedure
Sep-Pak® Alumina B, 3 cc/500 mg
A. 2 mL methanol
B. 2 mL ethyl acetate
C. 3 mL hexane
Load:
1 mL of hexane pre-extract
Wash:
A. 3 mL hexane
B. 1 mL ethyl acetate LC/MS spiked chilli powder (n=6, 80 μg/kg).
Analyte Recovery (%) RSD (%)

Elute: Sudan I 99 11
4 mL ethyl acetate/methanol (90:10)
Sudan II 91 11
Sudan III 93 6
Evaporate and reconstitute in 200 µL methanol
Sudan IV 122 11
Recovery data for spiked chilli powder (n=6, 80 μg/kg).
LC Conditions
Column: Atlantis ® dC18, 2.1 x 100 mm, 3 µm Ordering Information
B. acetonitrile Sep-Pak Alumina B, 3 cc/500 mg, 50/box WAT020825
Gradient: Time (min) A% B% Atlantis dC18, 2.1 x 100 mm, 3 µm 186001295

0.00 20 80 Qsert Vials, LCGC Certified Combination Packs
™
186001126C
10.00 50 95
Ref: Waters Applications 720001440EN
Injection Volume: 15 µL ©2011 Waters Corporation. Waters, Sep-Pak, Alliance, Atlantis, and Quattro micro are trademarks of Waters Corporation.
All other trademarks are property of their respective owners.
[ 77 ]
Su dan Dy e s in f r e sh c hil l is
INTRODUCTION Analyte MRM Transition

249→156
Sudan dyes are red dyes that are used for coloring solvents, oils, wax- Sudan I 249→93
es, petrol, shoe and floor polishes. Sudan dyes are not allowed to be 249→128
added to food in by the United States Food and Drug Administration 277→156
Sudan II 277→121
(US FDA), European Union (EU), and other countries.
277→106
353→77
Pretreatment Sudan III 353→120
353→196
1. Homogenize and extract 1 g of chilli with 10 mL acetone. 381→91
2. Dilute 1 mL aliquot to 5 mL with aqueous sodium hydroxide Sudan IV 381→106

38 →224
(adjust to pH 11).
SPE Procedure
Oasis® MAX, 3 cc/60 mg
Results
A. 2 mL ethyl acetate C. 1 mL 0.1 M sodium hydroxide
B. 2 mL methanol D. 2 mL water
Load:
5 mL of diluted acetone pre-extract
Wash:
A. 2 mL 70% methanol in water C. 2 mL methanol
B. 1 mL 1 M sodium hydroxide in water D. 1 mL ethyl acetate
Elute:
2 mL ethyl acetate/methanol/formic acid (89:9:2, v/v/v)
Evaporate and reconstitute in 200 µL acetonitrile/water (90:10, v/v) LC/MS spiked chilli sauce (n=6, 80 μg/kg), Oasis MAX method.
LC Conditions Analyte Recovery (%) RSD (%)

Sudan I 83 9
Sudan II 83 1
Column: Atlantis ® dC18, 2.1 x 100 mm, 3 µm
Sudan III 77 3
Sudan IV 75 4
B. acetonitrile
0.00 20 80
10.00 50 95
Injection Volume: 15 µL Ordering Information
Oasis MAX, 3 cc/60 mg, 60 µm, 100/box 186000368
MS Conditions
Atlantis dC18 2.1 x 100 mm, 3 µm 186001295
Instrument: Waters Quattro micro API
™
Qsert Vials, LCGC Certified Combination Packs
™
186001126C
Multiple reaction monitoring ©2011 Waters Corporation. Waters, Oasis, Atlantis, Sep-Pak, Alliance, and Quattro micro are trademarks of Waters
Corporation. All other trademarks are property of their respective owners.
[ 78 ]
Food Testing and QUALITY CONTROL (QC)
The methods in this section may be used for simple QC testing or to monitor for adulteration. The methods offer:
 Sample extraction
 Sample preparation
 Chromatographic conditions
Amino Ac ids in Anima l F e e d H yd ro lysat e s
INTRODUCTION Total Run Time: 9.5 min

Injection Volume: 1 µL, partial loop with needle overfill
Amino acid analysis has been used in the food and feed industries Detection: UV (TUV), 260nm
to verify and characterize materials and processes. The total amino
acid content, as well as the proportions of growth-limiting amino Amino Acid % RSD
acids, is an essential characteristic of the nutritional value of feeds. His 1.03
Ser 0.43
Samples Arg 0.60
Gly 0.39
Swine diet, poultry diet, whole soybean, and soybean meal samples
Asp 0.24
were acid-hydrolyzed in an independent laboratory as part of a
Glu 0.23
collaborative study. The samples were supplied at an estimated
Thr 0.26
concentration of 1.0 mg/mL in 0.1 M HCl and sealed under argon
Ala 0.27
in ampoules. Samples were stored at -80 °C until analysis. The
Pro 0.30
standard was NIST 2389 Amino Acids in 0.1 mol/L HCl Reference
Cys 0.18
Material, and it was diluted to 5, 100, and 250 pmol/μL.
Lys 0.23
Tyr 0.17
Sample Derivatization
Met 0.21
The samples were diluted 1:16 with 0.1 M HCl prior to derivatiza- Val 0.22
tion. The standard derivatization protocol was modified to include Ile 0.23
neutralization of excess acid with 0.1 M NaOH. Conditions for pre- Leu 0.24 Summary of retention times, in minutes, for
the hydrolysate standard throughout the five
column derivatization and analysis are described in detail in the Phe 0.23 days of analyses.
Waters UPLC Amino Acid Analysis Solution System Guide (P/N
®
71500129702). These derivatization conditions were modified to

AMQ
0.020 Poultry Diet

Glu
Glu
Peak
DerivPeak
0.025
Leu
Leu
include additional base.
Deriv
Lys
Lys
0.020
Val
Ala
Val
Asp
Ala
Asp
NH3
Pro
NH3
AU
Phe
Phe
0.015
Ile
Gly
Gly
Ile
0.015
Tyr
Met
Ser
Thr
Met
Ser
Arg
0.010
Cys
Cys
1. 60 μL AccQ•Tag™ Ultra Borate Buffer
His
His
0.005
0.010
0.000
AMQ
Glu
Glu
Peak
Leu
Leu
DerivPeak
0.025
Swine Diet
2. 10 μL diluted sample
Deriv
0.020 Lys
Lys
0.005
Ala
Val
Ala
Val
Asp
NH3
Pro
NH3
Asp
AU
Phe
Phe
Ile
0.015
Ile
Gly
Gly
Tyr
Ser
Thr
Ser
Arg
Met
0.010
Met
Cys
3. 10 μL 0.1 N NaOH
Cys
His
His
0.005
0.000
0.000
AMQ
Peak
Whole Soybean
DerivPeak
0.025
Lys
Glu
Lys
4. 20 μL reconstituted AccQ•Tag Ultra Reagent

Glu
Leu
Leu
Deriv
0.020
Asp
Asp
Val
Val
NH3
NH3
Ile
AU
Phe
Ile
Phe
0.015
Ala
Ala
Pro
Gly
Gly
Tyr
Arg
Ser
Ser
Thr
0.010
Met
Met
Cys
Cys
His
His
0.005
0.000
LC Conditions
AMQ
Glu
Glu
Peak
Soybean Meal
DerivPeak
0.025
Lys
Lys
Leu
Leu
Deriv
0.020
Asp
Asp
Val
Val
NH3
NH3
Ile
AU
Phe
Ile
Phe
Ala
0.015
Ala
Pro
Gly
Gly

Tyr
Arg
Thr
Ser
Ser
0.010
Met
Met
Cys
Cys
His
His
0.005
Column: AccQ•Tag Ultra, 2.1 x 100 mm, 1.7 µm 0.000 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 min
Column Temperature: 55 °C Animal feed hydrolysates, 6 ng on column, using the UPLC Amino Acid
Analysis Solution.
Flow Rate: 700 µL/min.
Mobile Phase A: 1:20 Dilution of AccQ•Tag Ultra Eluent A with
MilliQ ® water (prepared fresh daily)
Mobile Phase B: AccQ•Tag Ultra Eluent B
Gradient: AccQ•Tag Ultra Hydrolysate Method (provided in the
UPLC Amino Acid Analysis Solution)
[ 80 ]
Amino Ac ids in Anima l F e e d H yd ro lysat e s
Amino Acid Poultry Diet Swine Diet Whole Soybean Soybean Meal Mean Std. Dev.
His 2.513 2.504 2.526 2.534 2.521 0.016
Ser 3.363 3.358 3.370 3.373 3.367 0.008
Arg 3.546 3.544 3.553 3.555 3.551 0.006
Gly 3.665 3.661 3.671 3.673 3.668 0.006
Asp 3.990 t3.987 3.995 3.997 3.993 0.005
Glu 4.457 4.455 4.461 4.462 4.459 0.004
Thr 4.820 4.819 4.823 4.823 4.822 0.002
Ala 5.189 5.187 5.190 5.191 5.189 0.002
Pro 5.752 5.751 5.750 5.751 5.751 0.001
Cys 6.599 6.601 6.602 6.603 6.602 0.001
Lys 6.661 6.663 6.662 6.663 6.663 0.001
Tyr 6.799 6.800 6.801 6.801 6.801 0.001
Met 6.944 6.945 6.945 6.945 6.945 0.000
Val 7.064 7.065 7.064 7.066 7.065 0.001
Ile 7.787 7.788 7.787 7.787 7.787 0.001
Leu 7.868 7.869 7.868 7.869 7.869 0.001
Phe 7.985 7.985 7.985 7.986 7.985 0.001
Retention time summary, in minutes, for the different sample types from one day of analyses. Each reported value represents
the mean value of fifteen injections.

Amino Acid Analysis Kit 176001235
AccQ•Tag Ultra Column, 2.1 x 100 mm 186003837
Amino Acid Standard, Hydrolysate,
WAT088122
10 x 1 mL ampules

©2011 Waters Corporation. Waters, UPLC, ACQUITY UPLC, and AccQ•Tag are trademarks of Waters Corporation.
[ 81 ]
Amino Ac ids in T ea
Instrument: Waters ACQUITY UPLC® system with TUV
L-theanine is a free (non-protein) amino acid found almost exclu-
Column: AccQ•Tag Ultra, 2.1 x 100 mm, 1.7 µm
sively in tea plants. It is the predominant amino acid in green tea
leaves, giving tea its characteristic taste.
Flow Rate: 700 µL/min.
Sample Preparation
Mobile Phase A: 1:10 dilution of AccQ•Tag Ultra Eluent A
Free amino acids were analyzed in tea leaves. Tea leaves were concentrate with MilliQ ® water
standard consumer single serving products. The tea samples were Mobile Phase B: AccQ•Tag Ultra Eluent B
between 2.5-3.5 g. The tea leaves were extracted in 6 oz. of Gradient: AccQ•Tag Ultra Cell Culture Method (provided in the
UPLC Amino Acid Analysis Solution)
bottled water at an initial temperature of 72 °C for 2 hours, unless
Total Run Time: 9.5 min
otherwise specified. After a set period of time, the supernatant
Injection Volume: 1 µL, partial loop with needle overfill
of the mixture was transferred to a separate vial. Extracted tea
Detection: UV (TUV), 260nm
samples in water were stored at -20 °C until analysis.
0.06
Green Tea, Extracted for 2 h
Sample Derivatization 0.05
Deriv Peak
0.04
Theanine
0.03
The extracted tea samples were derivatized neat following the 0.02
GABA
NH3
Glu
Asp
0.01
Val
Ala
Phe
Ile
Gln
Lys
standard AccQ•Tag™ Ultra derivatization protocol. Conditions for
Asn
Pro
Ser
Thr
Arg
AU
Green Tea, Extracted for 18 h

pre-column derivatization and analysis are described in detail in
Theanine
0.05
Deriv Peak
0.04
the Waters UPLC® Amino Acid Analysis Solution System Guide
GABA
0.03
NH3
Glu
(P/N 71500129702). The derivatization conditions included:

0.02
Asp
Ala
Val
Tyr
0.01
Lys
Pro
Gln
Phe
Trp
AABA
Leu
NVa
Met
Ile
Asn
Ser
Arg
Thr
1. 70 µL of AccQ•Tag Ultra borate buffer 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 min
Two separate aliquots of a single type of green tea using the UPLC Amino
2. 10 µL of tea extract
Acid Analysis Solution. Each aliquot was extracted for 2 or 18 hours.
Theanine (5.1 min.) was confirmed by MS detection. All amino acid levels
3. 20 µl of derivatization reagent increase with duration of extraction.
AMQ
0.06
Lemon Tea
NH3
0.04
Asn
Deriv Peak
GABA
Pro
0.02
Asp
Ala
Met
Ser
Glu
Gly
0
AMQ
0.06
Theanine
Earl Grey
GABA
Deriv Peak
0.04
NH3
Asp
Glu
0.02
Phe
Val
Ala
Asn
Leu
Tyr
Gln
Lys
Ile
Trp
Ser
Pro
Arg
Thr
Tau
0
AU
0.06
AM
Decaf Earl Grey

Theanine
NH3
0.04
Deriv Peak
Asp
Glu
0.02
GABA
Ala
Val
Phe
Leu
Tyr
Asn
Ile
Lys
Ser
Gln
Trp
Orn
Pro
Arg
Thr
Tau
0
AMQ
0.06
Green Tea
Theanine
0.04
Deriv Peak
GABA
NH3
0.02
Glu
Asp
Ala
Val
Phe
Asn
Gln
Lys
Ile
Arg
Pro
Ser
Thr
0
1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 min
Extracted teas using the UPLC Amino Acid Analysis Solution. Theanine was O rd e ring Information
confirmed by MS detection. Amino acids levels vary with type of tea.

AccQ•Tag Ultra Column, 2.1 x 100 mm 186003837
Amino Acid Standard, Hydrolysate,
WAT088122
10 x 1 mL ampules
© 2011 Waters Corporation. Waters, AccQ•Tag, UPLC, ACQUITY UPLC are trademarks of Waters Corporation. All other
trademarks are the property of their respective owners..
[ 82 ]
Foo d Suga rs in B r an w it h R aisin C e r ea l
INTRODUCTION R ESU LT S
4 Raisin Bran Brand Cereal
Sugar analysis in foods is a standard food QC assay. 1200 4 mg/mL
1000
800
Sam pl e E x traction P roc edur e
LSU
600
400
200 2 3
0
1. Weigh out sample (~ 3g) into 50 mL centrifuge tube.
1200 Food Sugar Standard
2 4 1 mg/mL
1000 3
2. Add 25 mL of 50:50 acetonitrile/water and homogenize. 800 1
6
LSU
600 5
3. Centrifuge at 3200 rpm for 30 minutes. 400
200
0
4. Collect supernatant and filter using 0.45 µm 0 2 4 6 8 10 12 14 16 min
PVDF syringe filter.

COM POUNDS
LC CONDIT IONS
1. p-Toluamide 2. Fructose 3. Glucose
Instrument: Alliance System with 2424 EL SD
®
O NH 2 HO OH
HO O
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm OH HO O
Mobile Phase A: 80/20 acetonitrile/water with
HO OH
OH HO
0.2% triethylamine OH
Mobile Phase B: 30/70 acetonitrile/water with

0.2% triethylamine 4. Sucrose 5. Maltose
HO HO
Flow Profile: 90% A/10% B (75% acetonitrile with
O HO O
0.2% triethylamine) HO HO HO
HO HO HO O
Flow Rate: 1.0 mL/min O HO
O O
HO HO OH
Injection Volume: 15.0 µL OH HO
OH
Sample Concentration:1 mg/mL each
6. Lactose
Needle Wash: 75:25 acetonitrile:water
HO
Seal Wash: 50:50 acetonitrile:water HO OH O
O O
HO OH
HO HO HO
EL SD Conditions
Gain: 100
Pressure: 30 psi
Drift Tube Temperature: 50 ˚C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: 0.2 sec

XBridge Amide Column, 3.5 µm, 4.6 x 250 mm 186004870
Acrodisc LC 13 mm, 0.45 µm, 100/pkg
®
WAT200512
12 x 32 LCMS Certified Glass Screw Neck Vial 600000751CV
©2011 Waters Corporation. Waters , XBridge, and Alliance are trademarks of Waters Corporation. All other trademarks are
the property of their respective owners.
[ 83 ]
Foo d Suga rs in Mil k
1000 Low Fat Milk
This is a simple assay to monitor sugar levels in milk. There is 800 1% in 50:50
MeCN:H2O
a simple sample extraction procedure provide, followed by 600
LSU
400
6
LC/ELSD assay. 200
0
2 4
1000 Food Sugar Standard 3
SAM P L E EX T RAC T ION P ROC EDU R E 800 1 mg/mL 6
1
600 5
LSU
400
1. Dilute with 50:50 acetonitrile/water. 200
0
2. Filter using 0.45 µm PVDF syringe filter. 0 2 4 6 8 10 12 14 16 min
LC CONDIT IONS
Instrument: Alliance ® System with 2424 EL SD ST RU CTUR ES
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
1. p-Toluamide 2. Fructose 3. Glucose
Mobile Phase A: 80/20 acetonitrile/water with
O NH 2 HO OH
0.2% triethylamine HO O
OH HO O
Mobile Phase B: 30/70 acetonitrile/water with HO
OH OH
0.2% triethylamine HO
OH
Flow Profile: 90% A/10% B (75% acetonitrile
with 0.2% triethylamine)
4. Sucrose 5. Maltose
Flow Rate: 1.0 mL/min HO HO
Injection Volume: 15.0 µL O HO O
HO HO HO
Sample Concentration: 1 mg/mL each HO HO HO O
O HO
O O
Column Temperature: 35 ˚C HO HO OH
OH HO
OH
Seal Wash: 50:50 acetonitrile:water 6. Lactose
HO
EL SD Conditions HO OH O
O O
HO OH
HO HO HO
Gain: 100
Pressure: 30 psi
Drift Tube Temperature: 50 ˚C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: 0.2 seconds

Acrodisc LC 13 mm, 045 µm, 100/pkg
®
WAT200512
©2011 Waters Corporation. Waters, the Science of What’s Possible, XBridge, and Alliance are trademarks of Waters Corporation. All
other trademarks are the property of their respective owners.
[ 84 ]
Gins enosid e Rb1 in Gins eng Root P ow d e r E x t r ac t
0.30 Ginseng root powder extract
This application brief contains a simple extraction protocol Tailing factor = 0.95
0.25 USP plate count = 6170
and LC/UV conditions for ginsenoside Rb1 in ginseng root.
0.20
The gensenosides are a target of research in the root.
0.15
AU
Ginsenoside Rb1
SAM P L E EX T RAC T ION P ROC EDU R E 0.10
0.05
1. Weigh 200 mg ginseng root powder into an extraction vessel. 0.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 min
2. Add 1 mL 80% methanol, sonicate for 5 min.
3. Centrifuge at 10,000 rpm for 5 min. Structur e

4. Collect the supernatant. OH
HO O
5. Repeat steps 2-4 two more times. HO O

Ginsenoside Rb1 HO
HO O
HO O
6. Combine the extracts, mix well.

HO
OH
7. Filter through 13 mm nylon 0.2 µm filter for injection. OH

O
HO
HO O
OH
O O
HO
LC CONDIT IONS HO
HO
Instrument: Alliance ® System with 2998 PDA

Column: XBridge™ Amide, 3.5 µm, 4.6 x 150 mm
Mobile Phase: 80:20 acetonitrile/water
Isocratic Flow Rate: 1.4 mL/min
Injection Volume: 11.5 µL
Seal Wash: 10:90 acetonitrile:water
UV Wavelength: 203 nm
Sampling Rate: 20 Hz
Filter Time Constant: 0.1 sec

Acrodisc® LC 13 mm, 045 µm, 100/pkg WAT200512
©2011 Waters Corporation. Waters, XBridge, and Alliance are trademarks of Waters Corporation. All other trademarks are the
property of their respective owners.
[ 85 ]
[ Compound INDEX ]
1-Aminohydantion (AH).......................................................................... 23, 25 Dexamethasone............................................................................................... 20

3-Amino-2-oxazolidinone (AOZ)............................................................. 23, 25 Diaquat............................................................................................................ 70
1-Naphthol .................................................................................................... 41 Dichlorvos.................................................................... 56, 61, 63, 65, 67, 68
3-Hydroxycarbofuran ..................................................................................... 41 Dimethirimol ................................................................................................. 53
5-hydroxymethylfurfural................................................................................. 71 Dimethoate...................................................................................................... 58
Disulfoton........................................................................................................ 58
A
Disulfoton sulfone........................................................................................... 58
Abamectin ...................................................................................................... 53 Disulfoton sulfoxide........................................................................................ 58
ACRYLAMIDE.................................................................................................. 39 Doxycycline.............................................................................................. 34, 36
Aflatoxins........................................................................................................ 40
E
Alanine..................................................................................................... 80, 81
Aldicarb ......................................................................................................... 41 Endosulfan sulfate...................................................................... 55, 60, 62, 66
Aldicarb Sulfone............................................................................................. 41 Enkephalin ...................................................................................................... 51
Aldicarb Sulfoxide .......................................................................................... 41 Enrofloxacin..................................................................................................... 21
Ammelide........................................................................................................ 49 Ethiofencarbsulfone........................................................................................ 54
Ammeline........................................................................................................ 49 Ethiofencarbsulfoxide...................................................................................... 54
3-Amino-5-morpholino-methyl-1,3-oxazolidinone (AMOZ).................. 23, 25 Ethion .................................................................. 55, 57, 60, 62, 64, 66, 68
Anibfos ........................................................................................................... 53 Ethoprophos..................................................................................................... 58
Arginine................................................................................................... 80, 81 Etriticonazole.................................................................................................. 53
Asparagine............................................................................................... 80, 81
F
Atrazine.............................. 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 69
Azinphos-methyl .......................................................................................... 53 Fenoxycarb .................................................................................................... 53
Azoxystrobin.................................... 55, 56, 60, 61, 62, 63, 64, 65, 66, 67 Fensulfothion................................................................................................... 58
Fensulfothion sulfone...................................................................................... 58
B
Fensulfothion-oxon.......................................................................................... 58
BDMC............................................................................................................... 41 Fensulfothion-oxon-sulfone............................................................................ 58
Benzofenap ................................................................................................... 53 Ferimzone ..................................................................................................... 53
Bifenthrin................................................................................... 60, 62, 64, 66 Florfenicol ...................................................................................................... 19
Bromclenbuterol ............................................................................................. 18 Flufenacet........................................................................................................ 69
Bromobuterol .................................................................................................. 18 Formetanate hydrochloride ........................................................................... 53
Butafenacil ..................................................................................................... 53 Fructose.................................................................................................... 83, 84
Furathiocarb .................................................................................................. 53
C
G
Cadusafos........................................................................................................ 58
Carbaryl ................................... 41, 55, 56, 60, 61, 62, 63, 64, 65, 66, 67 Ginsenoside Rb1............................................................................................. 85
Carbendazim............................................................................................ 68, 69 Glucose..................................................................................................... 83, 84
Carbofuran ...................................................................................................... 41 Glutamine................................................................................................. 80, 81
Chloramphenicol ........................................................................................... 19 Glycine..................................................................................................... 80, 81
Chloridazon ................................................................................................... 53
H
Chlorpyrifos ....................................................................... 55, 60, 62, 66, 68
Chlorpyrifos-methyl ................................................... 55, 57, 60, 62, 64, 66 Hexachlorobenzene.................................................................................. 60, 64
Chlortetracycline........................................................................ 28, 34, 35, 36 Hexachonzol.................................................................................................... 68
Chlorothalonil.................................................................................................. 64 Histidine................................................................................................... 80, 81
Chromafenozide ............................................................................................. 53 I
Cimaterol......................................................................................................... 18
Cimbuterol ...................................................................................................... 18 Imazalil................................................................................ 56, 61, 63, 67, 69
Ciprofloxacin................................................................................................... 21 Imidacloprid ............................................................... 53, 56, 61, 63, 65, 67
Clenbuterol ..................................................................................................... 18 Indoxacarb ..................................................................................................... 53
Clenbuterol-D9 ............................................................................................... 18 Iprovalicarb ................................................................................................... 53
Clomeprop ..................................................................................................... 53 Isoleucine................................................................................................. 80, 81
Cloquintocet-mexyl ....................................................................................... 53 Isoxaflutole .................................................................................................... 53
Clothianidin ................................................................................................... 53 Isoxsuprine ..................................................................................................... 18
Cloxacillin ..................................................................................................... 28 K
Cyanuric Acid...................................................................... 45, 46, 47, 48, 49
Cyazofamid ................................................................................................... 53 Kresoxim-methyl................................................... 55, 60, 61, 62, 64, 65, 66
Cyflufenamid ................................................................................................. 53 L
Cylohalothrin................................................................................................... 57
Lactofen .......................................................................................................... 53
Cyprodinil......................................... 56, 60, 61, 62, 63, 64, 65, 66, 67, 69
Lactose..................................................................................................... 83, 84
Cysteine.................................................................................................... 80, 81
Leucine..................................................................................................... 80, 81
D Leucomalachite Green.............................................................................. 42, 43
DDD ............................................................................................................... 57 Linuron................................................................................. 56, 61, 63, 65, 67
Demeton-S-methyl.......................................................................................... 58 Lysine....................................................................................................... 80, 81
Demeton-S-methyl sulfone............................................................................. 58
[ 86 ]
[ Compound INDEX ]
M S
Mabuterol ....................................................................................................... 18 Salbutamol...................................................................................................... 18
Malachite Green....................................................................................... 42, 43 Salbutamol-D3 ............................................................................................... 18
Malathion........................................................................................................ 68 Semicarbizide........................................................................................... 23, 25
Maltose..................................................................................................... 83, 84 Serine....................................................................................................... 80, 81
Mapenterol ..................................................................................................... 18 Simeconazole ................................................................................................ 53
MCYST-LF........................................................................................................ 51 Spiramycin...................................................................................................... 29
MCYST-LR ....................................................................................................... 51 Streptomycin................................................................................................... 31
MCYST-LW....................................................................................................... 51 Sucrose..................................................................................................... 83, 84
MCYST-RR ...................................................................................................... 51 Sudan I...................................................................................................... 77, 78
Melamine............................................................................. 45, 46, 47, 48, 49 Sudan II..................................................................................................... 77, 78
Methamidophos............................................................ 54, 56, 61, 63, 65, 67 Sudan III.................................................................................................... 77, 78
Methiadathion................................................................................................. 68 Sudan IV................................................................................................... 77, 78
Methiocarb ..................................................................................................... 41 Sulfachloropyridazine ............................................................................ 28, 32
Methionine................................................................................................ 80, 81 Sulfadiazine ........................................................................................... 28, 32
Methomyl .................................................................... 41,56, 61, 63, 65, 67 Sulfadimethoxine .................................................................................... 28, 32
Methoxyfenozide ........................................................................................... 53 Sulfamerazine ......................................................................................... 28, 32
Mibemectin A3 ............................................................................................. 53 Sulfamethazine ...................................................................................... 28, 32
Mibemectin A4 ............................................................................................. 53 Sulfamethizole................................................................................................ 32
Sulfamethoxazole..................................................................................... 28, 32
N
Sulfamethoxypyridazine.......................................................................... 28, 32
Naproanilide .................................................................................................. 53 Sulfapyridine................................................................................................... 32
O Sulfapyridine ................................................................................................ 28
Sulfathiazole............................................................................................ 28, 32
Omethoate............................................................................................... 54, 58
Oryzalin ......................................................................................................... 53 T
Oxacillin ........................................................................................................ 28 Tebuconazole.................................... 55, 60, 61, 62, 63, 64, 65, 66, 67, 69
Oxamyl ........................................................................................................... 41 Terbufos........................................................................................................... 58
Oxycarboxin .................................................................................................. 53 Terbufos sulfone.............................................................................................. 58
Oxydemeton-methyl .............................................................................. 53, 58 Terbufos sulfoxide........................................................................................... 58
Oxydemeton-S-methyl sulfone....................................................................... 58 Terbutaline....................................................................................................... 18
Oxytetracycline ............................................................................... 34, 35, 36 Tetracycline................................................................................ 28, 34, 35, 36
Oxytetracydine .............................................................................................. 28 T heanine.......................................................................................................... 80
P T hiabendazole............................................................................ 56, 61, 63, 67
T hiacloprid .................................................................................................... 53
Paraquat........................................................................................................... 70 T hiamethoxam ............................................................................................... 53
Patulin............................................................................................................. 71 T hiamphenicol................................................................................................. 19
Permethrin.................................................................................. 60, 62, 64, 66 T hreonine................................................................................................. 80, 81
Penicillin G ............................................................................................. 27, 28 Tolyfluanid............................................... 55, 56, 60, 61, 62, 63, 64, 65, 67
Perfluorobutane Sulfonate.............................................................................. 72 Triabendazole.................................................................................................. 69
Perfluorooctane Sulfonate.............................................................................. 72 Tricyclazole..................................................................................................... 68
Phenmedipham ............................................................................................. 53 Tridemorph .................................................................................................... 53
Phenylalanine.......................................................................................... 80, 81 Trifluralin............................................................................. 55, 60, 62, 64, 66
o-Phenylphenol ........................................................................ 60, 62, 64, 66 Trizaphos......................................................................................................... 68
Phenylphenol ................................................................................................ 57 Tyrosine.................................................................................................... 80, 81
Phosalone........................................................................................................ 68
Procymidone............................................................................... 60, 62, 64, 66 V
Profenophos..................................................................................................... 68 Valine........................................................................................................ 80, 81
Proline...................................................................................................... 80, 81
Propham................................................................................................... 74, 76
Propoxur ........................................................................................................ 41
p-toluamide.............................................................................................. 83, 84
Pymetrozine........................................................................ 56, 61, 63, 65, 67
Pyrazolynate .................................................................................................. 53
Q
Quinal Phos..................................................................................................... 68
Quizalofop-P-tefuryl ..................................................................................... 53
R
Ractompamine ............................................................................................... 18
[ 87 ]
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[ 88 ]

Determinación de Acrilamida en Dieferentes Materiales

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Food T Est ing

A p p l i c at ion Not e boo k

Solid-Phase Extraction Strategies......................................................................................................................................................... 5

Sample Preparation Solutions............................................................................................................................................................... 9

Veterinary Drugs in Food.....................................................................................................16

β2-Agonists in Pork and Pig Liver Tissues............................................................................................................................................ 17

Enrofloxacin (Baytril®) in Chicken......................................................................................................................................................... 21

Penicillins, Tetracyclines, and Sulfonamides in Milk........................................................................................................................... 28

Sulfonamide Antibacterials in Milk....................................................................................................................................................... 32

Tetracyclines and Sulfonamides in Milk................................................................................................................................................ 34

Tetracyclines in Animal Tissues............................................................................................................................................................ 35

Pesticides and Contaminants................................................................................................38

Acrylamide in Fried Potato Products...................................................................................................................................................... 39

Aflatoxins in Produce Samples............................................................................................................................................................... 40

Carbamates in Fruits and Vegetables..................................................................................................................................................... 41

Malachite Green in Fish (HPLC/UV )........................................................................................................................................................ 42

Malachite Green in Fish (UPLC/MS/MS)................................................................................................................................................. 43

Melamine and Cyanuric Acid in Infant Formula using HPLC................................................................................................................. 45

Melamine and Cyanuric Acid in Infant Formula using UPLC................................................................................................................. 47

Melamine and Cyanuric Acid in Infant Formula using LC/MS/MS......................................................................................................... 49

Microcystins in Natural Waters............................................................................................................................................................... 51

Multi-Residue Analysis of Pesticides in Grain and Beans...................................................................................................................... 52

Multi-Residue Analysis of Pesticides in Vegetables and Fruits............................................................................................................. 53

Multi-Residue LC/MS/MS Determination of 52 Non-Gas Chromatography-Amenable

Multi-Residue Analysis of Pesticides by QuEChERS in:

Avocado by UPLC/MS/MS ............................................................................................................................................................... 56

Avocados and Grapes by GC/MS...................................................................................................................................................... 57

Baby Food by UPLC/MS/MS............................................................................................................................................................ 58

Rolled Oats by GC/MS..................................................................................................................................................................... 66

Rolled Oats by UPLC/MS/MS........................................................................................................................................................... 67

Vegetables and fruits by UPLC/MS/MS........................................................................................................................................... 69

Paraquat and Other Quaternary Ammonium Compounds in Water....................................................................................................... 70

Patulin in Apple Juice............................................................................................................................................................................. 71

PFOS and Related Compounds in Water and Tissue............................................................................................................................... 72

Propham in Potatoes by GC/MS.............................................................................................................................................................. 74

Propham in Potatoes by LC/MS............................................................................................................................................................... 76

Sudan Dyes in Chilli Oil and Powder...................................................................................................................................................... 77

Sudan Dyes in Fresh Chillis..................................................................................................................................................................... 78

FOOD TESTING AND QUALITY CONTROL (QC)...............................................................................79

Amino Acids in Animal Feed Hydrolysates........................................................................................................................................... 80

Amino Acids in Tea................................................................................................................................................................................. 82

Food Sugars in Bran with Raisin Cereal.................................................................................................................................................. 83

Food Sugars in Milk................................................................................................................................................................................ 84

Ginsenoside Rb1 in Ginseng Root Powder Extract Nitrofurans in Tissues.............................................................................................. 85

Retention-Cleanup-Elution Strategy SPE Procedure Steps

 Alternatively, evaporate the solvent and exchange to hexane.

Pass-Through Cleanup Strategy Wastewater

1. Sample is passed through For reversed-phase sorbents, preconditioning of the sorbent

Sample Size Oasis Cartridge 500–1000 mL 6 cc/500 mg (LP) or 6 cc/1 g

1–10 mL 1 cc/30 mg or 3 cc/60 mg

* LP=large particules (60 µm)

Table 3. Guidelines on the Various Types of Separation Mechanisms

Reversed Phase Normal Phase Ion Exchange

Matrix Aqueous Non-polar organic solvent Aqueous/low ionic strength

1. Solvate polar organic

Wash Aqueous/buffer Non-polar Low ionic strength buffer

Sorbent P rop e rties and T y pic a l Applic ations

Sorbent Properties & Applications

Normal or Reversed Phase

Product for Specific Applications

 Traditional SPE phases

S e p- Pak C a rtridg e S e pa ration Guid elin es

Packing Surface Polarity High Low High