Food TEsting. Application Notebook

Food TEsting Application Notebook

[ TAble of Contents ] Solid-Phase Extraction Strategies... 5 Sep-Pak and asis 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... 2 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... 4 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... 6 Flour by UPLC/MS/MS... 61 Grapes by GC/MS... 62 Grapes by UPLC/MS/MS... 63 ranges by GC/MS... 64 ranges by UPLC/MS/MS... 65 Rolled ats by GC/MS... 66 Rolled ats by UPLC/MS/MS... 67 Teas by UPLC/MS/MS... 68 Vegetables and fruits by UPLC/MS/MS... 69 Paraquat and ther Quaternary Ammonium Compounds in Water... 7 Patulin in Apple Juice... 71 PFS 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 il and Powder... 77 Sudan Dyes in Fresh Chillis... 78 FD TESTING AND QUALITY CNTRL (QC)...79 Amino Acids in Animal Feed Hydrolysates... 8 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 ]

Solid-Phase Extraction Strategies Retention-Cleanup-Elution Strategy As the sample is loaded onto the cartridge, the analytes of interest are retained by the sorbent. If needed, an optimized series of 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 than the load volume. SPE Procedure Steps The following section describes the steps involved in a complete solid-phase extraction procedure: 1. Pretreatment Solid samples (soil, tissue, etc.) Stationary Phase Particles Load Sample (Black) Step Elute 1 Step Elute 2 Step Elute 3 NTE: Different strength solvents can be used to separate the dyes. Shake, sonicate, or use soxhlet extraction. - 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. Non-aqueous liquid If the sample is soluble in water, dilute it with water for reversed-phase SPE. ne 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 Pass-through cleanup methods optimize matrix retention while the analytes of interest pass-through the cartridge unretained. No sample enrichment occurs during the solid-phase extraction (SPE) step. 1. Sample is passed through sorbent and collected. No sample enrichment. 2. Matrix interferences are retained on sorbent. Wastewater Filter or centrifuge as necessary. 2. Condition For reversed-phase sorbents, preconditioning of the sorbent with an organic solvent, such as methanol, acetonitrile, isopropanol, or tetrahydrofuran, is usually necessary to obtain reproducible results. Without this step, a highly aqueous solvent cannot penetrate the hydrophobic surface and wet the 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 ]

Solid-Phase Extraction Strategies 3. Load When the analytes of interest are not retained by the sorbent, this is called analyte breakthrough. For some methods, such as pass-through cleanup, analyte breakthrough is desirable and is maximized for those specific methods. However, in all other cases, analyte breakthrough is unwanted and contributes to poor recovery and method reproducibility. Breakthrough occurs when: There is too high an organic concentration in the load solution for very polar analytes. Dilute sample at least 1:1 with water or buffer prior to loading. The analytes are bound to proteins, they may pass through the sorbent. Ensure that analytes are not bound to proteins by acidifying or basifying the sample. Sorbent is overloaded by the matrix component. Therefore, it is important to choose the correct sorbent mass (see Tables 1 and 2). The flow rate of the load step is too fast. There is not enough contact time between the analytes and the sorbent. Look at the drops and adjust the vacuum so that you see discrete droplets, not a stream of liquid. Table 1. Choice of asis Cartridges Based on Sample Size Sample Size asis Cartridge 1 1 ml 1 cc/3 mg or 3 cc/6 mg 4. Wash The wash steps are designed to remove unwanted matrix components that remain from the loading step. The ideal wash solvent removes only the matrix while keeping the analytes bound to the sorbent. For complex samples this is impossible, so the wash steps are optimized using ph, solvent strength, and solvent polarity to remove as much matrix as possible while maintaining acceptable analyte recovery. 5. Elute nce the interferences are washed off the cartridge, a strong solvent is introduced to elute the analytes of interest. The volume and flow rate of the eluting solvents should be precisely controlled as in the load step to ensure reproducible results. Refer to Table 3 for guidelines on various types of separation mechanisms and recommended solvents. Table 2. Choice of Sep-Pak Cartridges Based on Sample Size Sample Size Sep-Pak Cartridge 1 1 ml 3 cc/2 mg or 6 cc/5 mg 1 5 ml 3 cc/2 mg or 6 cc/5 mg 5 1 ml 6 cc/5 mg (LP) or 6 cc/1 g 1 1 ml 3 cc/6 mg or 6 cc/2 mg 1 5 ml 6 cc/2 mg or 6 cc/5 mg (LP*) 5 1 ml 6 cc/5 mg (LP) or 12 cc/1 g (LP) * LP=large particules (6 µm) Table 3. Guidelines on the Various Types of Separation Mechanisms Reversed Phase Normal Phase Ion Exchange Analyte Low to moderate polarity/ hydrophobic Moderate to highly polar/uncharged Charged or ionized Matrix Aqueous Non-polar organic solvent Aqueous/low ionic strength Condition/Equilibrate 1. Solvate polar organic 2. Water Non-polar organic Low ionic strength buffer Wash Aqueous/buffer Non-polar Low ionic strength buffer Elute Increase polar organic content in steps Increase moderate to high polarity organic content in steps Stronger ionic strength buffers or ph to neutralize the charge [ 6 ]

Sep-pak and Asis Cartridges for rapid sample preparation Sorbent Properties and Typical Applications Separation Mode Sorbent Reversed Phase C 18 tc 18 C 8 Properties & Applications 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 peptides, trace organics in environmental water samples, organic acids in beverages; similar to reversedphase HPLC columns in elution behavior. Strongly hydrophobic silica-based bonded phase; trifunctional bonding chemistry gives it increased hydrolytic stability over C 18 ; applications similar to those of C 18. Moderately hydrophobic, silica-based bonded phase; use for methods requiring less retention than C 18. Typical applications include drugs and their metabolites in serum, plasma or urine, peptides in serum and plasma. tc 2 Silica-based bonded phase with low hydrophobicity; use for methods requiring less retention than C 8 ; applications are similar to C 18 and C 8. asis HLB 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 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 Aminopropyl [NH 2 ] Cyanopropyl [CN] Diol Normal Phase Silica Alumina A, N, B Florisil 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 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 applications or as less hydrophobic alternative to C 18 or C 8 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 hydrophobic phase in aqueous media; applications include antibiotics from cosmetics; isolation of proteins or peptides by hydrophobic-interaction chromatography [HIC]. 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 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 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 of Analytical Communities (AAC) and Environmental Protection Agency (EPA) methods; other applications include polychlorinated biphenyls in transformer oil. [ 7 ]

Sep-pak and Asis Cartridges for rapid sample preparation Ion Exchange Accell Plus QMA Accell Plus CM asis MCX asis WCX asis MAX asis WAX PSA 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 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 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 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. 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 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 applications 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 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. Sep-Pak PS2 Very hydrophobic copolymer designed for multi-residue pesticide analysis in water samples. Typical applications include JPMHLW official methods for pesticides in water and JPMHLW official methods for pesticides in food. Sep-Pak AC2 Sep-Pak Dry Carbon Black/PSA Highly hydrophobic, low ash content, activated carbon used to remove or enrich very polar organic molecules from water. Typical applications include JPMHLW official method for 1,4-dioxane analysis in water and pesticides, herbicides, especially highly polar small molecules. 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 an alternative selectivity compared to Aminopropyl. [ 8 ]

Sample preparation solutions 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. Sep-Pak Cartridge Separation Guidelines Chromatographic Mode Normal Phase Reversed Phase Ion Exchange Silica, Florisil, Alumina, C 18, tc 18, C 8, Diol, Accell Plus QMA, Separation Characteristic Diol, NH 2, CN PoraPak RDX, NH 2, CN Accell Plus CM, NH 2 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 Typical Elution Solvent Ethyl acetate, acetone, acetonitrile Methanol, acetonitrile, dichloromethane Buffers, salt solutions with high ionic strength Sample Elution rder Least polar sample components first Most polar sample components first Most weakly ionized sample components first Solvent Change Required to Elute Retained Compounds Increase solvent polarity Decrease solvent polarity Increase ionic strength or increase ph (anion exchange) or decrease ph (cation exchange) Certified Sep-Pak Cartridges ne 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 IS 13485 and IS 92-certified facility under strict GLP and cgmp guidelines. [ 9 ]

SAMPLE PREPARATIN SLUTINS Waters premium brand for SPE Cartridges, plates, and µelution technology Co-polymer, water wettable, reproducible utperforms C 18 for polar bases asis 2x4 Method The fastest, simplest, and cleanest approach to SPE method development Characterize your analyte (acid, base, pka) Choose 1 of 5 asis 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 [ 1 ]

SAMPLE PREPARATIN SLUTINS Easy and straightforward method to implement, requiring little training Conforms to the AAC and CEN official methods for determining pesticide residues in fruits in vegetables Cost effective Reliable, high quality product in a simple kit format DisQuE DISPERSIVE SAMPLE PREPARATIN KIT 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 AAC 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.AAC Int. 9(2) 27, 485-52. FILTERS 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. CERTIFIED VIALS 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. [ [ 11 11 ] ]

Intergrated solutions 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 AMIN ACID ANALYSIS 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. CARBAMATE ANALYSIS KIT Containing a Waters Carbamate column, asis HLB cartridges, vials, and Reference Standards, this kit is optimized to simplify your analysis while increasing your confidence in the results. SFT DRINK ANALYSIS KIT The soft drink mobile phase and soft drink standards separate caffeine, aspartame, benzoic acid, and sorbic acid. MELAMINE ANALYSIS PACKAGES 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. [ [ 12 12 ] ]

SEPARATIN SLUTINS Column Selection Guide 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 C 18 CSH Phenyl-Hexyl CSH Fluoro-Phenyl HSS C 18 HSS C 18 SB HSS T3 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 increased loading capacity for basic compounds. Bonding: Trifunctional C 18 ligand, fully end-capped, bonded to a CSH particle substrate. Selectivity Features: General purpose alternative selectivity ligand that provides pi-pi interactions with polyaromatic compounds, while maintianing excellent reproducibility at ph extremes. CSH technology enables superior peak shape and 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 using low-ph mobile phases. CSH technology enables superior peak shape and increased loading capacity for basic compounds. Bonding: Trifunctional propyl fluorophenyl ligand, non-endcapped, bonded to a CSH particle substrate. Selectivity Features: High performance C 18 chemistry, increased retention, superior peak shape, resists acid hydrolysis at low ph. Designed for UPLC separations where silica-based C 18 selectivities are desired. Bonding: High coverage trifunctional C 18, fully endcapped, bonded to High Strength Silica (HSS) HPLC particle substrate. Selectivity Features: Unique, non-endcapped C 18 chemistry designed specifically for method development scientists. ffers unique Selectivity for Bases (SB) when operating under low ph conditions and transferability between UPLC and HPLC separations. Bonding: Intermediate coverage trifunctionally bonded C 18, no endcapping, bonded to HSS HPLC particle substrate. Selectivity Features: Aqueous mobile-phase compatible HPLC column designed for extreme retention. Combines polar compound retention with transferability between UPLC and HPLC separations. Bonding: T3 (C 18 ) bonding and endcapping, bonded to HSS HPLC particle substrate. XBridge C 18 Shield RP18 C 8 Phenyl HILIC Selectivity Features: General purpose column ideally suited for method development due to extreme ph stability and applicability to the broadest range of compound classes. Bonding: Trifunctional C 18, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate. Selectivity Features: Alternate selectivity as compared to straight chain C 18, particularly with phenolic analytes. Compatible with 1% aqueous-phase composition. Bonding: Monofunctional embedded polar C 18, fully endcapped, bonded to substrate. Selectivity Features: General purpose column ideally suited for method development due to extreme ph stability and 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. 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 tested for HILIC separations using mobile phases containing high concentrations of organic solvent. Bonding: Unbonded BEH substrate. [ [ 13 13 ] ]

SEPARATIN SLUTINS XBridge continued Amide 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 temperature and high ph. Compatible with all modern detectors including MS, ELSD, UV and Fluorescence. Bonding: Trifuncional amide bonded to BEH substrate. Atlantis T3 HILIC C 18 Selectivity Features: Retention of polar compounds, compatible with 1% aqueous mobile phases, superior stability under low ph conditions. Specifically designed for enhanced retention of polar analytes. Bonding: T3 (C 18 ) bonding and endcapping, bonded to high purity silica substrate. Selectivity Features: Excellent for retention of very polar, basic, water-soluble analytes. Specifically designed and 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 1% aqueous mobile phases. Bonding: Difunctional C 18 bonding, fully endcapped, bonded to high purity silica substrate. SunFire C 18 C 8 Selectivity Features: General purpose method development column. Very high loading capacity, particularly for basic analytes in low ph mobile phases. Ideally suited for purification and impurity profile assays. Bonding: Difunctional C 18, fully endcapped, bonded to high purity silica substrate. Selectivity Features: General purpose method development column. Very high loading capacity, particularly for basic analytes in low ph mobile phases. Less hydrophobic, therefore, less retentive than C 18 for most analytes. Bonding: Difunctional C 8, fully endcapped, bonded to high purity silica substrate. ACQUITY UPLC CSH C 18 CSH Phenyl-Hexyl CSH Fluoro-Phenyl BEH C 18 BEH Shield RP18 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 increased loading capacity for basic compounds. Bonding: Trifunctional C 18 ligand, fully end-capped, bonded to a CSH particle substrate. Selectivity Features: General purpose alternative selectivity ligand that provides pi-pi interactions with polyaromatic compounds, while maintaining excellent reproducibility at ph extremes. CSH technology enables superior peak shape and 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 using low-ph mobile phases. CSH technology enables superior peak shape and increased loading capacity for basic compounds. Bonding: Trifunctional propyl fluorophenyl ligand, non-endcapped, bonded to a CSH particle substrate. Selectivity Features: General purpose column ideally suited for method development due to extreme ph stability and applicability to the broadest range of compound classes. Bonding: Trifunctional C 18, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate. Selectivity Features: Alternate selectivity as compared to straight chain C 18, particularly for phenolic analytes. Compatible with 1% aqueous-phase composition. Bonding: Monofunctional embedded polar C 18, fully endcapped, bonded to BEH substrate. [ 14 ]

SEPARATIN SLUTINS ACQUITY UPLC continued Selectivity Features: General purpose column ideally suited for method development due to extreme ph stability and 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 polyaromatic compounds. Unique level of ph stability for a phenyl-bonded phase. BEH Phenyl 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 BEH HILIC tested for HILIC separations using mobile phases containing high concentrations of organic solvent. Bonding: Unbonded BEH substrate. Selectivity Features: Ultra performance C 18 chemistry, increased retention, superior peak shape, resists acid hydrolysis BEH HSS C at low ph. Designed for UPLC separations where silica-based C 18 selectivities are desired. 18 Bonding: High coverage trifunctional C 18, fully endcapped, bonded to High Strength Silica (HSS) UPLC particle substrate. BEH Amide HSS C 18 HSS C 18 SB HSS T3 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 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 C 18 chemistry, increased retention, superior peak shape, resists acid hydrolysis at low ph. Designed for UPLC separations where silica-based C 18 selectivities are desired. Bonding: High coverage trifunctional CC 18, fully endcapped, bonded to HSS UPLC particle substrate. Selectivity Features: Unique, non-endcapped C 18 chemistry designed specifically for method development scientists. ffers unique Selectivity for Bases (SB) when operating under low ph conditions. Bonding: Intermediate coverage tri-functionally bonded C 18, no endcapping, bonded to HSS UPLC particle substrate. Selectivity Features: Aqueous mobile-phase compatible UPLC column designed for extreme retention. Combines polar compound retention with UPLC efficiencies and performance. Bonding: T3 (C 18 ) bonding and endcapping, bonded to HSS UPLC particle substrate. FD TESTING SPECIALITY CLUMNS 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 CLUMNS 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. ther applications in this section are for monitoring antibiotic residues for possible future regulation of their proper use.

β 2 -Agonists in pork and pig liver tissues β2-agonists, veterinary drugs such as albuterol, are used to force pigs to mature faster with a higher amount of lean meat. Trace levels of β2-agonists can cause palpitation, headaches, and even death in heart patients. β2-agonists have been banned as growth promoters in pork production. Pretreatment 1. Add 8 ml.2 M sodium acetate (ph 5.2) to 2 g of sample. Homogenize and take out supernatant. Add 5 μl β-glucuronidase/arylsulfatase and hydrolyze at 37 C overnight. 2. Shake the hydrolysate for 15 minutes. Centrifuge at 5 rpm for 1 minutes and take out 4 ml supernatant. 3. Add 1 μl of 1 ng/ml standards (clenbuterol-d 9, salbutamol-d 3 ) and mix. 4. Add 5 ml.1 M perchloric acid and adjust ph to 1 ±.3. 5. Centrifuge at 5 rpm for 1 minutes. 6. Collect supernatant and add 1 M sodium hydroxide to adjust ph to 11. 7. Add 1 ml saturated sodium chloride and 1 ml isopropanol-ethyl acetate (6:4, v/v). 8. After centrifugation, take organic layer and evaporate to dryness at 4 C under nitrogen gas. 9. Dissolve residue in 5 ml.2 M sodium acetate (ph 5.2). SPE Procedure LC Conditions Column: Guard Column: Flow Rate: Mobile Phase: Waters Alliance HPLC 2695 System Atlantis dc 18, 2.1 x 15 mm, 5 μm Atlantis dc 18, 2.1 x 1 mm, 5 μm.2 ml/min A..1% formic acid b..1% formic acid in acetonitrile Gradient: Time (min) A% B%. 96 4 2. 96 4 8. 77 23 21. 77 23 22. 5 95 25. 5 95 25.5 96 4 Injection Volume: 2 μl Column Temperature: 35 C asis MCX, 3 cc/6 mg Condition/Equilibrate: A. 2 ml methanol B. 2 ml water Load: 5 ml sample Wash: A. 2 ml water B. 2 ml (2:98, v/v) formic acid in water Dry cartridge by vacuum Elute: 2 ml 5% ammonia solution in methanol Evaporate to dryness at 4 C under nitrogen gas Add 2 μl.1% formic acid in methanol (5:95, v/v), ultrasonicate Centrifuge at 15 rpm for 1 minutes MS Conditions Waters Quattro Premier Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring [ 17 ]

β 2 -Agonists in pork and pig liver tissues Analyte MRM for Quantification MRM for Confirmation Salbutamol 24 148 24 222 Terbutaline 226 152 226 125 Cimaterol 22 16 22 143 Cimbuterol 234 16 234 143 Ractompamine 32 164 32 284 Clenbuterol 277 23 277 259 Bromclenbuterol 323 249 323 168 Bromobuterol 367 293 367 349 Isoxsuprine 32 15 32 284 Mabuterol 311 237 311 293 Mapenterol 325 237 325 217 Clenbuterol-D 9 (IS) 286 24 286 24 Salbutamol-D 3 (IS) 243 151 243 151 MRM method parameters. Results A A B C D E F G 7 β 2 -agonists by multiple reaction monitoring (MRM) scan mode (A) salbutamol-d 3 (B) salbutamol (C) terbutaline (D) cimaterol (E) cimbuterol (F) ractompamine (G) clenbuterol-d 9. B C Pig liver tissues were spiked with 11 β 2 -agonists standard mixture of concentrations.5 ng/g, 1 ng/g and 2 ng/g respectively. The SPE recoveries are between 89.4% and 11.5%, RSD are between 1% and 2.8%. D E F rdering Information asis MCX, 3 cc/6 mg, 3 μm, 1/box 186254 Atlantis dc 18, 2.1 x 15 mm, 5 μm 186131 6 β 2 -agonists by multiple reaction monitoring (MRM) scan mode (A) clenbuterol (B) bromclenbuterol (C) bromobuterol (D) isoxsuprine (E) mabuterol (F) mapenterol. Atlantis dc 18, 2.1 x 1 mm, 5 μm 1861379 Sentry 2.1 mm Guard Holder WAT97958 Qsert Vials, LCGC Certified Combination Packs 1861126C Ref: The determination of β 2 -agonists by LC/MS-MS JIN Yu-E1, GU 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) 211 Waters Corporation. Waters, asis, Atlantis, Sentry, Alliance and Quattro Premier are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 18 ]

Chloramphenicol in Honey Bee keeping and honey production is a world-wide industry. Trace levels of the antibiotic chloramphenicol have been detected in honey. The antibiotic can be detected when bee keepers apply the antibiotic on hives to control bacteria that affect bee larvae. Chloramphenicol is banned in food products. Pretreatment 1. Dissolve 5 g of honey (spiked with D5-CAP) in 5 ml water. 2. Extract with 15 ml ethyl acetate and centrifuge. 3. Transfer the supernatant to a clean tube and evaporate to dryness under nitrogen at 5 C. 4. Reconstitute the residue in 1 ml methanol and dilute with 2 ml water. MS Conditions Waters Quattro micro API Ionization Mode: Negative electrospray (ESI - ) Multiple reaction monitoring Analyte Chloramphenicol (CAP) Thiamphenicol (TAP) Florfenicol (FP) Internal Standard D 5 -CAP MRM method parameters. Results Chloramphenicol MRM Transition 321 152 321 257 354 185 354 29 356 336 356 185 327 157 SPE Procedure asis HLB, 6 cc/2 mg Condition/Equilibrate: A. 5 ml methanol B. 5 ml water Load: 2 ml of sample at 2 drops/second Thiamphenicol Florfenicol Wash: 5 ml water Elute: 2 x 2.5 ml of methanol Evaporate to dryness under nitrogen at 5 C Reconstitute residue with 9:1 water/methanol (5 µl) LC Conditions Waters Alliance HPLC 2695 System Column: Symmetry C 8, 2.1 x 5 mm, 3.5 µm Guard Column: Symmetry Sentry C 8, 2.1 x 1 mm, 3.5 µm Flow Rate: Mobile Phase:.3 ml/min A. water b. methanol Gradient: Time (min) A% B%. 9 1 8. 1 9 1. 1 9 1.1 9 1 15. 9 1 Injection Volume: 2 µl Column Temperature: 3 C verlays of chloramphenicol, thiamphenicol, florfenicol at 2 μg/kg for each MRM transition. Analyte Mean Recovery (%) RSD (%) Chloramphenicol 91.1 2.2 Thiamphenicol 91.9 5.9 Florfenicol 14.6 1.7 Recovery data for CAP, TAP and FP at.3 µg/kg (n = 5). rdering Information asis HLB, 6 cc/2 mg, 3 µm, 3/box Symmetry C 8, 2.1 x 5 mm, 3.5 µm Symmetry Sentry Guard C 8, 2.1 x 1 mm Sentry 2.1 mm Guard Holder Total Recovery Vials Ref: Waters Application Note 72115EN WAT1622 WAT2624 WAT16128 WAT97958 18675CV 211 Waters Corporation. Waters, asis, Symmetry, Sentry, Alliance, and Quattro micro are trademarks of Waters Corporation. [ 19 ]

Dexamethasone in Pork The European Union (EU) considers dexamethasone residues a high priority in food animals as they are synergistic with illegal growth promoters, such as beta-agonists or anabolic steroids. Pretreatment 1. Add 5 g of the ground pork into 3 ml of 95:5 acetonitrile:water (v/v). 2. Shake for 2 minutes, homogenize, and shake for 1 minutes. 3. Centrifuge for 5 minutes. 4. Repeat extraction. Gradient: Time (min) A% B%. 7 3 3. 1 9 3.1 7 3 4. 7 3 MS Conditions Results 1 Waters Quattro Premier XE MRM Transitions: 1. 393. 373. 2. 393. 393. Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring 5. Combined both extractions and load onto Sep-Pak Vac 3 cc (5 mg), Florisil cartridges and elute with 3 ml of 95% acetonitrile and bring up to 1 ml with acetonitrile. 6. Take out 2 ml of extract add 1 ml of hexane. Shake for 3 minutes and leave to stand. 7. Take acetonitrile layer and evaporate to dryness. % 1 % Blank.25.5.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 8 ppb spiked 8. Reconstitute with 4 ml of.2 M sodium phosphate buffer (ph 5) to a final volume of 6 ml with water. SPE Procedure LC Conditions Sep-Pak Plus C 18, 36 mg Waters ACQUITY UPLC System Column: ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 µm Flow Rate: Mobile Phase: Condition: A. 1 ml methanol B. 1 ml water C. 2 ml sodium phosphate buffer (ph 5) Wash: A. 5 ml sodium phosphate buffer (ph 5) B. 1 ml 25% methanol in water Elute: 1 ml 6% acetonitrile 4% water (6:4, v/v) Reconstitute in 2 μl in 1% acetonitrile and 9% water (1:9, v/v) 4 µl/min LAD A..1% formic acid in water b..1% formic acid in acetonitrile [ 2 ].25.5.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 min 5 ppb dexamethasone spiked in pork muscle. Compound Name: Dexamethasone 393 393 RT Area 5 ppb-spiked 1.64 333.3 5 ppb-spiked 1.63 322.59 5 ppb-spiked 1.63 548.12 5 ppb-spiked 1.63 56.48 5 ppb-spiked 1.63 386.79 RSD (%) 24.43 - Recovery (%) 9.9 - Compound Name: Dexamethasone 393 393 RT Area 5 ppb-spiked 1.64 27.38 5 ppb-spiked 1.64 25.18 5 ppb-spiked 1.63 53.14 5 ppb-spiked 1.63 44.46 5 ppb-spiked 1.63 327.81 RSD (%) 29.59 - Recovery (%) 89. - Recovery data for 5 ppb dexamethasone spiked in pork muscle. rdering Information Sep-Pak Vac, 3cc/5mg, Florisil Cartridges Sep-Pak Plus C 18, 36 mg WAT2815 WAT2515 ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 µm 1862353 Qsert Vials, LCGC Certified Combination Packs 1861126C 211 Waters Corporation. Waters, Sep-Pak, ACQUITY UPLC, and Quattro Premier. All other trademarks are the property of their respective owners.

Enrofloxacin (Baytril ) in Chicken The United States Food and Drug Administration (US FDA) banned the use of enrofloxacin for growth enhancement in poultry production. Ciprofloxacin is a degradant of enrofloxacin, therefore, both compounds need to be screened in the assay. Any detectable residue can be evidence of inappropriate poultry farming practice. Pretreatment 1. Extract 1.5 g homogenized sample with 3 ml ethanol/ acetic acid (99:1, v/v) and centrifuge at 4 rpm for 5 minutes. 2. Take 1 ml aliquot of the supernatant for SPE enrichment and cleanup. 3. For muscle samples, dilute 1 ml of supernatant with 5 ml water prior to SPE; liver samples are not diluted. SPE Procedure Cartridge II: Sep-Pak Accell QMA, 3 cc/5 mg UPLC Conditions CNDITIN/EQUILIBRATE: 3 ml 5% ammonia in methanol Attach Sep-Pak Accell QMA cartridge to outlet of MCX cartridge. Elute from MCX cartridge into Sep-Pak Accell QMA cartridge. (6 cc cartridge on top) Elute*: 3 ml (5:95, v/v) ammonia in methanol Remove asis MCX cartridge WASH: 3 ml ethanol ELUTE: 3 ml methanol in formic acid (98:2, v/v) Evaporate solvent and reconstitute in 15 µl of acetonitrile in water (15:85, v/v) * Note: As the analytes are eluted from cartridge I, they are subsequently retained by anion exchange on cartridge II. Therefore, the eluate from cartridge I becomes the load for cartridge II. Cartridge I: asis MCX, 6 cc/15 mg Waters ACQUITY UPLC System Condition/Equilibrate: A. 3 ml methanol B. 3 ml water C. 3 ml ethanol Load: 1 ml sample Wash: A. 3 ml 1% acetic acid/ethanol B. 3 ml water C. 3 ml methanol Column: Flow Rate: Mobile Phase: ACQUITY UPLC BEH C 18, 1 x 5 mm, 1.7 μm.12 ml/min A. 1% formic acid in water b. acetonitrile Gradient: Time (min) A% B%. 95 5 3. 5 5 6.5 5 5 1.5 95 5 15.5 95 5 Injection Volume: 1 μl Column Temperature: 3 C MS Conditions Waters Quattro micro API Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring Analyte Enrofloxacin Ciprofloxacin MRM method parameters. MRM Transition 36 342 36 316 332 314 332 288 [ 21 ]

Enrofloxacin (Baytril ) in Chicken 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%. asis MCX 6 cc/15 mg SPE cartridge Cation-exchange retention of bases. Neutrals or acids, are poorly retained and washed off before elution. Sep-Pak Accell QMA 3cc/5 mg SPE cartridge Anion-exchange retention of acids. Neutrals or bases are poorly retained and are washed off before elution. Tandem SPE cartridges setup. rdering Information asis MCX, 6 cc/15 mg, 6 μm, 3/box 186255 Sep-Pak Accell Plus QMA, 3 cc/5 mg, 5/box WAT285 ACQUITY UPLC BEH C 18, 1 x 5 mm, 1.7 μm 1862344 ACQUITY UPLC BEH C 18, 1 x 5 mm, 1.7 μm, 3/pk 176861 Qsert Vials 1861126 Ref: Waters application WA4326 211 Waters Corporation. Waters, asis, 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 ]

Nitrofurans in Honey Nitrofurans are a class of antibiotic drugs, used to treat bacterial infections, which may induce carcinogenic residues in animal tissues. Due to public health concerns, nitrofurans have been banned for use in food producing animals in many countries. Pretreatment 1. Dilute 2 g honey sample with 5 ml of.12 M hydrochloric acid. SPE Procedure SPE Step I: asis HLB, 3 cc/6 mg Condition/Equilibrate: A. 2 ml methanol B. 2 ml water Pass-through: 2 g prepared honey in 5 ml of.1 N hydrochloric acid Wash: 2 ml water Combine the solutions 1. Collect quantitatively the eluent from the pass-through and wash steps into a 15 ml capped sample tube. 2. Add 3 μl of 5 mm 2-nitrobenzaldehyde in dimethylsulfoxide. Hydrolyse and derivatize for 18 hours at 37 C. 3. Cool the sample to room temperature and adjust to ph 7 by adding 6 ml of.1 M dipotassium hydrogen phosphate. 4. Put sample through SPE Step II. LC Conditions Waters Alliance HPLC 2695 System Column: XTerra MS C 18, 2.1 x 1 mm, 3.5 µm Flow Rate: Mobile Phase: Injection Volume: 2 µl Column Temperature: 3 C MS Conditions SPE Step II: asis HLB, 3 cc/6 mg Condition/Equilibrate: A. 1 ml methanol B. 1 ml water Load: Prepared honey sample ph 7 Wash: A. 2 ml water B. 2 ml 3% methanol in water.2 ml/min DRY: 2 minutes Elute: 3 ml formic acid methyl-t-butyl/methanol/formic acid (79:9:2, v/v/v) Evaporate and reconstitute in 2 µl mobile phase Isocratic 7% 2 mm ammonium formate ph 4., 3% acetonitrile Waters Quattro micro API Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring Analyte AZ AMZ Semicarbizide (SC) 1-Aminohydantion (AH) MRM Transition 236 134 335 291 29 192 178 249 MRM method parameters. [ 23 ]

Nitrofurans in Honey Results AZ AMZ SC AH Spiked honey (4 ng/kg) metabolites as 2-nitrobenzaldehyde derivatives. Analytes RSD (%) Raw Wild Honey Buckwheat Honey Semicarbizide 9.8 9.7 AZ 13.9 9.6 AMZ 3.8 2.9 AH 14 3.8 Relative standard deviation obtained from two different lots of honey spiked at 5 ng/kg (ppt). Metabolite recovery was greater than 85% post-derivatization for each analyte. The samples used for spiking tested negative before the study. rdering Information asis HLB, 3 cc/6 mg, 3 µm, 1/box WAT94226 XTerra MS C 18, 2.1 x 1 mm, 3.5 µm 18644 LCMS Certified Vials 6751CV Ref: Waters Application Note 72134EN 211 Waters Corporation. Waters, asis, XTerra, Alliance, and Quattro micro are trademarks of Waters Corporation. [ 24 ]

Nitrofurans in Tissues The United States Food and Drug Administration (US FDA) banned Nitrofuran drugs are banned in food producing animals because they pose a public health risk. The rule went into effect as a result of evidence that the drugs may induce carcinogenic residues in animal tissues. Pretreatment 1. Homogenize 1 g of sample in 1 ml of.12 M hydrochloric acid. 2. Take 1 ml aliquot and treat with 4 µl of 5 mm 2-nitrobenzaldehyde in dimethylsulfoxide. LC Conditions Waters Alliance HPLC 2695 System Column: XTerra MS C 18, 2.1 x 1 mm, 3.5 µm Flow Rate: Mobile Phase: Injection Volume: 2 µl Column Temperature: 3 C MS Conditions.2 ml/min Isocratic 7% 2 mm ammonium formate ph 4, 3% acetonitrile Waters Quattro micro API Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring 3. Hydrolyze/derivatize the sample for 16 hours at 37 C. 4. Adjust the sample to ph 7.4 with potassium hydrogen phosphate. 5. Centrifuge sample for 5 minutes at 8 rpm. SPE Procedure asis HLB 3 cc/6 mg Condition/Equilibrate: A. 1 ml methanol B. 1 ml water Analyte AZ AMZ SC AH MRM method parameters. Results MRM Transition 236 134 335 291 29 192 249 178 AZ Load: Approximatively 1 ml of sample Wash: A. 2 ml water B. 2 ml 3% methanol in water AMZ DRY: 2 minutes SC Elute: 3 ml methyl-t-butyl/methanol/formic acid (89:9:2, v/v/v) AH Evaporate and reconstitute in 2 μl mobile phase Spiked chicken muscle (1 ng/g) metabolites as 2-nitrobenzaldehyde derivatives. [ 25 ]

Nitrofurans in Tissues STRUCTURES AZ N HC NH 2 N 2 N 2 N N H +,H 2, 37 o C, 18 hr H 2 N N 2 N AMZ N N N N N 2 1-Aminohydantion (AH) H N N NH 2 N N NH N 2 H Semicarbizide (SC) H 2 N N NH 2 H N N NH 2 Chemical structures of 2-nitrobenzaldehyde derivatives. rdering Information asis HLB, 3 cc/6 mg, 3 µm, 1/box WAT94226 XTerra MS C 18, 2.1 x 1 mm, 3.5 µm 18644 Qsert Vials, LCGC Certified Combination Packs 1861126C 211 Waters Corporation. Waters, asis, Alliance, XTerra, and Quattro micro are trademarks of Waters Corporation. All other trademarks are the property of their respective owners [ 26 ]

Penicillin G in Pork World organizations are concerned about the overuse of antibiotics and antibacterials levels in foods due to possible bacteria resistances and health concerns. Pretreatment 1 % 1 Blank Sample Spiked Sample 1. Homogenize 3 g of sample with 3 ml of 5% sodium tungstate, 3 ml of.17 M sulfuric acid and 3 ml of water. 2. Centrifuge 31 rpm for 1 minutes. 3. Filter with glass fiber filter. SPE Procedure LC Conditions Waters ACQUITY UPLC System Column: ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 µm Flow Rate: Mobile Phase: Sep-Pak Plus Short C 18, 36 mg 6 µl/min Load Wash: 3 ml water Elute: 4 ml 2% acetonitrile water Concentrate to 2.5 ml and adjust the volume to 6 ml water A..1% formic acid in water b..1% formic acid in acetonitrile Gradient: Time (min) A% B%. 9 1 5. 1 9 5.5 9 1 6. 9 1 %.25.5.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 min 5 ppb penicillin spiked in pork muscle. Compound Name: penicillin MRM 335 176 RT Area Pork blank 1 1.83 4.5 Pork blank 2 1.83 3.23 5 ppb spiked pork 1 1.83 23.3 5 ppb spiked pork 2 1.82 18.54 5 ppb spiked pork 3 1.83 21.82 5 ppb spiked pork 4 1.83 15.45 5 ppb spiked pork 5 1.82 21.5 RSD (%) 15.56 - Recovery (%) 58.26 - Compound Name: penicillin MRM 335 16 RT Area Pork blank 1 1.82 5.81 Pork blank 2 1.83 5.2 5 ppb spiked pork 1 1.83 42.97 5 ppb spiked pork 2 1.83 26.59 5 ppb spiked pork 3 1.83 21.89 5 ppb spiked pork 4 1.82 25.22 5 ppb spiked pork 5 1.83 39.3 RSD (%) 29.75 - Recovery (%) 78.3 - Recovery results for 5 ppb penicillin spiked in pork muscle. MS Conditions Waters Quattro Premier XE Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring MRM Transitions: 1: 33516 2: 335176 rdering Information Sep-Pak Plus Short C 18 WAT2515 ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 µm 186235 LCMS Certified Vials 6751CV 211 Waters Corporation. Waters, ACQUITY UPLC, Sep-Pak, and Quattro Premier are trademarks of Waters Corporation. [ 27 ]

Penicillins, Tetracyclines, and sulfonamides in milk Results World organizations are concerned about the overuse of antibiotics and antibacterials. This method can be used to monitor 1 1 2 antibacterials in milk. Pretreatment 3 Milk (1 ml) is first extracted with 3 ml acetonitrile (1 minute shake). The sample is centrifuged and the supernatant is collected. 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). % 1. Penicillin 2. xacillin 3. Cloxacillin The milk solids pellet is then extracted with 3 ml of ph 5 succinate/edta buffer. The sample is centrifuged and the supernatant is collected (extract 2, this step extracts the tetracyclines and any remaining sulfonamides). Extract 2 is combined with the evaporated extract 1 and the volume is made up to 1 ml with water. The combined extracts are then processed using an asis HLB cartridge. SPE Procedure asis HLB 1cc/3mg Condition/Equilibrate: 1 ml MeH, 1 ml water Load SAMPLE: From pretreatment Wash:.5 ml 5% methanol/water 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 min 1 ppb spiked in milk. Compound MRM Transition Recovery xytetracydine 461 426 78 Tetracycline 445 41 69 Chlortetracycline 479 444 76 Sulfadiazine 251 92 83 Sulfathiazole 256 92 87 Sulfapyridine 25 92 66 Sulfamerazine 265 92 95 Sulfamethazine 279 92 9 Sulfamethoxypyridazine 281 92 7 Sulfachloropyridazine 285 92 85 Sulfamethoxazole 254 92 97 Sulfadimethoxine 311 92 83 LC Conditions Elute: 6:4 methanol/water 6 mm ammonium acetate Penicillin G 335 16 93 xacillin 42 16 83 Cloxacillin 436 16 96 Column: Waters ACQUITY UPLC BEH Shield RP18, 2.1 x 1 mm, 1.7 µm Flow Rate: 4 ml/min Mobile Phase: A:.1% Formic acid in water b: Acetonitrile Gradient: Time (min) A% B% initial 85 15 2. 6 4 2.5 4 6 3. 1 9 4.5 1 9 4.6 85 15 5.5 85 15 MRM method parameters and recovery data. rdering Information asis HLB, 1 cc/3 mg, 1/box WAT94225 ACQUITY UPLC BEH Shield RP18, 2.1 x 1 mm, 1.7 µm 1862854 LCMS Certified Vials 6751CV 211 Waters Corporation. Waters, asis, and ACQUITY UPLC are trademarks of Waters Corporation. [ 28 ]

Spiramycin in Pork World health organizations are concerned about the antibiotics and antibacterials levels in food. This method can be used to monitor Spiramycin, a macrolide antibiotic, in pork. Pretreatment 1. Weigh 5 g of sample into 25 ml of water. Homogenize. 2. Centrifuge at 3 rpm for 1 minutes. 3. Collect supernatant. 4. Re-extract pellet with 15 ml 1.2% metaphosphoric acid/ methanol (5:5, v/v). 5. Vortex. 6. Centrifuge at 3 rpm for 1 minutes. Collect supernatant. 7. Combine both supernatants. 8. Filter sample using glass fiber filter. SPE Procedure LC Conditions Instrument Waters ACQUITY UPLC System Column: ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 µm Flow Rate: Mobile Phase: 4 µl/min A..1% formic acid in water b..1% formic acid in acetonitrile Gradient: Time (min) A% B%. 9 1 2. 6 4 2.1 9 1 3. 9 1 MS Conditions MRM transitions: Waters Quattro Premier XE 1: 422.5 11. 2: 422.5 174.1 Ionization mode: Positive electrospray (ESI + ) Multiple reaction monitoring Results 1 asis MCX, 6 cc/15 mg Condition: A. 3 ml phosphate buffer/methanol (1:9, v/v) B. 5 ml water % 1 Quantification Load: Sample (approximately 4 ml) % Confirmation.25.5.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 min 1 ppb spiramycin spiked sample in pork muscle. Wash: A. 3 ml.1 M dipotassium hydrogen phosphate (ph 3) B. 15 ml.1 M potassium phosphate/methanol (1:9, v/v) Evaporate: Approximately to 1 ml and bring up to 2 ml with 2% methanol SPE Buffer: Dissolve 8.7 g dipotassium hydrogen phosphate in 1 L of deion ized water, adjust to ph 3 with phosphoric acid. [ 29 ]

Spiramycin in Pork Compound Name: Spiramycin 174.1 RT Area 1 ppb spiked 1 1.34 92.79 1 ppb spiked 2 1.34 911.36 1 ppb spiked 3 1.34 141.24 1 ppb spiked 4 1.34 13.33 1 ppb spiked 5 1.34 111.27 RSD (%) - 7.97 Recovery (%) - 73.26 Compound Name: Spiramycin 11 RT Area 1 ppb spiked 1 1.34 163.65 1 ppb spiked 2 1.34 1267.22 1 ppb spiked 3 1.34 1368.84 1 ppb spiked 4 1.34 1227.1 1 ppb spiked 5 1.34 1379.4 RSD (%) - 5.96 Recovery (%) - 74.82 Recovery data for 1 ppb spiramycin spiked sample in pork muscle. rdering Information asis MCX, 6 cc/15 mg, 3/box 186256 ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 µm 186235 LCMS Certified Vials 6751CV 211 Waters Corporation. Waters, ACQUITY UPLC, asis, and Quattro Premier are trademarks of Waters Corporation. [ 3 ]

STREPTMYCIN IN HNEY Bee keeping and honey production is a world-wide industry. Trace levels of the Streptomycin, an aminoglycoside, has been detected in honey. Streptomycin is not permitted in food products. Pretreatment 1. Dissolve 2 g of honey in approximately 75 ml water. 2. Bring to a total volume of 1 ml with water. 3. Filter through a fluted filter to remove suspended solids. SPE Procedure Sep-Pak Vac 6 cc Accell Plus CM cartridge Condition: 2 x 5 ml 2% acetic acid MS Conditions Waters Quattro micro Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring Analyte Streptomycin Internal Standard MRM method parameters. Results 1 % 1 % Internal Standard Quantification Internal Standard Confirmation MRM Transition 582 263 582 176 584 263 584 246 RINSE: 2 x 5 ml water* LAD: 5 ml of honey solution at approximately 2 drops per second 1 % Streptomycin Quantification RINSE: 2 x 5 ml water 1 % Streptomycin Confirmation min LC Conditions Column: Guard Column: Flow Rate: Mobile Phase: Column Temperature: 3 C Injection Volume: ELUTE: 2 x 5 ml (8:2, 2% acetic acid/acetonitrile) Adjust to 1 ml with water * Do not allow the cartridge to dry out through the procedure Waters Alliance 2795 HPLC System Atlantis HILIC Silica, 2.1 x 5 mm, 3 μm Atlantis HILIC Silica, 2.1 x 1 mm, 3 μm.3 ml/min A. 2 mm ammonium formate in 1 mm formic acid b. 1mM Formic Acid in acetonitrile 2 μl Gradient: Time (min) A% B%. 1 9 6. 6 4 1. 6 4 1.1 1 9 16. 1 9 MRM for streptomycin and the internal standard. Spiked Concentration µg/kg Mean Std. Dev. RSD (%) 2 1.99.9 4.5 2 2.17.76 3.7 1 1.91 3.75 3.7 Method accuracy and precision over three days. rdering Information Sep-Pak Vac, 6 cc Accell Plus CM cartridge WAT54545 Atlantis HILIC Silica, 2.1 x 1 mm, 3 μm 18625 Atlantis HILIC Silica, 2.1 x 5 mm, 3 µm 186211 LCMS Certified Vials 6751CV Ref: Waters Application Note 72981EN 211 Waters Corporation. Waters,Sep-Pak, Quattro micro, Alliance, Accell and Atlantis are trademarks of Waters Corporation. [ 31 ]

Sulfonamide Antibacterials in Milk Sulfonamides are widely used for therapeutic and prophylactic purposes in animals. When sulfonamides are retained in food stuff, this may result in allergic or toxic reactions in sensitive consumers. This method can be used to monitor the presence of sulfonamides in milk. SPE Procedure asis MCX, 3 cc/6 mg Condition: A. 2 ml methanol B. 2 ml water Load: 5 ml whole homogenized milk Wash 1: 2 ml 5% methanol in water WASH 2: 1 ml.5 M aqueous hydrochloric acid WASH 3: 2 ml 2% methanol in water WASH 4: 1 ml methanol MS Conditions Compounds Sulfapyridine Sulfamethoxazole Sulfadiazine Sulfathiazole Sulfamerazine Sulfamethizole Sulfamethazine Sulfamethoxy-pyridazine Sulfachloropyridazine Sulfadimethoxine MRM method parameters. Waters Quattro Premier XE Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring Precusor (m/z) 249.8 91.9 249.8 155.8 253.7 91.9 253.7 155.8 25.8 91.9 25.8 155.9 255.7 91.9 255.7 155.9 264.8 92. 264.8 155.8 27.9 91.7 27.9 155.8 278.8 91.9 278.8 155.8 28.8 91.9 28.8 155.8 284.7 92. 284.7 155.9 31.8 91.9 31.8 155.9 ELUTE: 2.5 ml 9:1 methanol/water (2 mm ammonium bicarbonate ph 8.5) Reconstitute in.5 ml mobile phase LC Conditions Waters ACQUITY UPLC System Column: ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 µm Flow Rate: Mobile Phase:.2 ml/min A..5% formic acid/water b..5% formic acid/methanol Gradient: Time (min) A% B%. 9 1 3.25 8 2 3.26 9 1 Detector: ACQUITY UPLC PDA Detector [ 32 ]

Sulfonamide Antibacterials in Milk Results % 1 2 3 4 6 7 8 9 1 1- Sulfadiazine 2- Sulfathiazole 3- Sulfapyridine 4- Sulfamerazine 5- Sulfamethizole 6- Sulfamethazine 7- Sulfamethoxypyridine 8- Sulfachloropyridazine 9- Sulfamethoxazole 1-Sulfadimethoxine 5 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 1.2 4-Sulfamerazine 77 9.1 5-Sulfamethizole 73 1.1 6-Sulfamethazine 67 7. 7-Sulfamethoxypyridine 79 1. 8-Sulfachloropyridazine 6 7. 9-Sulfamethoxazole 71 9.1 1-Sulfadimethoxine 8 9.2 Recovery data for whole milk (5 ng/ml). rdering Information asis MCX, 3 cc/6 mg, 1/box 186254 ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm 1862352 LCMS Certified Vials 6751CV 211 Waters Corporation. Waters, asis, ACQUITY UPLC, and Quattro Premier are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 33 ]

Tetracyclines and Sulfonamides in Milk Tetracyclines and sulfonamides are widely used for therapeutic and prophylactic purposes in animal diseases. When these classes of veterinary drugs are retained in food stuff, this may result in allergic or toxic reactions in sensitive consumers. MS Conditions Results Waters Quattro Premier XE Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring Pretreatment 1 4 1. Mix 1.5 ml milk with 6 ml ph 4 McIivaine buffer. 1 2. Centrifuge. 3. Take supernatant/adjust to ph 1 with.75 ml 1 M NaH. % 2 1. xytetracycline 2. Tetracycline 3. Chlortetracycline 4. Doxycycline SPE Procedure 3 asis MAX, 1 cc/3 mg Condition/Equilibrate: 2 ml MeH, 2 ml water.2.4.6.8 1 1.2 1.4 1.6 1.8 2 min 1 ppb spiked sample. Load SAMPLE: From pretreatment Wash 1:.5 ml 5% NH 2 H/water WaSH 2:.5 ml methanol ELUTE:.5 ml 45.55 acetonitrile/75 mm oxalic acid. Dilute 1:3 with water for LC LC Conditions Column: Waters ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 µm Flow Rate: Mobile Phase: 4 ml/min A:.1% Formic acid/water b: Acetonitrile Gradient: time (min) A% B% initial 85 15 2.5 5 5 3.5 3 7 3.6 85 15 4. 85 15 rdering Information asis MAX, 1 cc/3 mg, 1/box 186366 ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 µm 186235 LCMS Certified Vials 6751CV 211 Waters Corporation. Waters, asis, and ACQUITY UPLC are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 34 ]

Tetracyclines in Animal Tissue Tetracyclines (TCs) are broad-spectrum antibiotics used in human and veterinary medicine. The use of TCs is increasing, as they are used not only in treatment, but also prevention of illnesses. Moreover, TCs are given to animals destined for human consumption to promote growth and may potentially result in the presence of residues in edible animal tissues, which can be toxic and dangerous for human health. LC Conditions Waters Alliance HPLC 2695 System Column: Nova-Pak C 8, 3.9 x 15 mm, 4 μm Flow Rate:.8 ml/min Mobile Phase: Acetonitrile: 5 mm oxalic acid (1:4) Injection Volume: 6 μl Detector: 2487 Dual Wavelength Detector UV Wavelength: 365 nm Pretreatment 1. Add 2 ml McIlvaines buffer-edta to 5 g homogenized sample in a 5 ml centrifuge tube and mix thoroughly. 1 2 1. xytetracycline (Spike level.5 µg/g) 2. Tetracycline 3. Chlortetracycline 2. Centrifuge at 3 rpm for 1 minutes. 3. Collect supernatant. 3 spiked pork 4. Repeat steps 1 and 2 with the residue. Collect supernatant. nonspiked sample 5. Combine both supernatant portions for SPE analysis. SPE Procedure.5 µg/g sample. 5 asis HLB, 6 cc/2 mg Condition/Equilibrate: A. 3 ml methanol B. 2 ml water Load: 4 ml sample (4 ml/min) Wash: 2 ml 5% methanol in water Elute: 3 ml methanol Evaporate to.2 ml Solutions McIlvaines Buffer 1. Thoroughly mix 1 ml.1 M citric acid with 625 ml.1 M disodium hydrogen phosphate dihydrate. 2. Adjust with sodium hydroxide or hydrochloric acid to ph 4 ±.5, if necessary. EDTA-McIlvaines Buffer 1. Add 6.5 g disodium EDTA to 1625 ml McIlvaines Buffer and mix thoroughly. Reconstitute with 1 ml 5 mm oxalic acid rdering Information asis HLB, 6 cc/2 mg, 6 µm, 3/box Nova-Pak C 8, 3.9 x 15 mm, 4 μm LCMS Certified Vials WAT1622 WAT35876 6751CV 211 Waters Corporation. Waters, asis, Alliance, and Nova-Pak are trademarks of Waters Corporation. [ 35 ]

Tetracyclines in Honey Tetracyclines (TCs), an antibiotic, is not permitted in apiculture. This method can monitors the presence of TC s in honey. Pretreatment 1. Add 3 ml EDTA-McIlvaines buffer to 6 g sample, mix thoroughly for 1 minute. 2. Centrifuge at 3 rpm for 5 minutes and collect supernatant for SPE. SPE Procedure Cartridge I: asis HLB, 6 cc/5 mg Condition/Equilibrate: A. 5 ml methanol B. 1 ml water Load: Honey sample (3 ml/min) Wash: 5 ml methanol/water (5:95, v/v) DRY: By vacuum for 2 minutes Elute: 15 ml ethyl acetate Solutions McIlvaines Buffer 1. Thoroughly mix 1 ml.1 M citric acid with LC Conditions Column: SunFire C 8, 2.1 x 15 mm, 3.5 µm Mobile Phase: Acetonitrile:methanol:.4% fomic acid (18:4:78) Flow Rate: Injection Volume:.2 ml/min 2 μl Column Temperature: 25 C MS Conditions 625 ml.1 ML disodium hydrogen phosphate dihydrate. 2. Adjust with sodium hydroxide or hydrochloric acid to ph 4 ±.5, if necessary. EDTA-McIlvaines Buffer 1. Add 6.5 g disodium EDTA to 1625 ml McIlvaines Buffer and mix thoroughly. Waters Quadrupole MS Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring Solution I Analyte MRM for Quantification MRM for Quantification Cartridge II: Sep-Pak Accell CM, 3 cc/5 mg Condition/Equilibrate: 5 ml ethyl acetate Load: Solution I (3 ml/min) Wash: 5 ml methanol DRY: By cartridge by vacuum for 5 minutes xytetracycline Tetracycline Chlortetracycline Doxycycline MRM method parameters. 461 426 461 443 461 381 445 41 445 154 445 428 479 444 479 154 479 462 445 428 445 41 445 154 461 426 445 41 479 444 445 428 Elute: 4 ml mobile phase [ 36 ]

Tetracyclines in Honey Results B A C A. xytetracycline, B. Tetracycline, C. Chlortetracycline and D. Doxycy cline standards. Analyte A. xytetracycline B. Tetracycline C. Chlortetracycline D. Doxycycline Concentration (mg/kg) Average Recovery.2 88..1 95.3.1 95.8.5 93.6.2 81.9.1 82.6.5 84.5.1 89.3.2 87.2.1 86..5 86.6.1 9.8.2 85.2.1 85.3.5 86.8.1 87.9 Recovery data for tetracyclines in honey. rdering Information asis HLB, 6 cc/5 mg, 6 µm 3/box 186115 Sep-Pak Accell CM, 3 cc/5 mg, 5/box WAT2855 SunFire C 8, 2.1 x 15 mm, 3.5 μm 1862712 LCMS Certified Vials 6751CV Ref: China GB/T 18932.23-23 211 Waters Corporation. Waters, Sep-Pak, Accell, asis, 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 723643en).

Acrylamide in Fried Potato Products Acrylamide, a chemical contaminant, is produced during the cooking of french fries, potato chips, and other processed foods. Acrylamide is considered to be a possible cancer causing agent. Pretreatment 1. 1 g crushed potato product was weighed into a centrifuge tube. 2. 15 ml of 2 M sodium chloride and 1 μl of internal standard (acrylamide-d 3 ) solution was added to the tube. Shake contents vigorously for 3 minutes. 3. Centrifuge at 1 x g for 12 minutes. 4. Take a 1.5 ml aliquot of the supernatant from the centrifuge tube for SPE extraction and cleanup. SPE Procedure Cartridge I: asis HLB, 6 cc/2 mg Condition: A. 2mL methanol B. 2mL 2 M sodium chloride Mobile Phase:.1% formic acid in water Injection Volume: 2 μl Column Temperature: 3 C MS Conditions Waters ZMD Mass Detector Ionization Mode: Positive Electrospray (ESI + ) Selected-Ion Recording (SIR) Compound: Mass Cone Voltage (V) Acrylamide 72 2 55 4 Acrylamide-D 3 75 2 58 4 Results abundance, m/z 72 Acrylamide 71 µg/kg Load: 1.5 ml potato extract Wash:.8 ml water 2 3 4 5 min LC/MS determination of acrylamide in potato chips. LC Conditions Column: Flow Rate: Elute: 3 ml 1% formic acid in methanol Cartridge II: asis MCX, 3 cc/6 mg Condition: 2 ml methanol A. Pass eluent from Part A. B. Rinse vial in.5 ml methanol, combine with passed eluent C. Collect in total Evaporate and Reconstitute:.4 ml water Waters Alliance HPLC 2695 System Atlantis dc 18, 2.1 x 15 mm, 5 μm.2 ml/min Fortification Level (µg/kg) Amount Found (μg/kg) RSD (%)* 1 96 12 2 211 8.7 5 488 5.8 1 11 8. 2 2 6.5 *Five replicate samples analyzed per level. rdering Information Atlantis dc 18, 2.1 x 15 mm, 5 μm 186131 asis HLB, 6 cc/2 mg WAT1622 asis MCX, 3 cc/6 mg 186254 Qsert Vials, LCGC Certified Combination Packs 1861126C Ref: Waters Application Note, 72688EN 211 Waters Corporation. Waters, asis, Alliance, and Atlantis are trademarks of Waters Corporation. All other trademarks are the property of their respective owners [ 39 ]

Aflatoxins in Produce SAmples Results 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 5 g ground sample with 5 g sodium chloride and place in blender jar. 2. Add 1 ml 8:2 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 6 ml of phosphate buffer saline. Mix well. 7. Filter extract through glass microfiber filter into a clean vessel. SPE Procedure AflaTest Affinity Column Pass 4 ml of filtered diluted extract (4 ml =.2 g sample equivalent) completely through AflaTest affinity column at a rate of about 1 2 drops/seconds until air comes through column Pass 1 ml of 2:8 methanol:water through the column at a rate of about 2 drops/second Repeat previous step once more until air comes through column Place glass cuvette under AflaTest column and add 1 ml HPLC grade methanol into glass syringe barrel Elute AflaTest column at a rate of 1 drop/second by passing methanol through the column and collecting all the sample eluate (1 ml) in a glass cuvette Add 1 ml of purified water to eluate. Inject 2-1 μl onto HPLC LC Conditions Waters Alliance HPLC System Column: XBridge C 18, 4.6 x 25 mm, 5 μm Flow Rate: 1 ml/min Mobile Phase: Acetonitrile/water/methanol (17:54:29, v/v/v) Injection: 1 µl Detector: 2475 Multi Wavelength Fluorescence Detection: Excitation Wavelength: 333 nm emmission Wavelength: 46 nm Aflatoxins in red pepper extract. Recovery: 76% at 2 ppb (7B1:1B2:3G1:1G2 aflatoxin mix). Action Levels for Aflatoxins United States (FDA) action levels (B1, B2, G2, G2, M1) Food Stuff Level Regulation All products, except milk, designated Policy Guides 712.26, 2 ng/g for humans 716.1, 7126.33 Milk.5 ng/g Policy Guides 712.26, 716.1, 7126.33 Corn for immature animals and Policy Guides 712.26, 2 ng/g dairy cattle 716.1, 7126.33 Corn for breeding beef cattle, swine Policy Guides 712.26, 1 ng/g and mature poultry 716.1, 7126.33 Corn for finishing swine 2 ng/g Policy Guides 712.26, 716.1, 7126.33 Corn for finishing beef cattle 3 ng/g Policy Guides 712.26, 716.1, 7126.33 Cottonseed meal (as feed ingredient) 3 ng/g Policy Guides 712.26, 716.1, 7126.33 All feedstuff other than corn 2 ng/g Policy Guides 712.26, 716.1, 7126.33 Aflatoxin regulatory action limits. rdering Information VICAM aflachra HPLC Columns, 25/box G117 VICAM Glass Cuvette 34 XBridge C 18, 4.6 x 25 mm, 5 μm 1863117 LCMS Certified Combination Packs 6751CV 211 Waters Corporation. Waters, Alliance, AflaTest, aflachra HPLC, Viacam and XBridge are trademarks of Waters Corporation. [ 4 ]

Carbamates in Fruits and Vegetables Carbamates have been identified as a health risk. They affect the nervous system by reducing the ability of cholinesterase, an enzyme, to function properly in regulating the neurotransmitter acetylcholine. Pretreatment 1. Add 5 ml of acetonitrile to 25 g of sample. Homogenize for 2 minutes and filter. 2. Collect 4-5 ml of filtrate into a flask containing 5 7 g sodium chloride. 3. Shake vigorously for 1 minute. Leave to stand at room temperature. Gradient: Time (min) A% B% C%. 88 12 5.3 88 12 5.4 68 16 16 14. 68 16 16 16.1 5 25 25 2. 5 25 25 22. 88 12 3. 88 12 Sample: 1 ng of each analyte on column Injection Volume: 4 μl Post Column Addition: PA*/NaH @.5 ml/min Detector: 2475 Multi Wavelength Fluorescence Detector Excitation Wavelength: 339 nm Emission Wavelength: 445 nm *PA: rthophthaldehyde 4. Take 1 ml aliquot from the acetonitrile layer and evaporate sample to dryness (8 C under nitrogen or air). 5. Reconstitute with 2 ml methanol/dichloromethane (1:99, v/v). 1 2 3 4 5 6 7 8 9 1 11 12 SPE Procedure LC Conditions Column: Flow Rate: Mobile Phase: Sep-Pak Aminopropyl, 6 cc/5 mg Condition/Equilibrate: 4 ml methanol/dichloromethane (1:99, v/v) PASS-THRUGH: 2 ml sample Collect the solution Elute: 2 ml methanol/dichloromethane (1:99, v/v) x 2 Combine the solutions Evaporate to dryness under nitrogen at 5 C Reconstitute in 2.5 ml methanol Waters Alliance HPLC 2695 System Carbamate Analysis Column, 3.9 x 15 mm 1.5 ml/min A. water b. methanol c. acetonitrile Aldicarb standards. Peak Analyte 4 µl 1 Aldicarb Sulfoxide 3.77 2 Aldicarb Sulfone 4.66 3 xamyl 5.17 4 Methomyl 6.3 5 3-Hydroxycarbofuran 9.83 6 Aldicarb 11.46 7 Propoxur 14.35 8 Carbofuran 14.94 9 Carbaryl 17.37 1 1-Naphthol 18.99 11 Methiocarb 22.2 12 BDMC 22.56 Expected retention times for aldicarb standards. rdering Information Sep-Pak Aminopropyl, 6 cc/5 mg, 3/box Carbamate Analysis Column, 3.9 x 15 mm LC Certified Vials WAT5456 WAT35577 186272C Ref: Ministry of Agriculture, China (NY/T 761.1 24 and NY/T761.3 24) 211 Waters Corporation. Waters, and Sep-Pak and Alliance are trademarks of Waters. Corporation. [ 41 ]

Malachite Green in Fish (HPLC/UV) Malachite Green (MG) is an effective and inexpensive fungicide used in aquaculture. During metabolism MG reduces to Leucomalachite Green (LMG), which has been shown to accumulate in fatty fish tissues. Both MG and LMG have demonstrated putative carcinogenic activity, and thus, have been banned for use in aquaculture by both the United States Food and Drug Administration (US FDA) and European Union (EU). Pretreatment 1. Weigh 1 g sample into a 3 ml centrifuge tube. 2. Add 5 μl TMPD* solution (1 mg/ml). 3. Add standard solutions (MG, LMG,.1 µg/ml) and internal standard, leave to stand for 1 minutes. 4. Add 1 ml McIlvaines Buffer (ph 2.6) /methanol (5:5, v/v) solution; homogenize for 45 seconds. 5. Centrifuge at 5 rpm for 2 minutes, transfer supernatant into centrifuge tube. 6. Repeat steps 4 and 5, combine the two portions of supernatant. A 2 ml aliquot will be used for SPE. *TMPD= N, N, N, M - Tetramethyl-1,4-phenylenediamine dihydrochloride SPE Procedure asis MCX 6cc/15 mg Condition/Equilibrate: A. 5 ml methanol b. 5 ml water c. 5 ml McIlvaines Buffer (ph 2.6) Load: 2 ml of sample Solutions McIlvaines Buffer (ph 2.6) : 1..1 M citric acid monohydrate (A) - Dissolve citric acid monohydrate (1.5 g) in water (5 ml). 2..2 M disodium hydrogen phosphate dihydrate (B) - Dissolve disodium hydrogen phosphate dihydrate (14.2 g) in water (5 ml). 3. Mix A (445.5 ml) and B (54.5 ml). McIlvaines Buffer (ph 2.6): methanol (5:5 v/v): Mix McIlvaines Buffer (ph 2.6) (25 ml) with methanol (25 ml). LC Conditions Column: Flow Rate: Waters Alliance HPLC 2695 System SunFire C 18, 4.6 x 15 mm, 5 μm Self-packed Pb 2 oxidising column: 4.6 x 2 mm, (Pb 2 : diatomaceous earth = 3:1); Both ends are fitted with 2 μm stainless steel frits.this column is connected between the analytical columns and the detector. 2 ml/min Mobile Phase: A. 125 mm ammonium acetate, adjust to ph 4.5 with formic acid b. acetonitrile Gradient: Time (min) A% B%. 45 55 7. 1 9 1. 1 9 Injection Volume: 5 µl Detector: UV Wavelength: 2487 Dual Wavelength UV Detector 619 nm Wash: A. 5 ml.1 N hydrochloric acid C. 4 ml 5% methanol/water B. 2 x 5 ml water D. 6 ml hexane, vacuum dry elute: 1 ml 5% ethyl acetate: 45% methanol: 5% ammonium hydroxide (v/v/v) Dry eluant at 5 C under nitrogen Reconstitute with 5% acetonitrile (1 µl) rdering Information asis MCX, 6 cc/15 mg, 3 μm, 3/box 186256 SunFire C 18, 4.6 x 15 mm, 5 μm 186255 Qsert Vials 1861126 Ref: Jointly developed by Waters China applications team and Beijing CIQ 211 Waters Corporation. Waters, asis, SunFire, and Alliance are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 42 ]

Malachite Green in Fish (UPLC/MS/MS) Malachite Green (MG) is an effective and inexpensive fungicide used in aquaculture. During metabolism MG reduces to Leucomalachite Green (LMG), which has been shown to accumlate in fatty fish tissues. Both MG and LMG have demonstrated putative carcinogenic activity, and thus, have been banned for use in aquaculture by both the United States Food and Drug Administration (US FDA) and European Union (EU). Pretreatment 1. Weigh 1 g sample into a 3 ml centrifuge tube. 2. Add 5 μl TMPD* solution (1 mg/ml). 3. Add standard solutions MG, LMG,.1 µg/ml and internal standard, leave to stand for 1 minutes. 4. Add 1 ml McIlvaines Buffer (ph 2.6) /methanol (5:5 v/v) solution; homogenize for 45 seconds. 5. Centrifuge at 5 rpm for 2 minutes, transfer supernatant into centrifuge tube. 6. Repeat steps 4 and 5, combine the two portions of supernatant. A 2 ml aliquot will be used for SPE. *TMPD= N, N, N, M - Tetramethyl-1,4-phenylenediamine dihydrochloride SPE Procedure asis MCX, 3 cc/6 mg Condition/Equilibrate: A. 2 ml methanol b. 2 ml water c. 2 ml McIlvaines Buffer (ph 2.6) Load: 2 ml of sample Wash: A. 2 ml.1n HCl B. 2 x 2.5 ml water C. 2 ml 5% methanol/water D. 3 ml hexane, vacuum dry elute: 5 ml 5% ethyl acetate:45% methanol:5% ammonium hydroxide (v/v/v) Solutions McIlvaines Buffer (ph 2.6) : 1..1 M citric acid monohydrate (A) - Dissolve citric acid monohydrate (1.5 g) in water (5 ml). 2..2 M disodium hydrogen phosphate dihydrate (B) - Dissolve disodium hydrogen phosphate dihydrate (14.2 g) in water (5 ml). 3. Mix A (445.5 ml) and B (54.5 ml). McIlvaines Buffer (ph 2.6): methanol (5:5 v/v): Mix McIlvaines Buffer (ph 2.6) (25 ml) with methanol (25 ml). LC Conditions Waters ACQUITY UPLC System Column: ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 µm Flow Rate: Mobile Phase:.4 ml/min A..1% formic acid in water b..1% formic acid in acetonitrile Gradient: Time (min) A% B%. 4 6 3. 5 95 5. 4 6 MS Conditions Waters Quattro Premier Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring Analyte MRM Transition 331.2 239.1 LMG 331.2 316.2 329.2 28.1 MG 329.2 313.1 MRM method parameters. Dry eluant at 5 C under nitrogen Reconstitute with 5% acetonitrile (1 µl) [ 43 ]

Malachite Green in Fish (UPLC/MS/MS) Results LMG MG The LD of LMG and MG are.2 ppb and.1 ppb, respectively. The recoveries of LMG and MG in fish is between 5-8%. rdering Information asis MCX, 3 cc/6 mg, 3 μm, 1/box 186254 ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 μm 186235 ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 μm, 3/pk 176863 Qsert Vials 1861126 211 Waters Corporation. Waters, asis, ACQUITY UPLC, and Quattro Premier are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 44 ]

Melamine and Cyanuric acid in infant formula using HPLC Responding to recent worldwide concern related to melamine in food, the United States Food and Drug Administration (US FDA) issued an interim method for the determination of residual melamine and cyanuric acid in foods using LC/MS/MS. Pretreatment 1. Weigh 5 g of liquid infant formula, or 1 g of dry infant formula, and add 4 ml of water. 2. Add 5 ng (5 μl of 1 μg/ml stock) of isotopicallylabeled melamine. 3. Add 25 ng (25 μl of 1 μg/ml stock) of isotopicallylabeled cyanuric acid. 4. Add 2 ml of 5:5 acetonitrile:water. 5. Shake 1-2 minutes. 6. Centrifuge for 1 minutes at 34 rpm. SPE Procedure Melamine SPE Cleanup asis MCX, 6 cc/15 mg Condition: A. 5 ml.1 M NaH in acetonitrile B. 5 ml.1m HCl in acetonitrile Equilibrate: A. 5 ml acetonitrile B. 5 ml 4% formic acid in water Load: A. 3 ml of 4% formic acid in water B. Add 2 ml of sample supernatant Wash: A. 5 ml acetonitrile B. 5 ml.2% diethylamine in acetonitrile Cyanuric Acid SPE Cleanup asis MAX cartridges, 6 cc/15 mg Condition: A. 5 ml.1 M HCl in acetonitrile B. 5 ml.1 M NaH in acetonitrile Equilibrate: A. 5 ml acetonitrile B. 5 ml 5% NH 4 H in water Load: A. 3 ml 5% NH 4 H in water B. Add 2 ml of sample supernatant WASH: 5 ml acetonitrile ELUTE: 2 ml 4% formic acid in acetonitrile Filter eluent into vial using.2 μm PTFE syringe filters and syringes LC Conditions Column: Mobile Phase A: Mobile Phase B: DILUTE: Cyanuric acid calibration standards accordingly Waters ACQUITY UPLC System Atlantis HILIC Silica 2.1 x 15 mm, 3 μm 1 mm Ammonium acetate in 5/5 Acetonitrile/H 2 1 mm Ammonium acetate in 95/5 Acetonitrile/H 2 Gradient Table: Time Flow Rate %A %B Curve (min) (ml/min) initial.5 1-2..5 1 6 3.5.5 6 4 6 5..5 6 4 6 5.2.8 1 6 11..8 1 6 11.1.5 1 6 14..5 1 6 Injection Volume: 2 μl ELUTE: 4 ml 2% diethylamine in acetonitrile Filter eluent into vials using.2 μm PTFE syringe filters and syringes [ 45 ]

Melamine and Cyanuric acid in infant formula using HPLC MS Conditions MS System: Software: Ionization Mode: Waters ACQUITY TQD MassLynx v.4.1 ESI Positive (melamine and 13 C 3 15 N 3 melamine) ESI negative (cyanuric acid and 13 C 3 15 N 3 cyanuric acid) 1 % Liquid Infant Formula Fortified Cyanuric Acid at 5 µg/kg Compound Ionization MRM Transitions Cone Voltage(V) Collision Energy (ev) Liquid Infant Formula Fortified Cyanuric Acid at 1 µg/kg.59 Melamine 13 C 3 15 N 3 Melamine Cyanuric Acid 13 C 3 15 N 3 Cyanuric Acid Positive Positive Negative Negative 127 85 4 17 127 68 4 25 133 89 4 17 133 45 4 26 128 42 3 13 128 85 3 11 134 44 3 13 134 89 3 11 MRM Conditions for Melamine, 13 C 3 15 N 3 Melamine, Cyanuric Acid, and 13 C 3 15 N 3 Cyanuric Acid. Results 1 Dry Infant Formula Fortified Melamine at 25 µg/kg 1.81 Liquid Infant Formula Blank.5.1.15.2.25.3.35.4.45.5.55.6.65.7.75.8.85 min Cyanuric Acid in Liquid Infant Formula at 1 ppb and 5 ppb using Atlantis HILIC Silica Column. Single Day Results Spiking Concentration Type Average Spike % Recovery ± % RSD (n) Multi-Day Results Average Spike % Recovery ± % RSD (n) 5 µg/kg Dry 113.3 ± 8.8 (n = 5) 19.2. ± 7.7 (n = 11) 25 µg/kg Dry 18.5 ± 5.5 (n = 5) - 1 µg/kg Liquid 11.2 ± 13.2 (n = 5) 14.7 ± 8.2 (n = 8) 1 µg/kg Liquid 113.8 ± 12.7 (n = 5) 116.4 ± 1.6 (n = 8) Melamine Spike % Recovery in Dry and Liquid Infant Formula using Atlantis HILIC Silica Column. Single Day Results Spiking Concentration Type Average Spike % Recovery ± % RSD (n) Multi-Day Results Average Spike % Recovery ± % RSD (n) 5 µg/kg Dry 115.7 ± 1.8 (n = 5) 113.9 ± 2.7 (n = 8) % 25 µg/kg Dry 14.7 ± 3.9 (n = 5) 14.7 ± 3.1 (n = 8) 1 µg/kg Liquid 19.2 ± 2.9 (n = 5) 18.4 ± 3.1(n = 8) 5 µg/kg Liquid 13.9 ± 2.1 (n = 5) 13.4 ± 3.3 (n = 8) Dry Infant Formula Fortified Melamine at 5 µg/kg Dry Infant Formula Blank MRM method parameters. 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 min Melamine in Dry Infant Formula at 5 ppb and 25 ppb using Atlantis HILIC Silica Column. rdering Information HPLC Melamine Analysis Package 1761773 asis MCX, 6 cc/15 mg, 1/box 186255 asis MAX, 6 cc/15 mg, 1/box 18637 Atlantis HILIC Silica, 2.1 x 15 mm, 3 µm 186215 LCMS Certified Vials 6751CV Ref: Waters Application Note 722862EN 211 Waters Corporation. Waters, asis, ACQUITY UPLC, ACQUITY, Atlantis, and MassLynx are trademarks of Waters Corporation. [ 46 ]

Melamine and Cyanuric acid in infant formula using UPLC Responding to recent worldwide concern related to melamine in food, the United States Food and Drug Administration (US FDA) issued an interim method for the determination of residual melamine and cyanuric acid in foods using LC/MS/MS. Pretreatment 1. Weigh 5 g of liquid infant formula, or 1 g of dry infant formula, and add 4 ml of water. 2. Add 5 ng (5 μl of 1 μg/ml stock) of isotopicallylabelled melamine. 3. Add 25 ng (25 μl of 1 μg/ml stock) of isotopicallylabelled cyanuric acid. 4. Add 2 ml of 5:5 acetonitrile:water. 5. Shake 1-2 minutes. 6. Centrifuge for 1 minutes at 34 rpm. SPE Procedure Melamine SPE Cleanup asis MCX, 6 cc/15 mg Condition: A. 5 ml.1 M NaH in acetonitrile B. 5 ml.1m HCl in acetonitrile Equilibrate: A. 5 ml acetonitrile B. 5 ml 4% formic acid in water Load: A. 3 ml of 4% formic acid in water B. Add 2 ml of sample supernatant Cyanuric Acid SPE Cleanup asis MAX cartridges, 6 cc/15 mg Condition: A. 5 ml.1 M HCl in acetonitrile B. 5 ml.1 M NaH in acetonitrile Equilibrate: A. 5 ml acetonitrile B. 5 ml 5% NH 4 H in water Load: 3 ml 5% NH 4 H in water Add 2 ml of sample supernatant WASH: 5 ml acetonitrile ELUTE: 2 ml 4% formic acid in acetonitrile Filter eluent into vial using.2 μm PTFE syringe filters and syringes LC Conditions Column: Mobile Phase A: Mobile Phase B: DILUTE: Cyanuric acid calibration standards accordingly Waters ACQUITY UPLC System ACQUITY UPLC BEH HILIC, 2.1 x 1 mm, 1.7 μm 1 mm Ammonium acetate 1 mm Ammonium acetate in 95/5 Acetonitrile/H 2 Gradient Table: Time Flow Rate %A %B Curve (min) (ml/min) initial.6 1 -.8.6 1 6 2.3.6 22 78 6 2.8.6 22 78 6 2.9.6 1 6 4..6 1 6 Injection Volume: 1 μl Wash: A. 5 ml acetonitrile B. 5 ml.2% diethylamine in acetonitrile ELUTE: 4 ml 2% diethylamine in acetonitrile Filter eluent into vials using.2 μm PTFE syringe filters and syringes [ 47 ]

Melamine and Cyanuric acid in infant formula using UPLC MS Conditions 1 MS System: Software: Ionization Mode: Waters ACQUITY TQD MassLynx v.4.1 ESI Positive (melamine and 13 C 3 15 N 3 melamine) ESI negative (cyanuric acid and 13 C 3 15 N 3 cyanuric acid) Liquid Infant Formula Fortified Cyanuric Acid at 5 µg/kg % Compound Melamine Ionization Positive MRM Transitions Cone Voltage (V) Collision Energy (ev) 127 85 4 17 127 68 4 25 Liquid Infant Formula Fortified Cyanuric Acid at 1 µg/kg.59 Liquid Infant Formula Blank 13 C 3 15 N 3 Melamine Cyanuric Acid Positive Negative 133 89 4 17 133 45 4 26 128 42 3 13 128 85 3 11.5.1.15.2.25.3.35.4.45.5.55.6.65.7.75.8.85 min Cyanuric Acid in Liquid Infant Formula at 1 ppb and 5 ppb using Atlantis HILIC Silica Column. 13 C 3 15 N 3 Cyanuric Acid Negative 134 44 3 13 134 89 3 11 Spiking Concentration Type Single Day Results Average Spike % Recovery ± % RSD (n) Multi-Day Results Average Spike % Recovery ± % RSD (n) MRM method parameters. Results 1 Dry Infant Formula Fortified Melamine at 25 µg/kg 1.81 5 µg/kg Dry 115. ± 4.7 (n = 5) 11.7 ± 6.9 (n = 11) 25 µg/kg Dry 19.6 ± 3.1 (n = 5) - 1 µg/kg Liquid 13.9 ± 1.5 (n = 5) 14.7 ± 8.2 (n = 8) 1 µg/kg Liquid 15.7 ± 3.2 (n = 5) 15.1 ± 4.5 (n = 8) Melamine spike % recovery in dry and liquid infant formula using BEH HILIC column. % Spiking Concentration Type Single Day Results Average Spike % Recovery ± % RSD (n) Multi-Day Results Average Spike % Recovery ± % RSD (n) Dry Infant Formula Fortified Melamine at 5 µg/kg Dry Infant Formula Blank 5 µg/kg Dry 114.9 ± 3.9 (n = 5) 116.1 ± 4.8 (n = 8) 25 µg/kg Dry 19.6 ± 3.1 (n = 5) 14.9 ± 4.8 (n = 8) 1 µg/kg Liquid 117.7 ± 4. (n = 5) 115. ± 5. (n = 8) 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 min tmelamine in Dry Infant Formula at 5 ppb and 25 ppb using Atlantis HILIC Silica Column. 5 µg/kg Liquid 13.8 ± 5.9 (n = 5) 13.1 ± 2.9 (n = 8) Cyanuric Acid Spike % Recovery in Dry and Liquid Infant Formula using BEH HILIC Column. rdering Information UPLC Melamine Analysis Package 1761791 asis MCX, 6 cc/15 mg, 3/box 186255 asis MAX, 6 cc/15 mg, 3/box 18637 ACQUITY UPLC BEH HILIC, 2.1 x 1 mm, 1.7 µm 1863461 LCMS Certified Vials 6751CV Ref: Waters Application Note 722862EN 211 Waters Corporation. Waters, asis, ACQUITY UPLC, ACQUITY, UPLC, Atlantis, and MassLynx are trademarks of Waters Corporation. [ 48 ]

Melamine and Cyanuric acid in infant formula using lc/ms/ms Responding to recent worldwide concern related to melamine in food, the United States Food and Drug Administration (US FDA) issued an interim method for the determination of residual melamine and cyanuric acid in foods using LC/MS/MS. Sample Extraction 1. Weigh 1 g infant formula. 2. Add 9 ml of sample extraction buffer into a 5 ml centrifuge tube. 3. Shake or vortex the solution for 1 minute. 4. Centrifuge for 15 minutes at 35 rpm Melamine SPE Cleanup asis MCX, 6 cc/15 mg Condition: A.5 ml.1 M NaH in acetonitrile b.5 ml.1m HCl in acetonitrile CNDITIN: A. 5 ml methanol b. 5 ml.5n HCl LAD: A. 3 ml of.5n HCl b. 2 ml of sample supernatant WASH: A. 5 ml water b. 2 ml acetonitrile ELUTE: 4 ml 5% ammonium hydroxide in acetonitrile LC Conditions Waters ACQUITY UPLC System Column: ACQUITY UPLC BEH HILIC 2.1 x 15 mm, 1.7 µm Mobile Phase A: 1 mm ammonium acetate at ph 3.9 in 5/5 water/acetonitrile Mobile Phase B: 1 mm ammonium acetate at ph 3.9 in 11.5/88.5 water/acetonitrile Gradient Table: Time Flow Rate %A %B Curve (min) (ml/min) initial.45. 1. - 5..45. 1. 6 6..45. 1. 11 Injection Volume: 1 µl MS Conditions MS System: Ionization Mode: Compounds Melamine Ammelide Ammeline Cyanuric Acid MRM method parameters. Waters ACQUITY TQD ESI Positive (melamine, ammelide and ammeline) esi Negative (cyanuric acid) MRM Transitions Dwell (sec) Cone Voltage (V) Collision Energy (ev) 127 85.7 3 18 127 68.7 3 22 129 87.7 3 14 129 43.7 3 18 128 86.8 3 15 128 43.8 3 2 128 42.55 25 12 128 85.55 25 6 Cyanuric Acid SPE Cleanup Sep-Pak Aminopropyl (NH 2 ), 6 cc/5 mg Condition: 5 ml 5% diethylamine in acetonitrile LAD: A. 2 ml 5% NH 4 H in water B. 3 ml of sample supernatant ELUTE: 5 ml 1:1:8 water:formic acid:acetonitrile [ 49 ]

Melamine and Cyanuric acid in infant formula using lc/ms/ms 1 % Ammeline 2.24 1 % Melamine 1.87 1 % Ammelide 1.58 1.41 1.75 1 % Cyanuric Acid 1.1.2.4.6.8 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. µg/g. Compound Recovery Average Spike % Recovery ± % RSD (n) verall Recovery Average % verall Recovery (n) Cyanuric Acid 12.8 ± 12.8 (n = 5) 74.4 (n = 5) Ammelide 97.4 ± 2.5 (n = 5) 6.2 (n = 5) Melamine 16.8 ± 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. rdering Information asis MCX, 6 cc/15 mg 186256 Sep-Pak Aminopropyl (NH 2 ), 6 cc/5 mg WAT5456 ACQUITY UPLC BEH HILIC 2.1 x 15 mm, 1.7 µm 1863462 LCMS Certified Vials 6751CV Ref: Waters Application Note, 72688EN 211 Waters Corporation. Waters, asis, Sep-Pak, ACQUITY UPLC, and ACQUITY are trademarks of Waters Corporation. [ 5 ]

Microcystins in Natural Waters Microcystin-LR is a potent mammalian toxin which is known to have been responsible for the deaths of domesticated animals, livestock loss, and the potential presence in potable water supplies. Pretreatment 1. Filter water sample through.45 μm membrane filter. 2. Add 1 µl of enkephalin (concentration 1 µg/l) to 1 ml filtered water sample and mix thoroughly. SPE Procedure LC Conditions Waters Alliance HPLC 2695 System Column: Symmetry3 C 18, 4.6 x 75 mm, 3.5 µm Flow Rate: Mobile Phase:.2 ml/min A..2% formic acid in water b..2% formic acid in methanol Gradient: Time (min) A% B%. 45 55 12. 1 9 12.5 1 15. 1 15.1 45 55 25. 45 55 Injection Volume: 1 µl Column Temperature: 3 C asis HLB, 3 cc/6 mg Condition/Equilibrate: A. 3 ml methanol B. 6 ml water Load: 1 ml sample (1 ml/min) Wash: A. 3 ml water B. 5 ml 2% methanol Dry cartridge by vacuum for 1 minute elute: 5 ml methanol Evaporate to dryness at 5 C under nitrogen stream Reconstitute residue with 1 ml 5% methanol MS Conditions Waters Quattro Ultima Pt Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring Characteristic Analyte MRM MW [M+H] + [M+H] 2+ Ion Fragment 278. Enkephalin 556.1 278. 555.6 556.1 N.D 397.1 MCYST-LR 519.9 135. 994.5 995.7 498.4 135. 861.5 MCYST-RR 498.4 135. 137.6 138.4 519.9 135. 62. MCYST-LW 125.8 891.7 124.5 125.8 N.D 897.1 583.2 MCYST-LF 986.8 852.5 985.5 986.8 N.D 852.5 544. MRM method parameters. Results Analyte Concentration (μg/l) Average Recovery (%) RSD (%).1 1. 6.45 MCYST-RR.2 95.2 4.2.4 9. 4.35.2 15. 5.4 MCYST-LR.5 96. 4.53.8 93.8 4.22.4 13.8 5.3 MCYST-LW 1. 12.7 5.87 1.6 93.8 5.67.2 13. 7.3 MCYST-LF.5 19.8 5.69.8 12.3 4.57 Recovery data for spiked samples at various concentrations. rdering Information asis HLB, 3 cc/6 mg, 3 μm, 1/box WAT94226 Symmetry3 C 18, 4.6 x 75 mm, 3.5 μm 186189 Nylon Filter.45 μm LCMS Certified Vials WAT2524 6751CV Ref: Determination of Microcystins in Natural Water by Liquid Chromatography Tandem Mass Spectrometry, Chen Qi, Huang Baifen, Zhang Jing, Ren Yiping; Zhejiang Provincial Center for Disease Prevention and Control 211 Waters Corporation. Waters, asis, Symmetry3, Alliance, and Quattro Ultima Pt are trademarks of Waters Corporation. [ 51 ]

Multi-residue analysis of pesticides in Grain and Beans This application brief shows the step wise procedure of the Japan Ministry of Health, Labor and Welfare (JPMHLW) fficial Method for multi-residue analysis of pesticides for grain and bean samples. Pretreatment 1. Extract 1 g sample with 2 ml of water. Add 5 ml of acetonitrile. 2. Homogenize sample. Centrifuge and collect supernatant. 3. Repeat extraction with 2 ml of acetonitrile. 4. Collect acetonitrile layer and dilute to 1 ml with acetonitrile. 5. Sample 2 ml of diluted acetonitrile extract, add 1 g sodium chloride and 2 ml of.5 M of phosphate buffer. Shake for 1 minutes. Sep-Pak Vac Carbon Black/Aminopropyl, 6 cc/5 mg/5 mg CNDITIN: 1 ml of toluene:acetonitrile (1:3, v/v) Load the sample extract ELUTE: 2 ml of toluene:acetonitrile (1:3, v/v) Collect eluate Evaporate to dryness Dissolve the residue with 1 ml of acetone:n-hexane (1:1, v/v) 6. Collect acetonitrile layer for SPE treatment. SPE Procedure Sep-Pak Vac C 18, 1 g/6 cc CNDITIN: 1 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) rdering Information Sep-Pak Vac C 18, 1 g/6 cc Sep-Pak Vac Carbon Black/Aminopropyl, 6 cc/5 mg/5 mg LCMS Certified Vials WAT3695 1863369 6751CV 211 Waters Corporation. Waters, and Sep-Pak are trademarks of Waters Corporation. [ 52 ]

Multi-residue analysis of pesticides in vegetables and fruits This application brief shows the Japan Ministry of Health, Labor and Welfare (JPMHLW) Method for multi-residue analysis of pesticides in vegetables and fruit. This sample preparation method calls for an extract from the commodity, followed by a SPE extract from a Sep-Pak Vac Carbon Black/Aminopropyl column. SPE Procedure Sep-Pak Vac Carbon Black/Aminopropyl, 6 cc/5 mg/5 mg CNDITIN: Toluene/acetonitrile 1 ml (1:3 v/v). Do not allow to dry LAD: Rinse vials with toluene/acetonitrile (1:3 v/v) solution and load 2 ml of extracted sample ELUTE : 2 ml toluene/acetonitrile (1:3 v/v). Flow: 2 ml/min LC Conditions Column Flow Rate: Mobile Phase: Waters Alliance HPLC 2695 System XTerra MS C 18, 2.1 x 15 mm, 3.5 μm.2 ml/min A. water b. methanol c. 1 mm ammonium acetate in water Gradient: Time (min) A% B% C%. 8 15 5 1. 55 4 5 3.5 55 4 5 6. 45 5 5 8. 4 55 5 17.5 95 5 3. 8 15 5 47. 8 15 5 Injection Volume: 5 µl Temperature: MS Conditions 4 C Waters Quattro Premier XE Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring Pesticides* Spike Conc./μg/g Recovery (%) Abamectin.1 12. Anibfos.1 111.7 Azinphos-methyl.1 17.6 Benzofenap.1 139.5 Butafenacil.1 14.5 Chloridazon.1 16. Chromafenozide.1 18.2 Clomeprop.1 14.4 Cloquintocet-mexyl.1 18.7 Clothianidin.1 11.5 Cyazofamid.1 18.3 Cyflufenamid.1 11.1 Dimethirimol.1 11. Fenoxycarb.1 18.7 Ferimzone.1 112.6 Formetanate hydrochloride.1 86.7 Furathiocarb.1 1.5 Imidacloprid.1 111.8 Indoxacarb.1 121.2 Iprovalicarb.1 16.2 Isoxaflutole.1 99.5 Lactofen.1 16.8 Methoxyfenozide.1 13.3 Mibemectin A3.1 114.5 Mibemectin A4.1 11.2 Naproanilide.1 115.9 ryzalin.1 13.8 xycarboxin.1 85.1 xydemeton-methyl.1 18. Phenmedipham.1 12.2 Pyrazolynate.1 72.7 Quizalofop-P-tefuryl.1 145.3 Simeconazole.1 16. Thiacloprid.1 19.2 Thiamethoxam.1 18.3 Tridemorph.1 94.6 Etriticonazole.1 113.3 *Five replicate samples analyzed per level. rdering Information Sep-Pak Vac Carbon Black/Aminopropyl, 6 cc/5 mg/5 mg 1863369 XTerra MS C 18, 2.1 x 15 mm, 3.5 μm 18656 LCGC Certified Vials 186272C 211 Waters Corporation. Waters, Alliance, XTerra, Sep-Pak and Quattro Premier are trademarks of Waters Corporation. [ 53 ]

Multi-residue LC/MS/MS determination of 52 non-gas chromatography-amenable pesticides and metabolites in Fruits and Vegetables This multi-residue pesticide sample preparation shows the steps used to process fruit and vegetable samples, extract and concentrate the extract by an asis HLB SPE method. Pretreatment 1. Samples (lemon, raisin, tomato and avocado) were cut into small pieces. 2. A 2 g portion of homogenized sample was mixed with 6 ml.1% formic acid in methanol:water (8:2, v/v). 3. Extraction for 2 minutes with Ultra-Turrax at 8 rpm. 4. Filtration and dilution with methanol:water.1% formic acid (8:2, v/v) to a final volume of 1 ml. 5. 2.5 ml aliquot diluted to 2 ml with.1% formic acid in water. Load 5 ml of the diluted extract onto the SPE cartridge. LC Conditions Column: Flow Rate: Mobile Phase: Waters Alliance HPLC 2695 System Atlantis dc 18, 2.1 x 1 mm, 5 μm.2 ml/min A..1% formic acid in water b..1% formic acid in methanol Gradient: Time (min) A% B%. 95 5 1. 95 5 8.5 5 5 25. 1 9 28. 1 9 29. 95 5 Injection Volume: 2 µl MS Conditions Waters Quattro micro Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring SPE Procedure asis HLB, 6cc/2mg Results Methamidophos methoate Condition: A. 5 ml methanol B. 5 ml methanol: MTBE* (1:9).1% formic acid C. 5 ml methanol.1% formic acid Equilibrate: 5 ml water.1% formic acid Ethiofencarbsulfone Ethiofencarbsulfoxide LAD: 5 ml diluted extract DRY: By passing air for through cartridge 1 hour LC/MS/MS data for 4 representative pesticides. ELUTE: 5 ml methanol:mtbe (1:9, v/v).1% formic acid, by means of gravity Sample post-treatment:.5 ml water added to the extract. Evaporate with nitrogen at 4 C until.5 ml. Adjust final volume to 1 ml with methanol:water (1:9, v/v). *MBTE: methyl-t-buthyl ether rdering Information asis HLB, 6 cc/2 mg, 3/box WAT1622 Atlantis dc 18, 2.1 x 1, 5 µm 1861297 LCMS Certified Combination Packs 6751CV Ref: Journal of Chromatography A, 119 (26) 242-252 211 Waters Corporation. Waters, asis, Alliance, Atlantis, and Quattro micro are trademarks of Waters Corporation. [ 54 ]

Multi-residue Analysis of pesticides by QuEChERS in: Avocado by gc/ms 18 16 Avocado is a fruit that contains fat; therefore, the recommended selection of the dispersive solid-phase extraction (SPE) tube should include C 18 to remove the fats. % Recovery 14 12 1 8 6 4 Extraction Procedure 1. Add 15 ml 1% acetic acid in acetonitrile into the 5 ml DisQuE extraction tube containing 1.5 g of sodium acetate and 6 g of magnesium sulfate. 2 Atrazine Azoxystrobin Carbaryl Chlorpyrifos Chlorpyrifos-methyl Cyprodinil Pesticides in Avocados by GC/MS. Endosulfan sulfate Pesticide Ethion Kresoxim-methyl Tebuconazole Tolyfluanid Trifluralin 2. Add 15 g of homogenized sample into the 5 ml tube. 3. Add any internal standards and standard mixture. 4. Shake vigorously for 1 minute and centrifuge > 15 rcf for 5 minutes. 5. Transfer 1 ml of the acetonitrile extract into the 2 ml clean-up tube containing 5 mg PSA, 15 mg MgS 4, and 5 mg C 18. 6. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. 7. Transfer.5 ml extract into a tube. 8. Add any post-extraction internal standards. 9. Add.25 ml toluene. 1. Evaporate at 5 C with N 2 to <.1 ml. 11. Bring volume up to.2 ml with toluene. 12. Transfer to vial with insert for analysis. TEST CNDITINS GC Conditions Column: Carrier Gas: Flow Rate: Agilent 689N GC RTX-5MS, 3 x.25 mm, (.25 µm film) Helium 1. ml/min Temperature Program: Initial 1 C, hold 1 min, then 1 C/min to 32 C, hold for 7 minute Injection Volume: MS Conditions Ionization: Acquisition: 2 µl splitless Waters Quattro micro GC-MS Electron Impact (7 ev) Single Ion Recording (SIR) Mode RDERING INFRMATIN* DisQuE 5 ml Tube-AAC/Acetate 1864571 DisQuE 2 ml Tube-AAC/C 18 186483 LCGC Certified Vials 186272C Insert 3 µl with Poly Spring WAT9417 * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) 211 Waters Corporation. Waters, DisQuE and Quattro micro are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 55 ]

Multi-residue Analysis of pesticides by QuEChERS in: Avocado by lc/ms/ms Avocado is a fruit that contains fat; therefore, the recommended selection of the dispersive solid-phase extraction (SPE) tube should include C 18 to remove the fats. Extraction Procedure 1. Add 15 ml 1% acetic acid in acetonitrile into the 5 ml DisQuE extraction tube. 2. Add 15 g of homogenized sample into the 5 ml tube. 3. Add any internal standards and standard mixture. 4. Shake vigorously for 1 minute and centrifuge > 15 rcf for 5 minute. 5. Transfer 1 ml of the acetonitrile extract into the 2 ml clean-up tube containing 5 mg PSA, 15 mg MgS 4, and 5 mg C 18. 6. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. 7. Transfer 1 μl of final extract into a 1.5 ml centrifuge tube. 8. Add any post-extraction internal standards. LC Conditions LC System: Column: Column Temperature: 4 C Sample Temperature: 4 C Flow Rate: Mobile Phase A: Mobile Phase B: Waters ACQUITY UPLC System ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm.3 ml/min. Water +.1% formic acid Methanol +.1% formic acid Gradient: Time Flow Rate A% B%..3 75 25.25.3 75 25 7.75.3 5 1 8.5.3 1 8.51.5 75 25 1.5.5 75 25 11..3 75 25 Injection Volume: MS Conditions 15 μl, Partial loop injection Waters ACQUITY TQ Detector Ionization: Positive electrospray (ESI + ) Acquisition: Multiple reaction monitoring (MRM) 9. Dilute as needed with an appropriate buffer or solvent. 1. Centrifuge > 16 rcf for 5 minutes. 11. Transfer to autosampler vial. TEST CNDITINS 16 14 12 % Recovery 1 8 6 4 2 Atrazine Azoxystrobin Carbaryl Cyprodinil Dichlorvos Imazalil Imidacloprid Linuron Pesticides Pesticides in Avocados by UPLC /MS/MS Methamidophos Methomyl Pymetrozine Thiabendazole Tolyfluanid RDERING INFRMATIN* DisQuE 5 ml Tube-AAC/Acetate 1864571 DisQuE 2 ml Tube-AAC/C 18 186483 ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm 1862352 LCMS Certified Vials 6749CV * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) 211 Waters Corporation. Waters, The Science of What s Possible, DisQuE, ACQUITY UPLC, and ACQUITY are trademarks of Waters Corporation. [ 56 ]

Multi-residue Analysis of pesticides by QuEChERS in: Avocado and grapes by gc/ms Avocado is a fruit that contains fat; therefore, the recommended selection of the dispersive SPE tube should include C 18 to remove the fats. Extraction Procedure 1. Add 15 g of homogenized sample to the 5 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 > 15 rcf for 1 minute. 5. Transfer 1 ml of the acetonitrile extract into the 2 ml DisQuE extraction tube containing 5 mg PSA and 15 mg of magnesium sulphate. 6. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. 7. Transfer 25 μl of final extract into an autosampler vial. 8. Add any post-extraction internal standards. 9. Dilute as needed with an appropriate buffer or solvent. GC Conditions Column: Carrier Gas: Agilent 689N GC system Restek Rtx-5MS, 3 x.25 mm i.d.,.25 μm df Helium Temperature Program: Initial temp at 8 C, hold for 1 min, 1 C/min to 32 C, hold for 5 min Flow Rate: Injection Volume: MS Conditions Ionization Mode: GC/MS (SIR) 1. ml/min Split/splitless mode with.5 min purge time and 1 μl injection Waters Quattro micro GC mass spectrometer Positive electrospray (7 ev) Selected-Ion Recording (SIR) Channel Mass Mass Phenylphenol 17 141 Atrazine 2 173 Chlorpyrifos methyl 286 19 DDD 235 165 Ethion 231 153 Cylohalothrin 197 181 SIE method parameters. Chlorpyrifos Methyl Phenylphenol DDD Ethion Cyclohalothrin Thiabendazole %Recovery (± %RSD) in Grape 97.7 % (± 4.98%) 15.19% (± 1.31%) 19.42% (± 3.1%) 13.19% (± 2.1%) 12.27% (± 7.33%) 13.58% (± 3.99%) %Recovery (± %RSD) in Avocado 17.45% (± 1.93%) 19.64% (± 4.45%) 96.85% (± 3.4%) 99.65% (± 9.3%) 95.62% (± 12.56%) 11.46% (± 3.86%) GC separation of five basic/neutral pesticides. Compounds (1) phenylphenol, (2) atrazine (IS), (3) chloropyrifos methyl, (4) DDD, (5) ethion, (6) cyclohalothrin. rdering Information* DisQuE Dispersive Sample Preparation Kit (1/pk) 1761676 LCGC Certified Vials 186272C * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) Ref: Waters Application Note 722755EN 211 Waters Corporation. Waters, DisQuE, and Quattro micro are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 57 ]

Multi-residue Analysis of pesticides by QuEChERS in: BAby food by uplc/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. Extraction Procedure 1. Add 15 g of homogenized sample to the 5 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 > 15 rcf for 1 minute. 5. Transfer 1 ml of the acetonitrile extract into the 2 ml DisQuE extraction tube containing 5 mg PSA and 15 mg of magnesium sulphate. 6. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. 7. Transfer 25 μl of final extract into an autosampler vial. 1 LC Conditions LC System: Column: Column Temperature: 4 C Sample Temperature: 4 C Flow Rate: Mobile Phase A: Mobile Phase B: Waters ACQUITY UPLC System ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 μm.7 ml/min Water +.1% formic acid Methanol +.1% formic acid Gradient: Time (min) A% B%. 99 1 5. 1 99 6. 1 99 6.1 99 1 8. 99 1 Total Run Time: Injection volume: MS Conditions MS System: 8 min 5 μl, full loop injection Waters Xevo TQ MS Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring 8. Add any post-extraction internal standards. 9. Dilute as needed with an appropriate buffer or solvent. 1.35 % 17 1.2.4.6.8 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 ]

Multi-residue Analysis of pesticides by QuEChERS in: BAby food by uplc/ms/ms Peak Pesticide RT 1 methoate.97 2 xydemeton-s-methyl 1.35 3 Demeton-S-methyl sulfone 1.39 4 Dimethoate 1.79 5 Fensulfothion-oxon 2.32 6 Fensulfothion-oxon-sulfone 2.39 7 Demeton-S-methyl 2.63 8 Disulfoton sulfoxide 2.93 9 Disulfoton sulfone 2.98 1 Fensulfothion 3.1 11 Fensulfothion sulfone 3.17 12 Terbufos sulfone 3.3 13 Terbufos sulfoxide 3.32 14 Ethoprophos 3.68 15 Disulfoton 4.3 16 Cadusafos 4.9 17 Terbufos 4.28 Xevo TQ MS MRM method parameters. MRM Transitions 214 183 214 155 247 169 247 19 263 169 263 121 23 125 23 171 293 237 293 265 39 253 39 175 231 89 231 61 291 185 291 97 37 97 37 115 39 281 39 157 325 269 325 297 321 171 321 115 35 187 35 131 243 131 243 173 275 89 275 61 271 159 271 131 289 13 289 233 Dwell Time (s) Cone Voltage (V) Collision Energy (ev).8 16.4 18.4 2.1 12.4 22.4 19.1 12.4 15.2 16.2 25.2 19.3 19.3 1.1 18.8 14.2 16.6 12 12 15 14 18 16 16 2 14 18 13 15 25 12 22 13 32 28 23 14 24 15 11 11 28 11 27 19 14 1 32 14 22 9 5 rdering Information* DisQuE Dispersive Sample Preparation Kit (1/pk) 1761676 ACQUITY UPLC BEH C 18, 2.1 x 5, 1.7 µm 186235 LCMS Certified Vials 6751CV * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) Ref: Waters Application Note 722812EN 211 Waters Corporation. Waters, DisQuE, ACQUITY UPLC, and Xevo are trademarks of Waters Corporation. [ 59 ]

Multi-residue Analysis of pesticides by QuEChERS in: flour by gc/ms Flour is low water content commodity that requires the addition of water and soak time as a pretreatment step before extraction. Sample Pretreatment: 1. Add 5 g of flour and 1 ml of water in a tube and soak for 1 minutes. GC Conditions Column: Carrier Gas: Flow Rate: Agilent 689N GC RTX-5MS, 3 x.25 mm, (.25 µm film) Helium 1. ml/min Temperature Program: Initial 1 C, hold 1 min, then 1 C/min to 32 C, hold for 7 minute Injection Volume: 2 µl splitless Extraction Procedure MS Conditions 1. Add 15 ml 1% acetic acid in acetonitrile into the 5 ml DisQuE extraction tube 1 containing 1.5 g of sodium acetate and 6 g of magnesium sulfate. Ionization: Acquisition: Waters Quattro micro GC-MS Electron Impact (7 ev) Single Ion Recording (SIR) Mode 2. Add soaked flours sample into the 5 ml tube. 16 14 3. Add any internal standards and standard mixture. 4. Shake vigorously for 1 minute and centrifuge > 15 rcf for 5 minute. 5. Transfer 1 ml of the acetonitrile extract into the 2 ml DisQuE clean-up tube 2, containing 5 mg PSA and 15 mg of magnesium sulphate. 6. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. % Recovery 12 1 8 6 4 2 Atrazine Azoxystrobin Bifenthrin Carbaryl Chlorpyrifos Chlorpyrifos-methyl Pesticides in Flour by GC/MS. Cyprodinil Endosulfan sulfate Ethion Hexachlorobenzene Pesticides Kresoxim-methyl Permethrin o-phenylphenol Procymidone Tebuconazole Tolyfluanid Trifluralin 7. Transfer.5 ml extract into a tube. 8. Add any post-extraction internal standards. 9. Add.25 ml toluene. 1. Evaporate at 5 C with N2 to <.1 ml. 11. Bring volume up to.2 ml with toluene. rdering Information* DisQuE Dispersive Sample Preparation Kit (1/pk) 1761676 LCGC Certified Vials Insert 3 µl with Poly Spring 186272C WAT9417 * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) 211 Waters Corporation. Waters, DisQuE and Quattro micro are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 6 ]

Multi-residue Analysis of pesticides by QuEChERS in: flour by uplc/ms/ms Flour is low water content commodity that requires the addition of water and soak time as a pretreatment step before extraction. Sample Pretreatment: 1. Add 5 g of flour and 1 ml of water in a tube and soak for 1 minutes. Extraction Procedure % Recovery MS Conditions Waters ACQUITY TQ Detector Ionization: Positive electrospray (ESI + ) Acquisition: Multiple reaction monitoring (MRM) 16 14 12 1 8 6 4 2 1. Add 15 ml 1% acetic acid in acetonitrile into the 5 ml DisQuE extraction tube 1 containing 1.5 g of sodium acetate and 6 g of magnesium sulfate. 2. Add soaked flours sample into the 5 ml tube. Pesticides in Flour by UPLC/MS/MS. Atrazine Azoxystrobin Carbaryl Cyprodinil Dichlorvos Imazalil Imidacloprid Kresoxim-methyl Linuron Methamidophos Pesticides Methomyl Pymetrozine Tebuconazole Thiabendazole Tolyfluanid 3. Add any internal standards and standard mixture. 4. Shake vigorously for 1 minute and centrifuge > 15 rcf for 5 minute. 5. Transfer 1 ml of the acetonitrile extract into the 2 ml DisQuE clean-up tube 2, containing 5 mg PSA and 15 mg of magnesium sulphate. LC Conditions LC System: Column: Column Temperature: 4 C Sample Temperature: 4 C Flow Rate: Mobile Phase A: Mobile Phase B: Waters ACQUITY UPLC System ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm.3 ml/min. Water +.1% formic acid Methanol +.1% formic acid Gradient: Time Flow Rate A% B%..3 75 25.25.3 75 25 7.75.3 5 1 8.5.3 1 8.51.5 75 25 1.5.5 75 25 11..3 75 25 Injection Volume: 15 μl, Partial loop injection RDERING INFRMATIN* DisQuE Dispersive Sample Preparation Kit (1/pk) 1761676 ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm 1862352 LCMS Certified Vials 6749CV * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) 211 Waters Corporation. Waters, DisQuE, ACQUITY UPLC, and ACQUITY are trademarks of Waters Corporation. [ 61 ]

Multi-residue Analysis of pesticides by QuEChERS in: Grapes by GC/ms MS Conditions Grapes are a commodity containing an ample amount of water. This sample uses the standard Association of Analytical Communities (AAC) extraction and clean-up tubes. Ionization: Acquisition: Waters Quattro micro GC-MS Electron Impact (7 ev) Single Ion Recording (SIR) Mode Extraction Procedure 14 12 1 1. Add 15 ml 1% acetic acid in acetonitrile into the 5 ml DisQuE extraction tube 1 containing 1.5 g of sodium acetate and 6 g of magnesium sulfate. 2. Add 15 g of homogenized sample into the 5 ml tube. 3. Add any internal standards and standard mixture. 4. Shake vigorously for 1 minute and centrifuge > 15 rcf for 5 minute. % Recovery 8 6 4 2 Atrazine Azoxystrobin Bifenthrin Carbaryl Chlorpyrifos Chlorpyrifos-methyl Cyprodinil Pesticides in Grapes by GC/MS. Endosulfan sulfate Ethion Kresoxim-methyl Pesticide Permethrin o-phenylphenol Procymidone Tebuconazole Tolyfluanid Trifluralin 5. Transfer 1 ml of the acetonitrile extract into the 2 ml DisQuE clean-up tube 2, containing 5 mg PSA and 15 mg of magnesium sulphate. 6. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. 7. Transfer.5 ml extract into a tube. 8. Add any post-extraction internal standards. 9. Add.25 ml toluene. 1. Evaporate at 5 C with N 2 to <.1 ml. 11. Bring volume up to.2 ml with toluene. 12. Transfer to vial with insert for analysis. GC Conditions Column: Carrier Gas: Flow Rate: Agilent 689N GC RTX-5MS, 3 x.25 mm, (.25 µm film) Helium 1. ml/min Temperature Program: Initial 1 C, hold 1 min, then 1 C/min to 32 C, hold for 7 minute Injection Volume: 2 µl splitless rdering Information* DisQuE Dispersive Sample Preparation Kit (1/pk) 1761676 LCGC Certified Vials Insert 3 µl with Poly Spring 186272C WAT9417 * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) 211 Waters Corporation. Waters, DisQuE and Quattro micro are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 62 ]

Multi-residue Analysis of pesticides by QuEChERS in: Grapes by uplc/ms/ms Grapes are a commodity containing an ample amount of water. This sample uses the standard Association of Analytical Communities (AAC) extraction and clean-up tubes. MS Conditions Waters ACQUITY TQ Detector Ionization: Positive electrospray (ESI + ) Acquisition: Multiple reaction monitoring (MRM) 16 Extraction Procedure 14 12 1. Add 15 ml 1% acetic acid in acetonitrile into the 5 ml DisQuE extraction tube 1 containing 1.5 g of sodium acetate and 6 g of magnesium sulfate. % Recovery 1 8 6 4 2 2. Add 15 g of homogenized sample into the 5 ml tube. 3. Add any internal standards and standard mixture. 4. Shake vigorously for 1 minute and centrifuge > 15 rcf Atrazine Azoxystrobin Carbaryl Cyprodinil Dichlorvos Imazalil Imidacloprid Linuron Methamidophos Pesticides Methomyl Pymetrozine Tebuconazole Thiabendazole Tolyfluanid for 5 minute. Pesticides in Grapes by UPLC /MS/MS. 5. Transfer 1 ml of the acetonitrile extract into the 2 ml DisQuE clean-up tube 2, containing 5 mg PSA and 15 mg of magnesium sulphate. 6. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. 7. Transfer 1 μl of final extract into an autosampler vial. 8. Add any post-extraction internal standards. 9. Dilute as needed with an appropriate buffer or solvent. LC Conditions LC System: Column: Waters ACQUITY UPLC System ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm Column Temperature: 4 C Sample Temperature: 4 C Flow Rate: Mobile Phase A: Mobile Phase B:.3 ml/min. Water +.1% formic acid Methanol +.1% formic acid Gradient: Time Flow Rate A% B%..3 75 25.25.3 75 25 7.75.3 5 1 8.5.3 1 8.51.5 75 25 1.5.5 75 25 11..3 75 25 Injection Volume: 15 μl, Partial loop injection rdering Information* DisQuE Dispersive Sample Preparation Kit (1/pk) 1761676 ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm 1862352 LCMS Certified Vials 6749CV * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) 211 Waters Corporation. Waters, The Science of What s Possible, DisQuE, ACQUITY UPLC, and ACQUITY are trademarks of Waters Corporation. [ 63 ]

Multi-residue Analysis of pesticides by QuEChERS in: ranges by gc/ms MS Conditions ranges are a commodity containing an ample amount of water. This sample uses the standard Association of Analytical Communities (AAC) extraction and clean-up tubes. Ionization: Acquisition: Waters Quattro micro GC/MS Electron Impact (7 ev) Single Ion Recording (SIR) Mode Extraction Procedure 1. Add 15 ml 1% acetic acid in acetonitrile into the 5 ml DisQuE extraction tube 1 containing 1.5 g of sodium acetate and 6 g of magnesium sulfate. 2. Add 15 g of homogenized sample into the 5 ml tube. 3. Add any internal standards and standard mixture. 4. Shake vigorously for 1 minute and centrifuge > 15 rcf for 5 minute. 5. Transfer 1 ml of the acetonitrile extract into the 2 ml DisQuE clean-up tube 2, containing 5 mg PSA and 15 mg of magnesium sulphate. 6. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. 7. Transfer.5 ml extract into a tube. 8. Add any post-extraction internal standards. 9. Add.25 ml toluene. 1. Evaporate at 5 C with N 2 to <.1 ml. 11. Bring volume up to.2 ml with toluene. 12. Transfer to vial with insert for analysis. GC Conditions % Recovery 18 16 14 12 1 8 6 4 2 Atrazine Azoxystrobin Bifenthrin Carbaryl Chlorothalonil Chlorpyrifos-methyl Cyprodinil Ethion Hexachlorobenzene Pesticides in ranges by GC/MS. Pesticide Kresoxim-methyl Permethrin o-phenylphenol Procymidone Tebuconazole Tolyfluanid Trifluralin Column: Carrier Gas: Flow Rate: Temperature Program: Injection Volume: Agilent 689N GC RTX-5MS, 3 x.25 mm, (.25 µm film) Helium 1. ml/min Initial 1 C, hold 1 min, then 1 C/min to 32 C, hold for 7 minute 2 µl splitless rdering Information* DisQuE 5 ml Tube-AAC/Acetate 1864571 DisQuE 2 ml Tube-AAC/C 18 186483 LCGC Certified Vials 186272C Insert 3 µl with Poly Spring WAT9417 * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) 211 Waters Corporation. Waters, DisQuE and Quattro micro are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 64 ]

Multi-residue Analysis of pesticides by QuEChERS in: ranges by uplc/ms/ms ranges are a commodity containing an ample amount of water. This sample uses the standard Association of Analytical Communities (AAC) extraction and clean-up tubes. MS Conditions Waters ACQUITY TQ Detector Ionization: Positive electrospray (ESI + ) Acquisition: Multiple reaction monitoring (MRM) Extraction Procedure 16 14 1. Add 15 ml 1% acetic acid in acetonitrile into the 5 ml DisQuE extraction tube 1. 2. Add 15 g of homogenized orange with skin into the 5 ml tube. 3. Add any internal standards and standard mixture. 4. Shake vigorously for 1 minute and centrifuge > 15 rcf for 5 minute. Recovery (%) 12 1 8 6 4 2 Atrazine Azoxystrobin Carbaryl Cyprodinil Dichlorvos Imidacloprid Kresoxim-methyl Linuron Pesticides Methamidophos Methomyl Pymetrozine Tebuconazole Tolyfluanid 5. Transfer 1 ml of the acetonitrile extract into the 2 ml clean-up Pesticides in ranges by UPLC /MS/MS. tube containing 5 mg PSA, 15 mg MgS 4, and 5 mg C 18. 6. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. 7. Transfer 1 μl of final extract into a 1.5 ml centrifuge tube. 8. Add any post-extraction internal standards. 9. Dilute as needed with an appropriate buffer or solvent. 1. Centrifuge > 16 rcf for 5 minutes. 11. Transfer to autosampler vial. LC Conditions LC System: Column: Column Temperature: 4 C Sample Temperature: 4 C Flow Rate: Mobile Phase A: Mobile Phase B: Waters ACQUITY UPLC System ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm.3 ml/min. Water +.1% formic acid Methanol +.1% formic acid Gradient: Time Flow Rate A% B%..3 75 25.25.3 75 25 7.75.3 5 1 8.5.3 1 8.51.5 75 25 1.5.5 75 25 11..3 75 25 Injection Volume: 15 μl, Partial loop injection rdering Information* DisQuE 5 ml Tube-AAC/Acetate 1864571 DisQuE 2 ml Tube-AAC/C 18 186483 ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm 1862352 LCMS Certified Vials 6749CV * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) 211 Waters Corporation. Waters, DisQuE, ACQUITY UPLC, UPLC, and ACQUITY are trademarks of Waters Corporation. [ 65 ]

Multi-residue Analysis of pesticides by QuEChERS in: rolled ats by gc/ms MS Conditions Rolled oats are a low water content commodity that requires the addition of water and soak time as a pretreatment step before extraction. Ionization: Acquisition: Waters Quattro micro GC/MS Electron Impact (7 ev) Single Ion Recording (SIR) Mode Sample Pretreatment: 14 12 1 1 Add 7.5 g of ground rolled oats and 15 ml of water in a tube and soak for 1 minutes. Extraction Procedure: 1. Add 15 ml 1% acetic acid in acetonitrile into the 5 ml DisQuE extraction tube 1 containing 1.5 g of sodium acetate and 6 g of magnesium sulfate. % Recovery 8 6 4 2 Pesticides in Rolled ats by GC/MS. Atrazine Azoxystrobin Bifenthrin Carbaryl Chlorpyrifos Chlorpyrifos-methyl Cyprodinil Endosulfan sulfate Ethion Kresoxim-methyl Pesticide Permethrin o-phenylphenol Procymidone Tebuconazole Trifluralin 2. Add soaked oat sample into the 5 ml tube. 3. Add any internal standards and standard mixture. 4. Shake vigorously for 1 minute and centrifuge > 15 rcf for 5 minute. 5. Transfer 1 ml of the acetonitrile extract into the 2 ml DisQuE clean-up tube 2, containing 5 mg PSA and 15 mg of magnesium sulphate. 6. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. 7. Transfer.5 ml extract into a tube. 8. Add any post-extraction internal standards. 9. Add.25 ml toluene. 1. Evaporate at 5 C with N 2 to <.1 ml. 11. Bring volume up to.2 ml with toluene. 12. Transfer to vial with insert for analysis. GC Conditions Column: Carrier Gas: Flow Rate: Agilent 689N GC RTX-5MS, 3 x.25 mm, (.25 µm film) Helium 1. ml/min Temperature Program: Initial 1 C, hold 1 min, then 1 C/min to 32 C, hold for 7 minute Injection Volume: 2 µl splitless rdering Information* DisQuE Dispersive Sample Preparation Kit (1/pk) 1761676 LCGC Certified Vials Insert 3 µl with Poly Spring 186272C WAT9417 * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) 211 Waters Corporation. Waters, DisQuE and Quattro micro are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 66 ]

Multi-residue Analysis of pesticides by QuEChERS in: Rolled oats by uplc/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 1. Add 15 ml 1% acetic acid in acetonitrile into the 5 ml DisQuE extraction tube 1 containing 1.5 g of sodium acetate and 6 g of magnesium sulfate. 2. Diluted 7.5 g ground rolled oats with 15 ml water and soak for 1 min. 3. Add sample into the 5 ml tube. 4. Add any internal standards and standard mixture. 5. Transfer 1 ml of the acetonitrile extract into the 2 ml DisQuE clean-up tube 2, containing 5 mg PSA and 15 mg of magnesium sulphate. 6. Transfer 1 ml of the acetonitrile extract into the clean-up tube 2. 7. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. LC Conditions LC System: Waters ACQUITY UPLC System Column: ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm Column Temperature: 4 C Sample Temperature: 4 C Flow Rate:.3 ml/min. Mobile Phase A: Water +.1% formic acid Mobile Phase B: Methanol +.1% formic acid Gradient: Time Flow Rate A% B%..3 75 25.25.3 75 25 7.75.3 5 1 8.5.3 1 8.51.5 75 25 1.5.5 75 25 11..3 75 25 Injection Volume: 15 μl, Partial loop injection MS Conditions Waters ACQUITY TQ Detector Ionization: Positive electrospray (ESI + ) Acquisition: Multiple reaction monitoring (MRM) 8. Transfer 1 μl of final extract into a 1.5 ml centrifuge tube. 14 12 1 9. Add any post-extraction internal standards. 1. Dilute as needed with an appropriate buffer or solvent. % Recovery 8 6 4 2 11. Centrifuge > 16 rcf for 5 minutes. 12. Transfer to autosampler vial. Atrazine Azoxystrobin Carbaryl Cyprodinil Dichlorvos Imazalil Imidacloprid Linuron Methamidophos Methomyl Pymetrozine Thiabendazole Tebuconazole Tolyfluanid Pesticide Pesticides in Rolled ats by UPLC /MS/MS. rdering Information* DisQuE Dispersive Sample Preparation Kit (1/pk) 1761676 ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm 1862352 LCMS Certified Vials 6749CV * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) 211 Waters Corporation. Waters, DisQuE, ACQUITY UPLC, UPLC, and ACQUITY are trademarks of Waters Corporation. [ 67 ]

Multi-residue Analysis of pesticides by QuEChERS in: teas by UPLC/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. Sample Preparation Procedure 1. Tare weigh an empty beaker. 2. Weigh out 1 g of tea leaves in the beaker. 3. Add in 6 g of hot water at 8-85 C to the beaker. Brew the tea for 2 minutes. 4. Weigh the beaker with water and tea. 5. Calculate the weight of water loss due to evaporation. Add water to the beaker to make up for the loss of water. 6. Homogenize the sample until it reaches consistent texture. LC Conditions LC System: Column: Column Temperature: 4 C Sample Temperature: 4 C Flow Rate: Mobile Phase A: Mobile Phase B: Waters ACQUITY UPLC System ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm.3 ml/min. Water +.1% formic acid Methanol +.1% formic acid Gradient: Time Flow Rate A% B%..3 75 25.25.3 75 25 7.75.3 5 1 8.5.3 1 8.51.5 75 25 1.5.5 75 25 11..3 75 25 Injection Volume: 15 μl, Partial loop injection Extraction Procedure 1. Transfer 15 g of homogenized sample into an empty 5 ml tube. 2. Add any internal standards and standard mixture. 3. Add 15 ml 1% acetic acid in acetonitrile into the 5 ml DisQuE extraction tube 1 containing 1.5 g of sodium acetate and 6 g of magnesium sulfate. 4. Transfer all the powder in the DisQuE extraction tube 1 into the 5 ml containing sample and solvent. 5. Shake vigorously for 1 minute and centrifuge > 15 rcf for 5 minute. 6 Transfer 1 ml of the acetonitrile extract into the 2 ml DisQuE clean-up tube 2, containing 5 mg PSA and 15 mg of magnesium sulphate. MS Conditions Waters ACQUITY TQ Detector Ionization: Positive electrospray (ESI + ) Acquisition: Multiple reaction monitoring (MRM) % Recovery 225 2 175 15 125 1 75 5 25 Carbendazim Dichlorvos Tricyclazole Pesticides in Teas by UPLC /MS/MS. Malathion Methidathion Ethion Profenophos Phosalone Chloropyrifos Hexachonzol Trizaphos Quinal Phos Pesticide Green Tea Black Tea 7. Shake for 3 seconds and centrifuge >16 rcf for 5 minute. 8. Transfer 1 μl of final extract into an autosampler vial. 9. Add any post-extraction internal standards. 1. Dilute as needed with an appropriate buffer or solvent. rdering Information* DisQuE Dispersive Sample Preparation Kit (1/pk) 1761676 ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm 1862352 LCMS Certified Vials 6749CV * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) 211 Waters Corporation. Waters, The Science of What s Possible, DisQuE, ACQUITY UPLC, UPLC, and ACQUITY are trademarks of Waters Corporation. [ 68 ]

Multi-residue Analysis of pesticides by QuEChERS in: vegetables and fruits by uplc/ms/ms Most fruits and vegetables are commodities containing an ample amount of water. These samples use the standard Association of Analytical Communities (AAC) extraction and clean-up tubes. Extraction Procedure 1. Add 15 g of homogenized sample to the 5 ml DisQuE extraction tube containing 1.5 g of sodium acetate and 6 g of magnesium sulphate. MS Conditions 1 % Waters ACQUITY TQ Detector Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring 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 >15 rcf for 1 minute..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 DisQuE extraction tube containing 5 mg PSA and 15 mg of magnesium sulphate. 6. Shake for 3 seconds and centrifuge >15 rcf for 1 minute. 7. Transfer 25 μl of final extract into an autosampler vial. 8. Add any post-extraction internal standards. 9. Dilute as needed with an appropriate buffer or solvent. 42 Pesticide Residues In ne 1 Minute Run In Injection Solvent. Recovery (%) 12 Baby Food Mango Avocado 1 8 6 4 2 Atrazine Carbendazim Cyprodinil Flufenacet Imazalil Tebuconazole Triabendazole LC Conditions LC System: Column: Column Temperature: 4 C Sample Temperature: 4 C Flow Rate: Mobile Phase A: Mobile Phase B: Waters ACQUITY UPLC System ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 μm.45 ml/min 98:2 water:.1% formic acid in methanol Methanol +.1% formic acid Gradient: time (min) A% B%. 9 1.25 9 1 7.75 1 8.5 1 8.51 9 1 Total Run Time: Injection Volume: 1 min 2 μl, full loop injection Pesticide Recovery Data for Three Types of Sample Matrices. rdering Information* DisQuE Dispersive Sample Preparation Kit (1/pk) 1761676 ACQUITY UPLC BEH C 18, 2.1 x 1, 1.7 µm 1862352 LCMS Certified Vials 6751CV * For all AAC and CEN QuEChERS methods, refer to the DisQuE Dispersive Sample Preparation Brochure (lit code: 72348EN) Ref: Waters Application Note 722578EN 211 Waters Corporation. Waters, DisQuE, ACQUITY UPLC, and ACQUITY are trademarks of Waters Corporation. [ 69 ]

Paraquat and other Quaternary Ammonium Compounds in Water Paraquat is one of the most widely used herbicides in the world. As with many herbicides it is dangerously poisonous to humans if swallowed. Pretreatment 1. Adjust sample to ph 7 by adding 1 M ammonium phosphate buffer dropwise to 2 ml river water. SPE Procedure Analyte Paraquat Diaquat MRM method parameters. Results 1 MRM Transition 171 77 171 155 183 157 183 168 Paraquat asis WCX, 3cc/6mg Condition/Equilibrate: A. 1 ml methanol B. 1 ml water Load: 2 ml sample Wash: A. 1 ml 1 M ph 7 phosphate buffer B. 1 ml water C. 1 ml methanol 1 1 Diquat ELUTE: 1.5 ml acetonitrile/water/trifuoroacetic acid (84:14:12, v/v/v) Reconstitute in.5 ml mobile phase 1 1 2 3 4 5 6 7 LC/MS separation of paraquat and diquat at.5 μg/l. LC Conditions Column: Flow Rate: Mobile Phase: Injection Volume: Waters Alliance HPLC 2695 System Atlantis HILIC, 2.1 x 15 mm, 3.5 μm.4 ml/min 4% acetonitrile 6% 25 mm ammonium formate (ph 3.7) 2 μl Column Temperature: 3 C MS Conditions Waters Quattro micro Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring Paraquat Diquat Day 1 1.8 μg/l (8.1% RSD) 1.5 μg/l (2.9% RSD) Day 4 1.1 μg/l (8.% RSD) 1.9 μg/l (5.9% RSD) Day 5.95 μg/l (7.1% RSD) 1.8 μg/l (4.4% RSD) Intraday results obtained from spiked water samples (spike level 1. µg/l). rdering Information asis WCX, 3 cc/6 mg, 6 µm, 1/box 1862497 Atlantis HILIC, 2.1 x 15 mm, 3.5 μm 186215 75 μl Polypropylene Vials 1862635 Ref: Waters Application Note WA4524 211 Waters Corporation. Waters, asis, Atlantis, Alliance, and Quattro micro are trademarks of Waters Corporation. [ 7 ]

Patulin in Apple Juice Patulin is a mycotoxin that is produced by certain species of 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. SPE Procedure asis HLB, 3cc/6mg Condition: A. 3 ml methanol B. 3 ml water MS Conditions Analytes Patulin 5-hydroxymethylfurfural (HMF) MRM method parameters. Results Waters ACQUITY TQ Detector Ionization Mode: Negative electrospray (ESI - ) Multiple reaction monitoring MRM Transition 153 19 153 81 125 95 HMF Load: 2.5 ml sample 3. e - 1 2.5e - 1 Wash 1: 3 ml 1% sodium bicarbonate (1g/1mL) AU 2.e - 1 1.5e - 1 1.e - 1 Patulin Wash 2: 1 ml.1% acetic acid DRY: Using vacuum manifold 5.e - 2.2.4.6.8 1 1.2 1.4 1.6 1.8 2 2.2 min Apple juice extract at 5 µg/kg containing patulin and 5-hydroxymethylfurfural (HMF) at 276 nm. Elute: 2 x 1.5 ml 1% ethyl acetate in methyl t-butyl ether (MTBE) 1 % Patulin Reconstitute: 5 µl water 1 HMF 7528 LC Conditions Waters ACQUITY UPLC System Column: ACQUITY UPLC BEH Shield RP18, 2.1 x 1 mm, 1.7 µm Flow Rate: Mobile Phase: 6 µl/min A..1% aqueous ammonium hydroxide b..1% ammonium hydroxide in acetonitrile Gradient: Time (min) A% B%. 99 1 1.8 99 1 2.3 1 9 2.8 1 9 2.81 99 1 Injection Volume: Column Temperature: 4 C Sample Temperature: 4 C Detector: Detection: 2 µl, Full loop injection ACQUITY UPLC PDA 276 nm % Apple juice extract at 5 µg/kg containing patulin and 5-hydroxymethylfurfural in negative electrospray mode. Concentration Average Recovery (%RSD) 5 µg/kg 86.1% (13.6) 5 µg/kg 95.4% (5.9) 5 µg/kg 89.9% (17.5) Recovery data obtained from asis HLB extraction of patulin in apple juice. Four data points were measured at each level. rdering Information.6.8 1 1.2 1.4 1.6 1.8 min asis HLB, 3cc/6mg, 1/box ACQUITY UPLC BEH Shield RP18, 2.1 x 1 mm, 1.7 μm WAT94226 1862854 LCMS Certified Vials 6749CV Ref: Developed by Vural Gökmen, Food Engineering Department, Hacettepe University, Ankara, Turkey and John Martin, Waters Corporation 211 Waters Corporation. Waters, asis, ACQUITY UPLC, and ACQUITY are trademarks of Waters Corporation. [ 71 ]

PFS and Related Compounds in Water and Tissue Perfluorinated compounds (PFCs) such as perfluorooctanesulfonic acid (PFS) and perfluorooctanoic acid are persistent organic pollutants (PPs). PFCs may be toxic and have bioaccumulative properties. There is growing interest in the development of analytical methods for PFCs in food, drinking water, tissue, plasma, and blood. Pretreatment Water samples 1. Adjust 1 ml of sample to ph 3 with formic acid prior to SPE. Chicken liver samples 1. Homogenize 1 g of sample and extract with 1 ml of 1 mm potassium hydroxide in methanol. Shake for 16 hours. 2. Centrifuge the sample for 1 minutes at 8 rpm. 3. Dilute 1 ml aliquot of supernatant to 2 ml with water and adjust the ph to 4-5 using 2% formic acid. SPE Procedure asis WAX, 3 cc/6 mg Condition/Equilibrate: A. 2 ml methanol B. 2 ml water Load: 1 ml water or 2 ml diluted tissue sample Wash: A.1 ml 2% formic acid B. 2 ml methanol Elute: 2 ml 1% ammonia in methanol Note: The SPE eluate is collected in polypropylene test tubes, diluted with 2 ml of 2% aqueous formic acid and brought to 5 ml with water. Alternatively, the eluate may be evaporated and reconstituted in 1 ml mobile phase prior to analysis. Polypropylene labware should be used exclusively. LC Conditions PFC PFBS (Perfluorobutane Sulfonate) PFS (Perfluorooctane Sulfonate) C3 C4 C5 C6 C7 C8 C9 C1 C11 MRM method parameters. Waters ACQUITY UPLC System Column: ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 µm Flow Rate:.4 ml/min Mobile Phase: A. 2 mm ammonium acetate in water/acetonitrile (9:1) b. acetonitrile/methanol Gradient: Time (min) A% B%. 85 15 8. 5 95 Injection Volume: Column Temperature: 4 C MS Conditions 1 µl (full loop injection mode) Waters Quattro Premier XE Ionization Mode: Negative electrospray (ESI - ) Multiple reaction monitoring MRM 299 8 499 8 163 119 213 169 263 219 313 269 363 319 413 369 463 419 513 469 563 519 [ 72 ]

PFS and Related Compounds in Water and Tissue UPLC /MS/MS of 12 PFCs spiked at 1 μg/kg in chicken liver. Recovery from Drinking Water (%) Spike Level µg/l PFBS PFS C3 C4 C5 C6 C7 C8 C9 C1 C11.1 122 19 18 119 97 184 17 83 121 11 11.3 11 117 95 132 15 11 119 126 137 118 94.7 12 98 91 17 93 118 1 78 13 126 119 1. 113 94 128 16 98 13 1 88 1 11 117 4. 14 86 11 99 99 12 12 92 115 99 84 1 14 1 98 11 1 87 89 82 13 99 11 Recovery data for PFCS from drinking water. Recovery data for PFCs from chicken liver. Recovery from Chicken Liver (%) Spike Level µg/kg PFBS PFS C3 C4 C5 C6 C7 C8 C9 C1 C11 2 LQ 81 LQ 18 132 165 1 97 5 98 LQ 138 148 LQ 97 1 133 89 73 LQ 1 93 5 12 134 121 99 87 123 95 11 33 2 12 5 128 144 94 96 11 117 9 8 25 3 87 51 14 12 124 89 86 13 91 84 2 5 92 54 92 92 118 94 97 97 86 86 22 rdering Information PFC Analysis Kit 1761744 PFC Column Kit 1761692 PFC QC Standard 1864597 asis WAX, 3 cc/6 mg, 6 µm, 1/box 1862492 ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 µm 186235 ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 µm, 3/pk 176863 75 μl Polypropylene Vial 1862635 Ref: Waters Application Note 721817EN 211 Waters Corporation. Waters, asis, ACQUITY UPLC, UPLC, Quattro Premier are trademarks of Waters Corporation. [ 73 ]

Propham in Potatoes by GC/MS Propham is the active substance used as herbicides and potato sprout inhibitor. This analytical method can be used to monitor Propham residues in potatoes. Pretreatment 1. Add 15 g of ground potatoes into a 5 ml centrifuge tube. 2. Add 15 ml 1% acetic acid in acetonitrile with 1.5 g anhydrous sodium acetate and 6 g anhydrous magnesium sulfate. 3. Shake vigorously for 1 minute. 4. Centrifuge >15 rcf for 1 minute. 5. Take out 7.5 ml extract and dilute to 1 ml with 2.5 ml toluene. SPE Procedure Sep-Pak Vac Carbon Black/Aminopropyl, 6 cc/5 mg/5 mg CNDITIN: 1 ml 25:75 toluene: actonitrile (v/v) Add 2 mg anhydrous magnesium sulfate to top of cartridge to remove water LAD: Extract (collect) ELUTE: 1 ml 25:75 toluene: acetonitrile (collect) Combine both collected fractions and adjust volume to exactly 2 ml by addition of toluene:acetonitrile (25:75, v/v) Take 5 ml and evaporate to just below 1 ml and bring up to 1 ml with toluene. Inject onto GC/MS. GC Conditions Agilent 689 GC Column: DB-5ms, 3m x.25mm (i.d.),.25 µm film. Direct connection of column to injection-port liner Transfer Line to MS: 3 C Source Temperature: 2 C Injection Volume: 1 µl splitless Injection Port Temperature: 18 C Initial Temperature: 8 C Time at Initial Temperature: 1 min. Then Program at 1 C/ min to 2 C Then at 25 C minute to 3, hold 5 minutes GC/MS Conditions Ionization Mode: HP689 GC Flow 1 Initial Flow: GC/MS (SIR) Waters Quattro micro GC Electron Impact (7 ev) Selected-Ion Recording (SIR) 1 ml/min time Rate Final Flow (min) (ml/min) (ml/min). 5 3.5 5 3.6 5 1 Channel Mass 1 (Quantification) 92.8 2 (Confirmation) 119 3 (Confirmation) 12 SIR method parameters. [ 74 ]

Propham in Potatoes by GC/MS Results Blank % 1 9.79 % 1 ppm spike 1 4 6 8 1 12 14 min 1 µg/g spiked potato sample. Compound Name: propham 92.8 RT Area 1 ppm spiked 1 9.78 379. 1 ppm spiked 2 9.82 382. 1 ppm spiked 3 9.8 458. 1 ppm spiked 4 9.79 399. 1 ppm spiked 5 9.75 421. Mean - 47.8 RSD (%) - 8.1 Recovery (%) - 95.73 Recovery data for 1 μg/g spiked potato sample. rdering Information Sep-Pak Vac Carbon Black/Aminopropyl, 6 cc/5 mg/5 mg LCMS Certified Vials 1863369 6751CV 211 Waters Corporation. Waters, Quattro micro, and, Sep-Pak are trademarks of Waters Corporation. All other trademarks are property of their respective owners. [ 75 ]

Propham in Potatoes by lc/ms Results Propham is the active substance used as herbicides and potato sprout inhibitor. This analytical method can be used to monitor Propham residues in potatoes. 1 % Blank Pretreatment 1. Add 15 g of ground potatoes to 5 ml centrifuge tube. 2. Add 15 ml 1% acetic acid in acetonitrile with 1.5 g anhydrous 1 1 ppm spiked sample 2.15 sodium acetate and 6 g anhydrous magnesium sulfate. 3. Shake vigorously for 1 minute. % 4. Centrifuge >15 rcf for 1 minute. SPE Procedure Sep-Pak Light NH 2 Transfer 5 ml to another tube and add.5 mg of anhydrous magnesium sulfate Vortex and allow powder to settle Pass through 2 ml of prepared sample extract Take out 2 µl dilute with 8 µl water.5 1 1.5 2 2.5 3 3.5 4 4.5 5 min Pass through 1 μg/g spiked potato sample. Compound 1: propham 138.3 RT Area 1 ppm spiked 1 2.15 5895.34 1 ppm spiked 2 2.15 6424.93 1 ppm spiked 3 2.15 6996.63 1 ppm spiked 4 2.15 7557.8 1 ppm spiked 5 2.15 7567.6 Mean - 6888.46 RSD (%) - 1.57 Recovery (%) - 84.43 LC Conditions Inject onto LC/MS (1 µl) Waters ACQUITY UPLC System Column: ACQUITY UPLC BEH C 18, 2.1 x 1 mm, 1.7 µm Mobile Phase: A..1% formic acid in water b..1% formic acid in acetonitrile Gradient: Time (min) A% B%. 8 2 3. 2 8 3.2 8 2 5. 8 2 Compound 2: propham 12.3 RT Area 1 ppm spiked 1 2.15 1849.85 1 ppm spiked 2 2.15 195.71 1 ppm spiked 3 2.15 291.4 1 ppm spiked 4 2.15 2276.4 1 ppm spiked 5 2.15 236.56 Mean - 294.98 RSD (%) - 9.5 Recovery (%) - 81.35 Recovery data for of 1 μg/g spiked potato sample. MS Conditions Waters Quattro micro Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring MRM Transitions: 1. 18.3 12.3 2. 18.3 138.3 rdering Information Sep-Pak Light NH 2 WAT23513 ACQUITY UPLC BEH C 18, 2.1 x 1 mm 1862352 211 Waters Corporation. Waters, ACQUITY UPLC, Quattro micro and Sep-Pak are trademarks of Waters Corporation. [ 76 ]

SUDAN DYES IN CHILLI IL AND PWDER Sudan dyes are red dyes that are used for coloring solvents, oils, waxes, petrol, shoe and floor polishes. Sudan dyes are not allowed to be added to food in by the United States Food and Drug Administration (US FDA), European Union (EU), and other countries. Pretreatment For Chilli il 1. Dilute.1 g chilli oil in 1 ml with hexane. For Chilli Powder 1. Homogenize and extract 1 g chilli powder with 1 ml acetone. 2. Centrifuge. 3. A 1 ml aliquot is evaporated to complete dryness and the residue is taken up in 1 ml hexane. MS Conditions Waters Quattro micro API Ionization mode: Positive electrospray (ESI + ) Multiple reaction monitoring Analyte Sudan I Sudan II Sudan III Sudan IV Results MRM Transition 249 156 249 93 249 128 277 156 277 121 277 16 353 77 353 12 353 196 381 91 381 16 381 224 SPE Procedure Sep-Pak Alumina B, 3 cc/5 mg Condition/Equilibrate: A. 2 ml methanol B. 2 ml ethyl acetate C. 3 ml hexane Load: 1 ml of hexane pre-extract LC Conditions Wash: A. 3 ml hexane B. 1 ml ethyl acetate Elute: 4 ml ethyl acetate/methanol (9:1) Evaporate and reconstitute in 2 µl methanol Waters Alliance HPLC 2695 System Column: Atlantis dc 18, 2.1 x 1 mm, 3 µm Flow Rate:.4 ml/min Mobile Phase: A..1% formic acid in water b. acetonitrile Gradient: Time (min) A% B%. 2 8 1. 5 95 Injection Volume: 15 µl Column Temperature: 3 C LC/MS spiked chilli powder (n=6, 8 μg/kg). Analyte Recovery (%) RSD (%) Sudan I 99 11 Sudan II 91 11 Sudan III 93 6 Sudan IV 122 11 Recovery data for spiked chilli powder (n=6, 8 μg/kg). rdering Information Sep-Pak Alumina B, 3 cc/5 mg, 5/box WAT2825 Atlantis dc 18, 2.1 x 1 mm, 3 µm 1861295 Qsert Vials, LCGC Certified Combination Packs 1861126C Ref: Waters Applications 72144EN 211 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 ]

Sudan Dyes in fresh chillis Sudan dyes are red dyes that are used for coloring solvents, oils, waxes, petrol, shoe and floor polishes. Sudan dyes are not allowed to be added to food in by the United States Food and Drug Administration (US FDA), European Union (EU), and other countries. Pretreatment 1. Homogenize and extract 1 g of chilli with 1 ml acetone. 2. Dilute 1 ml aliquot to 5 ml with aqueous sodium hydroxide (adjust to ph 11). SPE Procedure asis MAX, 3 cc/6 mg Condition/Equilibrate: A. 2 ml ethyl acetate C. 1 ml.1 M sodium hydroxide B. 2 ml methanol D. 2 ml water Analyte Sudan I Sudan II Sudan III Sudan IV MRM method parameters. Results MRM Transition 249 156 249 93 249 128 277 156 277 121 277 16 353 77 353 12 353 196 381 91 381 16 38 224 Load: 5 ml of diluted acetone pre-extract Wash: A. 2 ml 7% 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 2 µl acetonitrile/water (9:1, v/v) LC/MS spiked chilli sauce (n=6, 8 μg/kg), asis MAX method. LC Conditions Waters Alliance HPLC 2695 System Column: Atlantis dc 18, 2.1 x 1 mm, 3 µm Flow Rate: Mobile Phase:.4 ml/min A..1% formic acid in water b. acetonitrile Gradient: Time (min) A% B%. 2 8 1. 5 95 Injection Volume: 15 µl Column Temperature: 3 C MS Conditions Waters Quattro micro API Ionization Mode: Positive electrospray (ESI + ) Multiple reaction monitoring Analyte Recovery (%) RSD (%) Sudan I 83 9 Sudan II 83 1 Sudan III 77 3 Sudan IV 75 4 rdering Information asis MAX, 3 cc/6 mg, 6 µm, 1/box 186368 Atlantis dc 18 2.1 x 1 mm, 3 µm 1861295 Qsert Vials, LCGC Certified Combination Packs 1861126C Ref: Waters Application Note 72144EN 211 Waters Corporation. Waters, asis, 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 CNTRL (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 Acids in Animal Feed Hydrolysates Amino acid analysis has been used in the food and feed industries to verify and characterize materials and processes. The total amino acid content, as well as the proportions of growth-limiting amino acids, is an essential characteristic of the nutritional value of feeds. Samples Swine diet, poultry diet, whole soybean, and soybean meal samples were acid-hydrolyzed in an independent laboratory as part of a collaborative study. The samples were supplied at an estimated concentration of 1. mg/ml in.1 M HCl and sealed under argon in ampoules. Samples were stored at -8 C until analysis. The standard was NIST 2389 Amino Acids in.1 mol/l HCl Reference Material, and it was diluted to 5, 1, and 25 pmol/μl. Sample Derivatization The samples were diluted 1:16 with.1 M HCl prior to derivatization. The standard derivatization protocol was modified to include neutralization of excess acid with.1 M NaH. Conditions for precolumn derivatization and analysis are described in detail in the Waters UPLC Amino Acid Analysis Solution System Guide (P/N 71512972). These derivatization conditions were modified to include additional base. 1. 6 μl AccQ Tag Ultra Borate Buffer 2. 1 μl diluted sample 3. 1 μl.1 N NaH 4. 2 μl reconstituted AccQ Tag Ultra Reagent LC Conditions Waters ACQUITY UPLC System Column: AccQ Tag Ultra, 2.1 x 1 mm, 1.7 µm Column Temperature: 55 C Sample Temperature: 2 C Flow Rate: 7 µl/min. Mobile Phase A: 1:2 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) AU AU AU AU Total Run Time: Injection Volume: Detection: Amino Acid.25.2.2 AMQ.15.1.5..1.25.2 AMQ.15.5.1.5...25.2.15.1.5..25.2.15.1.5. AMQ AMQ % RSD His 1.3 Ser.43 Arg.6 Gly.39 Asp.24 Glu.23 Thr.26 Ala.27 Pro.3 Cys.18 Lys.23 Tyr.17 Met.21 Val.22 Ile.23 Leu.24 Phe.23 NH3 NH3 NH3 NH3 Poultry Diet His Swine Diet His Whole Soybean His Soybean Meal His 9.5 min 1 µl, partial loop with needle overfill UV (TUV), 26nm Ser Arg Gly Ser Arg Gly Ser Arg Gly Ser Arg Gly Asp Summary of retention times, in minutes, for the hydrolysate standard throughout the five days of analyses. Asp Asp Asp Animal feed hydrolysates, 6 ng on column, using the UPLC Amino Acid Analysis Solution. Glu Glu Glu Glu Thr Thr Thr Thr 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. min Ala Ala Ala Ala Pro Pro Pro Pro Deriv Peak Deriv Peak Deriv Peak Deriv Peak Cys Cys Cys Cys Lys Lys Lys Lys Tyr Met Tyr Met Tyr Tyr Met Met Val Val Val Val Ile Ile Ile Ile Leu Leu Leu Leu Phe Phe Phe Phe [ 8 ]

Amino Acids in Animal Feed Hydrolysates Amino Acid Poultry Diet Swine Diet Whole Soybean Soybean Meal Mean Std. Dev. His 2.513 2.54 2.526 2.534 2.521.16 Ser 3.363 3.358 3.37 3.373 3.367.8 Arg 3.546 3.544 3.553 3.555 3.551.6 Gly 3.665 3.661 3.671 3.673 3.668.6 Asp 3.99 t3.987 3.995 3.997 3.993.5 Glu 4.457 4.455 4.461 4.462 4.459.4 Thr 4.82 4.819 4.823 4.823 4.822.2 Ala 5.189 5.187 5.19 5.191 5.189.2 Pro 5.752 5.751 5.75 5.751 5.751.1 Cys 6.599 6.61 6.62 6.63 6.62.1 Lys 6.661 6.663 6.662 6.663 6.663.1 Tyr 6.799 6.8 6.81 6.81 6.81.1 Met 6.944 6.945 6.945 6.945 6.945. Val 7.64 7.65 7.64 7.66 7.65.1 Ile 7.787 7.788 7.787 7.787 7.787.1 Leu 7.868 7.869 7.868 7.869 7.869.1 Phe 7.985 7.985 7.985 7.986 7.985.1 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. rdering Information Amino Acid Analysis Kit 1761235 AccQ Tag Ultra Column, 2.1 x 1 mm 1863837 Amino Acid Standard, Hydrolysate, 1 x 1 ml ampules LCMS Certified Vials WAT88122 6751CV Ref: Waters Application Note 72284EN 211 Waters Corporation. Waters, UPLC, ACQUITY UPLC, and AccQ Tag are trademarks of Waters Corporation. [ 81 ]

Amino Acids in Tea L-theanine is a free (non-protein) amino acid found almost exclusively in tea plants. It is the predominant amino acid in green tea leaves, giving tea its characteristic taste. LC Conditions Waters ACQUITY UPLC system with TUV Column: AccQ Tag Ultra, 2.1 x 1 mm, 1.7 µm Column Temperature: 6 C Sample Temperature: 2 C Sample Preparation Free amino acids were analyzed in tea leaves. Tea leaves were standard consumer single serving products. The tea samples were between 2.5-3.5 g. The tea leaves were extracted in 6 oz. of bottled water at an initial temperature of 72 C for 2 hours, unless otherwise specified. After a set period of time, the supernatant of the mixture was transferred to a separate vial. Extracted tea samples in water were stored at -2 C until analysis. Flow Rate: Mobile Phase A: Mobile Phase B: Gradient: Total Run Time: Injection Volume: Detection: 7 µl/min. 1:1 dilution of AccQ Tag Ultra Eluent A concentrate with MilliQ water AccQ Tag Ultra Eluent B AccQ Tag Ultra Cell Culture Method (provided in the UPLC Amino Acid Analysis Solution) 9.5 min 1 µl, partial loop with needle overfill UV (TUV), 26nm Sample Derivatization The extracted tea samples were derivatized neat following the standard AccQ Tag Ultra derivatization protocol. Conditions for pre-column derivatization and analysis are described in detail in the Waters UPLC Amino Acid Analysis Solution System Guide (P/N 71512972). The derivatization conditions included: 1. 7 µl of AccQ Tag Ultra borate buffer 2. 1 µl of tea extract 3. 2 µl of derivatization reagent AU.6.5.4.3.2.1.5.4.3.2.1 NH3 Green Tea, Extracted for 2 h Asn Ser Gln Arg Asp Green Tea, Extracted for 18 h NH3 Asn Ser Gln Arg Asp Glu Glu Thr Thr Ala Ala 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 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 increase with duration of extraction. GABA GABA Theanine Theanine Pro Pro AABA Deriv Peak Deriv Peak Lys Lys Tyr Val Met Val NVa Ile Ile Leu Phe Phe Trp.6.4.2 AMQ NH3 Asn Ser Gly Asp Glu Ala GABA Pro Deriv Peak Met Lemon Tea.6.4.2 AMQ NH3 Asn Tau Ser Gln Arg Asp Glu Thr Ala GABA Theanine Pro Deriv Peak Lys Tyr Val Earl Grey Ile Leu Phe Trp AU.6.4 AM NH3 Theanine Decaf Earl Grey.2 Asn Tau Ser Gln Arg Asp Glu Thr Ala GABA Pro rn Deriv Peak Lys Tyr Val Ile Leu Phe Trp.6.4.2 AMQ NH3 Asn Ser Gln Arg Asp Glu Thr Ala 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 min GABA Theanine Pro Deriv Peak Lys Val Green Tea Extracted teas using the UPLC Amino Acid Analysis Solution. Theanine was confirmed by MS detection. Amino acids levels vary with type of tea. Ile Phe rdering Information AccQ Tag Ultra Column, 2.1 x 1 mm 1863837 Amino Acid Standard, Hydrolysate, 1 x 1 ml ampules WAT88122 LCMS Certified Vials 6751CV 211 Waters Corporation. Waters, AccQ Tag, UPLC, ACQUITY UPLC are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.. [ 82 ]

Food Sugars in Bran with Raisin Cereal Sugar analysis in foods is a standard food QC assay. Sample Extraction Procedure 1. Weigh out sample (~ 3g) into 5 ml centrifuge tube. 2. Add 25 ml of 5:5 acetonitrile/water and homogenize. 3. Centrifuge at 32 rpm for 3 minutes. 4. Collect supernatant and filter using.45 µm PVDF syringe filter. LC CNDITINS Column: Mobile Phase A: Mobile Phase B: Flow Profile: Flow Rate: Alliance System with 2424 EL SD XBridge Amide, 3.5 μm, 4.6 x 25 mm 8/2 acetonitrile/water with.2% triethylamine 3/7 acetonitrile/water with.2% triethylamine 9% A/1% B (75% acetonitrile with.2% triethylamine) 1. ml/min Injection Volume: 15. µl Sample Concentration:1 mg/ml each Column Temperature: 35 C Needle Wash: Seal Wash: ELSD Conditions 75:25 acetonitrile:water 5:5 acetonitrile:water LSU LSU RESULTS 12 1 8 6 4 2 12 1 8 6 4 2 1 2 4 6 8 1 12 14 16 min CMPUNDS 2 3 2 Raisin Bran Brand Cereal 4 mg/ml Food Sugar Standard 1 mg/ml 1. p-toluamide 2. Fructose 3. Glucose H H H NH 2 4. Sucrose H H H H H H H 3 H H H H H H H H 6. Lactose H H H H H 4 4 H H H 5. Maltose H H 5 H H 6 H H H H Gain: 1 Pressure: 3 psi Drift Tube Temperature: 5 C Nebulizer: Data Rate: Cooling 1 pps Filter Time Constant:.2 sec rdering Information XBridge Amide Column, 3.5 µm, 4.6 x 25 mm 186487 Acrodisc LC 13 mm,.45 µm, 1/pkg WAT2512 12 x 32 LCMS Certified Glass Screw Neck Vial 6751CV 211 Waters Corporation. Waters, XBridge, and Alliance are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. [ 83 ]

Food Sugars in Milk This is a simple assay to monitor sugar levels in milk. There is a simple sample extraction procedure provide, followed by LC/ELSD assay. SAMPLE EXTRACTIN PRCEDURE 1. Dilute with 5:5 acetonitrile/water. 2. Filter using.45 µm PVDF syringe filter. RESULTS 1 8 6 4 2 LSU LSU 1 8 6 4 2 Low Fat Milk 1% in 5:5 MeCN:H 2 Food Sugar Standard 1 mg/ml 1 2 3 2 4 6 8 1 12 14 16 min 4 5 6 6 LC CNDITINS Column: Mobile Phase A: Mobile Phase B: Flow Profile: Flow Rate: Alliance System with 2424 EL SD XBridge Amide, 3.5 μm, 4.6 x 25 mm 8/2 acetonitrile/water with.2% triethylamine 3/7 acetonitrile/water with.2% triethylamine 9% A/1% B (75% acetonitrile with.2% triethylamine) 1. ml/min Injection Volume: 15. µl Sample Concentration: 1 mg/ml each Column Temperature: 35 C Needle Wash: Seal Wash: 75:25 acetonitrile:water 5:5 acetonitrile:water ST RU CTUR ES 1. p-toluamide 2. Fructose 3. Glucose NH 2 H H H H H H H H H 4. Sucrose 5. Maltose H H H H H H H H H H H H H H H 6. Lactose H H ELSD Conditions Gain: 1 H H H H H H H H Pressure: 3 psi Drift Tube Temperature: 5 C Nebulizer: Data Rate: Cooling 1 pps Filter Time Constant:.2 seconds rdering Information XBridge Amide Column, 3.5 µm, 4.6 x 25 mm 186487 Acrodisc LC 13 mm, 45 µm, 1/pkg WAT2512 12 x 32 LCMS Certified Glass Screw Neck Vial 6751CV 211 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 ]

Ginsenoside Rb1 in Ginseng Root Powder Extract This application brief contains a simple extraction protocol and LC/UV conditions for ginsenoside Rb1 in ginseng root. The gensenosides are a target of research in the root. SAMPLE EXTRACTIN PRCEDURE 1. Weigh 2 mg ginseng root powder into an extraction vessel. 2. Add 1 ml 8% methanol, sonicate for 5 min. 3. Centrifuge at 1, rpm for 5 min. AU RESULTS.3.25.2.15.1.5. Ginsenoside Rb1 Ginseng root powder extract Tailing factor =.95 USP plate count = 617..5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. min Structure 4. Collect the supernatant. 5. Repeat steps 2-4 two more times. 6. Combine the extracts, mix well. Ginsenoside Rb1 H H H H H H H H 7. Filter through 13 mm nylon.2 µm filter for injection. LC CNDITINS H H H H H H H Alliance System with 2998 PDA Column: XBridge Amide, 3.5 µm, 4.6 x 15 mm Mobile Phase: 8:2 acetonitrile/water Isocratic Flow Rate: 1.4 ml/min Injection Volume: 11.5 µl Column Temperature: 6 C Sample Temperature: 1 C Needle Wash: 95:5 acetonitrile:water Seal Wash: 1:9 acetonitrile:water UV Wavelength: 23 nm Sampling Rate: 2 Hz Filter Time Constant:.1 sec rdering Information XBridge Amide Column, 3.5 µm, 4.6 x 15 mm 1864869 Acrodisc LC 13 mm, 45 µm, 1/pkg WAT2512 12 x 32 LCMS Certified Glass Screw Neck Vial 6751CV 211 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 3-Amino-2-oxazolidinone (AZ)...23, 25 1-Naphthol...41 3-Hydroxycarbofuran...41 5-hydroxymethylfurfural...71 A Abamectin...53 ACRYLAMIDE...39 Aflatoxins...4 Alanine...8, 81 Aldicarb...41 Aldicarb Sulfone...41 Aldicarb Sulfoxide...41 Ammelide...49 Ammeline...49 3-Amino-5-morpholino-methyl-1,3-oxazolidinone (AMZ)...23, 25 Anibfos...53 Arginine...8, 81 Asparagine...8, 81 Atrazine... 55, 56, 57, 6, 61, 62, 63, 64, 65, 66, 67, 69 Azinphos-methyl...53 Azoxystrobin... 55, 56, 6, 61, 62, 63, 64, 65, 66, 67 B BDMC...41 Benzofenap...53 Bifenthrin...6, 62, 64, 66 Bromclenbuterol...18 Bromobuterol...18 Butafenacil...53 C Cadusafos...58 Carbaryl... 41, 55, 56, 6, 61, 62, 63, 64, 65, 66, 67 Carbendazim...68, 69 Carbofuran...41 Chloramphenicol...19 Chloridazon...53 Chlorpyrifos... 55, 6, 62, 66, 68 Chlorpyrifos-methyl... 55, 57, 6, 62, 64, 66 Chlortetracycline...28, 34, 35, 36 Chlorothalonil...64 Chromafenozide...53 Cimaterol...18 Cimbuterol...18 Ciprofloxacin...21 Clenbuterol...18 Clenbuterol-D9...18 Clomeprop...53 Cloquintocet-mexyl...53 Clothianidin...53 Cloxacillin...28 Cyanuric Acid... 45, 46, 47, 48, 49 Cyazofamid...53 Cyflufenamid...53 Cylohalothrin...57 Cyprodinil... 56, 6, 61, 62, 63, 64, 65, 66, 67, 69 Cysteine...8, 81 D DDD...57 Demeton-S-methyl...58 Demeton-S-methyl sulfone...58 Dexamethasone...2 Diaquat...7 Dichlorvos... 56, 61, 63, 65, 67, 68 Dimethirimol...53 Dimethoate...58 Disulfoton...58 Disulfoton sulfone...58 Disulfoton sulfoxide...58 Doxycycline...34, 36 E Endosulfan sulfate...55, 6, 62, 66 Enkephalin...51 Enrofloxacin...21 Ethiofencarbsulfone...54 Ethiofencarbsulfoxide...54 Ethion... 55, 57, 6, 62, 64, 66, 68 Ethoprophos...58 Etriticonazole...53 F Fenoxycarb...53 Fensulfothion...58 Fensulfothion sulfone...58 Fensulfothion-oxon...58 Fensulfothion-oxon-sulfone...58 Ferimzone...53 Florfenicol...19 Flufenacet...69 Formetanate hydrochloride...53 Fructose...83, 84 Furathiocarb...53 G Ginsenoside Rb1...85 Glucose...83, 84 Glutamine...8, 81 Glycine...8, 81 H Hexachlorobenzene...6, 64 Hexachonzol...68 Histidine...8, 81 I Imazalil... 56, 61, 63, 67, 69 Imidacloprid... 53, 56, 61, 63, 65, 67 Indoxacarb...53 Iprovalicarb...53 Isoleucine...8, 81 Isoxaflutole...53 Isoxsuprine...18 K Kresoxim-methyl... 55, 6, 61, 62, 64, 65, 66 L Lactofen...53 Lactose...83, 84 Leucine...8, 81 Leucomalachite Green...42, 43 Linuron... 56, 61, 63, 65, 67 Lysine...8, 81 [ 86 ]

[ Compound INDEX ] M Mabuterol...18 Malachite Green...42, 43 Malathion...68 Maltose...83, 84 Mapenterol...18 MCYST-LF...51 MCYST-LR...51 MCYST-LW...51 MCYST-RR...51 Melamine... 45, 46, 47, 48, 49 Methamidophos... 54, 56, 61, 63, 65, 67 Methiadathion...68 Methiocarb...41 Methionine...8, 81 Methomyl... 41,56, 61, 63, 65, 67 Methoxyfenozide...53 Mibemectin A3...53 Mibemectin A4...53 N Naproanilide...53 methoate...54, 58 ryzalin...53 xacillin...28 xamyl...41 xycarboxin...53 xydemeton-methyl...53, 58 xydemeton-s-methyl sulfone...58 xytetracycline...34, 35, 36 xytetracydine...28 P Paraquat...7 Patulin...71 Permethrin...6, 62, 64, 66 Penicillin G...27, 28 Perfluorobutane Sulfonate...72 Perfluorooctane Sulfonate...72 Phenmedipham...53 Phenylalanine...8, 81 o-phenylphenol...6, 62, 64, 66 Phenylphenol...57 Phosalone...68 Procymidone...6, 62, 64, 66 Profenophos...68 Proline...8, 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 S Salbutamol...18 Salbutamol-D3...18 Semicarbizide...23, 25 Serine...8, 81 Simeconazole...53 Spiramycin...29 Streptomycin...31 Sucrose...83, 84 Sudan I...77, 78 Sudan II...77, 78 Sudan III...77, 78 Sudan IV...77, 78 Sulfachloropyridazine...28, 32 Sulfadiazine...28, 32 Sulfadimethoxine...28, 32 Sulfamerazine...28, 32 Sulfamethazine...28, 32 Sulfamethizole...32 Sulfamethoxazole...28, 32 Sulfamethoxypyridazine...28, 32 Sulfapyridine...32 Sulfapyridine...28 Sulfathiazole...28, 32 T Tebuconazole... 55, 6, 61, 62, 63, 64, 65, 66, 67, 69 Terbufos...58 Terbufos sulfone...58 Terbufos sulfoxide...58 Terbutaline...18 Tetracycline...28, 34, 35, 36 Theanine...8 Thiabendazole...56, 61, 63, 67 Thiacloprid...53 Thiamethoxam...53 Thiamphenicol...19 Threonine...8, 81 Tolyfluanid... 55, 56, 6, 61, 62, 63, 64, 65, 67 Triabendazole...69 Tricyclazole...68 Tridemorph...53 Trifluralin... 55, 6, 62, 64, 66 Trizaphos...68 Tyrosine...8, 81 V Valine...8, 81 [ 87 ]

Sales ffices Austria and European Export (Central South Eastern Europe, CIS and Middle East) 43 1 877 18 7 Australia 61 2 9933 1777 Belgium 32 2 726 1 Brazil 55 11 4134 3788 Canada 1 8 252 4752 China 86 1 5293 6688 Czech Republic 42 2 617 11384 Denmark 45 46 59 88 Finland 358 9 5659 6288 France 33 1 3 48 72 Germany 49 6196 4 6 Hong Kong 852 2964 18 Hungary 36 1 35 586 Norway 47 6 384 65 Poland 48 22 833 44 Puerto Rico 1 787 747 8445 Russia/CIS 7 495 727 449/ 29 9737 Singapore 65 6593 71 Spain 34 93 6 93 Sweden 46 8 555 115 Switzerland 41 56 676 7 Taiwan 886 2 251 9928 United Kingdom 44 28 238 61 All other countries: Waters Corporation U.S.A. 1 58 478 2 1 8 252 4752 www.waters.com India 91 8 2837 19 Ireland 353 1 448 15 Italy 39 2 265 983 Japan 81 3 3471 7191 Korea 82 2 63 48 Mexico 52 55 52 186 The Netherlands 31 76 58 72 www.waters.com/ft The quality management system of Waters manufacturing facilities in Taunton, Massachusetts and Wexford, Ireland complies with the International Standard IS 91:28 Quality Management and Quality Assurance Standards. Waters quality management system is periodically audited by the registering body to ensure compliance. 211 Waters Corporation. Waters, The Science of What s Possible, Accell, ACQUITY, ACQUITY UPLC, aflachra HPLC, Alliance, Atlantis, Nova-Pak, asis, Quattro micro, Quattro Premier, DisQuE, PoraPak, UPLC, AflaTest, Quattro Ultima Pt, Sentry, Sep-Pak, SunFire, Symmetry, Symmetry3, XBridge, XTerra, INSIGHT, Empower, AccQ-Tag, VanGuard, Guard-Pak, MassLynx, and Xevo are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. 722565EN June 211 VW-PDF [ 88 ]