APPLICATION NOTEBOOK FOR SOLID PHASE EXTRACTION

APPLICATION NOTEBOOK FOR SOLID PHASE EXTRACTION 215

INTRODUCTION AFFINISEP offers a comprehensive range of sorbents for the challenging fields of sample preparation, sample clean-up and extraction, from conventional to more sophisticated sorbents. So, for very specific and challenging applications, AFFINISEP has developed AFFINIMIP SPE products, SPE cartridges based on Molecularly Imprinted Polymers (MIP) which require ready-to-use protocols. AFFINISEP has also developed AttractSPE TM products, SPE cartridges based on classical polymeric sorbents. In addition, our SPE products experience is continuously enriched with customer interactions and an endless analytical development for new applications. This experience is communicated through Application notes (available on website and via newsletters). For your convenience, this application notebook will be permanently updated with new protocols and results. Please regularly visit our website www.affinisep.com for the latest version of the Application Notebook. Moreover, we have as well evaluated our products through interlaboratories proficiency testing such as FAPAS and BIPEA. For more information, please contact us at contact@affinisep.com. This Application notebook will be an essential tool to address your technical issues. TECHNICAL SUPPORT AFFINISEP has fully integrated technologies platform with specialized teams in organic chemistry, polymer chemistry, analytical and bioanalytical chemistry who are at your disposal to help you in your challenges. At AFFINISEP, we are committed to providing the best technical support possible. Our Technical Support Group is a team of highly qualified M.Sc. and PhD Chemists, who are at your disposal to resolve your problem and to answer to your queries. For technical inquiries, feel free to contact us either by email: tech.support@affinisep.com We are also very thankful to customer s feedback about our products, protocols and customer services by email to: contact@affinisep.com

TABLE OF CONTENTS Analysis of Mycotoxins 6 Simultaneous determination of Aflatoxins, Fumonisins, Ochratoxin A, HT-2, T-2, Zearalenone and Deoxynivalenol in Wheat 7 Fumonisins B1 + B2 and Zearalenone in Maize-based baby food 8 Fumonisins B1 + B2 and Zearalenone in Maize Flour 9 Patulin in Baby food apple juice 1 Patulin in Apple juice 11 Patulin in Baby food apple puree 12 Patulin in Apple puree 13 Patulin in Apple puree 14 Patulin in Apple Fruit puree 15 Patulin in WHOLE apple 16 Patulin in Cider 17 Patulin in Alcohol Pommeau and Liquor 18 Patulin in Tomato Ketchup and Tomato Powder 19 Patulin in Blueberry juice 2 Patulin in Thick Juice and Concentrate Juice 21 Patulin in Dried apple 22 Ochratoxin A in Cereals 23 Ochratoxin A in Paprika 24 Ochratoxin A in Red and White wine 25 Zearalenone in Maize and Rice 26 Zearalenone in Cereal-based Baby food 27 Zearalenone in Edible corn oil 28 Zearalenone in Meat 29 Deoxynivalenol in cereals for food (Water extraction) 3 Deoxynivalenol in babyfood cereals 31 Deoxynivalenol in cereals for animal feeds 32 Deoxynivalenol in Meat 33 Deoxynivalenol, 3-AcetylDON and 15-AcetylDON in cereals (Hydro-organic extraction) 34 AFFINIMIP SPE VS IMMUNOAFFINITY COMPARATIVE STUDY 35 Analysis of Endrocrine disrupting compounds (EDC) 37 Determination of estrogens in plasma 38 Protocol comparative - AFFINIMIP SPE Estrogens vs usual protocol 39 www.affinisep.com 3

TABLE OF CONTENTS Bisphenol A in Liquid infant formula 4 Bisphenol A in powdered infant formula 41 PROTOCOL COMPARATIVE WITH A COMPETITOR POWDERED INFANT FORMULA 42 Bisphenol A in canned food (liquid form) 43 Bisphenol A in canned food (Vegetable) 44 Bisphenol A in Beer 45 Bisphenol A in White/Red wines 46 Bisphenol A in Cola drinks 47 Bisphenol A and BADGE in Milk 48 Total Bisphenol A in Human Urine 49 Parabens in cosmetic product 5 Analysis of Antibiotics and drug residues 51 Chloramphenicol in Honey 52 Chloramphenicol in Bovine urine 53 Chloramphenicol in Shrimp 54 Amphetamines in Human urine 55 Amphetamines in Human serum 56 Tetracyclines (Oxytetracycline, Chlortetracycline, Tetracycline), theirs epimers and Doxycycline in Milk and Salmon 57 Tetracyclines and theirs epimers in Meat 58 Antibacterial aminoglycosides on Milk or Meat 59 Multiresidue analysis of antibiotics in Kidney 6 Multiresidue analysis of NSAID in muscle tissue 6 Penicillin based antibacterials on Muscles 61 Erythromycin and Clindamycin 61 Quinoxaline-2-Carboxylic acid and 3-methyl quinoxaline-2- carboxylic acid in Muscle, Liver and Kidneys 62 Multi-class method of antibiotics in Milk 63 Vancomycin in Fish 63 Glucocorticoids 64 Sulfonamides in Milk 64 Multi-class method of antibiotics in Distiller Grains 65 Praziquantel and Tiamulin 65 Cephalexin in Fish 66 Valnemulin and Tiamulin in Fish 66 Pharmaceuticals and Personal care products (Group 1 compounds) of EPA Method 1694 67 Miscellaneous drugs in Waste water 67 www.affinisep.com 4

TABLE OF CONTENTS Analysis of Pesticides 68 Aminopyralid, Clopyralid and Picloram in Compost and Water 69 Glyphosate and AMPA in water 7 16 pesticides from Groundwater 71 Pesticides from Surface Water 71 Acetamide Herbicides in Drinking Water 72 Herbicides in Drinking Water 72 Analysis of other residues and miscellaneous 73 Methanephrines in Plasma comparison with WCX cartridges 74 Guaïacol in Wine 75 Carnosic acid in meat 76 Artificial sweeteners in Water 77 Cocaine and main metabolides in Waste Water 77 Melamine in Milk 78 Cyanuric acid in Milk 78 Melamine in Food 79 Polycyclic Aromatic Hydrocarbons from Drinking Water 79 Product list 8 AFFINIMIP SPE PRODUCT LIST FOR MYCOTOXIN ANALYSES 81 AFFINIMIP SPE PRODUCT LIST (MISCELLANEOUS) 82 AttractSPE TM PRODUCT LIST 83 MYCOTOXIN STANDARDS & SPE ACCESSORIES PRODUCT LIST 84 LIST OF PUBLICATIONS AND POSTERS 85 ABOUT AFFINISEP - ORDERING INFORMATION 88 www.affinisep.com 5

Analysis of MYCOTOXINS www.affinisep.com 6 6

AFFINIMIP SPE Multimyco LCMSMS SIMULTANEOUS DETERMINATION OF MULTIMYCOTOXINS IN WHEAT Aflatoxin B1, Zearalenone, Ochratoxin A, HT-2, T-2, Fumonisin B1, Deoxynivalenol 25g of ground wheat were extracted with 1mL of Acetonitrile/Water (5/5, v/v/v) for 2 min using a blender. The extract was filtered through a folded filter paper and 2mL of the filtrate were diluted with 18mL of water. Then, this solution was filtered through a filter paper. Cleanup with a 6mL/1mg AFFINIMIP SPE Multimyco LCMSMS cartridge 3mL Acetonitrile 3mL water 6mL of loading solution Washing of interferences (W1) 6mL Water/Acetic acid 1% 3mL Water/Acetonitrile (95/5 v/v) Drying by applying vacuum 3 minutes 3mL Methanol/Ethyl Acetate/Formic acid (48.5/48.5/3, v/v/v) The elution fraction was then evaporated and dissolved in Acetontrile /Water with.1% Formic acid (15/85 v/v) before HPLC analysis. Recovery yield Recovery of multimycotoxins extracted from wheat and analyzed after AFFINIMIP SPE Multimyco LCMSMS cleanup Compound WHEAT C µg/kg Mean µg/kg R% Aflatoxin B1 2 1.6 85 Fumonisin B1 1 937 94 HT-2 1 119 119 T-2 5 56.5 113 Zearalenone 5 54 18 Deoxynivalenol 125 125 82 Ochratoxin A 3 2.6 88 UFLC Method Column: Phenomenex Kinetix XB-C18 Detection: LC-MS/MS Injection volume: 2µL. Ordering information: FS118-2 for 25 cartridges, 3mL FS118-3 for 5 cartridges, 3mL FS118-4 for 1 cartridges, 3mL FS118-2B for 25 cartridges, 6mL FS118-3B for 5 cartridges, 6mL www.affinisep.com 7

AFFINIMIP SPE FumoZON DETERMINATION OF FUMONISINS B1 / B2 AND ZEARALENONE IN MAIZE-BASED BABY FOOD Regulations for maize-based baby food: Zearalenone Europe (EC 1126/27) : 2µg/Kg Fumonisins Europe (EC 1126/27) : 2µg/Kg Cleanup with a 3mL/1mg AFFINIMIP SPE FumoZON cartridge 25g of ground samples were extracted with 1 ml of Acetonitrile/Methanol/deionized Water (25/25/5, v/v/v) for 3 min using a blender. The extract was filtered through a folded filter paper and 1 ml of the filtrate were diluted with 1 ml of deionized water. Then, this solution was filtered through a filter paper. This solution was used as the loading solution. 2mL Acetonitrile 2mL Water 6mL of loading solution Washing of interferences 6mL 6/4 Water/ACN 2mL Methanol 2% Acetic Acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. HPLC Method with MS detection Column: Hypersil Gold C18 column 5mm x 2.1mm Mobile phase ZON AND FB1: Water-Formic Acid.1%/ACN (73/27) Mobile phase FB2: Water-Formic Acid.1%/ACN (65/35) Flow rate:.2ml/min MS detection: m/z 722 for Fumonisin B1 (ESI + ) m/z 76 for Fumonisin B2 (ESI + ) m/z 317 for Zearalenone (ESI - ) Injection volume: 2µL. Recovery of Zearalenone, Fumonisins B1 and B2 in maize-based baby food after AFFINIMIP SPE FumoZON clean-up and relative standard deviation calculated from results generated under reproducibility conditions. Sample C µg/kg Mean µg/kg 3mL-1mg sorbent FS19-2 for 25 cartridges FS19-3 for 5 cartridges Recoveries % Zearalenone 2 16.9 84.4 Fumonisin B1 2 168.6 84.3 Fumonisin B2 2 185.6 92.8 ION SUPPRESSION EVALUATION % RSD R 1.6 (n=4) 1.4 (n=3) 1.9 (n=3) Ion suppression phenomenon can induce an erroneous quantification. To evaluate the ionsuppression, blank maize-based baby food samples were cleaned up with AFFINIMIP SPE FumoZON. The SPE extracts were spiked with a mixture of Fumonisin B1 and Zearalenone at 2 different concentrations. The standard calibration curves were compared to the matrix SPE extracts. The use of AFFINIMIP SPE FumoZON strongly reduces ion-suppression phenomena with a maximum of 15% observed for Fumonisins. Ion suppression percentage obtained in Maizebased baby food (tested twice). Analyte C µg/kg Ion suppression % Zearalenone 1 1% and 5% Zearalenone 5 % and 5% Fumonisin B1 1 8% and 11% Fumonisin B1 5 12% and 14% www.affinisep.com 8

Intensity Intensity Intensity 3.52 AFFINIMIP SPE FumoZON DETERMINATION OF FUMONISINS B1 / B2 AND ZEARALENONE IN MAIZE FLOUR Regulations for cereal flour: Zearalenone Europe (EC 1126/27) : 75µg/Kg Fumonisins Europe (EC 1126/27) : 1µg/Kg for maize flour USA: FDA advisory 2µg/Kg RT:.8 RT:.8-2. 22 21 2 19 18 17 16 15 14 13 12 11 1 9 8 7 6 5 4 3 2 1 RT:.8-3. 4 35 Fumonisin B2 2 4 6 8 1 12 14 16 18 2 Time (min) 6.19 3 25 2 15 1 Fumonisin B1 5 12 11 1 Cleanup with a 3mL/1mg AFFINIMIP SPE FumoZON cartridge 25g of ground samples were extracted with 1 ml of Acetonitrile/Methanol/deionized Water (25/25/5, v/v/v) for 3 min using a blender. The extract was filtered through a folded filter paper and 1 ml of the filtrate were diluted with 1 ml of deionized water. Then, this solution was filtered through a filter paper. This solution was used as the loading solution. 2mL Acetonitrile 2mL Water 6mL of loading solution Washing of interferences 6mL 6/4 Water/ACN 2mL Methanol 2% Acetic Acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. HPLC Method with MS detection Column: Hypersil Gold C18 column 5mm x 2.1mm Mobile phase ZON AND FB1: Water-Formic Acid.1%/ACN (73/27) Mobile phase FB2: Water-Formic Acid.1%/ACN (65/35) Flow rate:.2ml/min MS detection: m/z 722 for Fumonisin B1 (ESI + ) m/z 76 for Fumonisin B2 (ESI + ) m/z 317 for Zearalenone (ESI - ) Injection volume: 2µL. 9 8 7 6 5 4 3 2 1 Chromatograms obtained after AFFINIMIP SPE FumoZON Clean-up of a maize flour spiked at 38µg/kg with Zearalenone, 248µk/kg with Fumonisin B1 and 63µg/kg with Fumonisin B2. Recovery of Zearalenone, Fumonisins B1 and B2 in maize flour after AFFINIMIP SPE FumoZON clean-up and relative standard deviation calculated from results generated under reproducibility conditions Sample C µg/kg Mean µg/kg Recoveries % Zearalenone 38 39.2 13.2 Fumonisin B1 Fumonisin B1 Fumonisin B2 248 22.2 83.1 4 41. 1.2 63 684.6 18.7 3mL-1mg sorbent FS19-2 for 25 cartridges FS19-3 for 5 cartridges Zearalenone 26.95 5 1 15 2 25 3 Time (min) % RSD R 8.5 (n=8) 1.3 (n=8) - (n=2) 11.5 (n=3) www.affinisep.com 9

4 35 3 25 2 15 1 5-5 -1-15 -2-25 9 1 11 12 13 14 15 16 17 18 19 2 Minutes 4 35 3 25 2 15 1 5-5 -1-15 -2-25 AFFINIMIP SPE Patulin DETERMINATION OF PATULIN IN BABY FOOD APPLE JUICE Regulations for apple juice: Europe (EC 1881/26) : 5µg/Kg USA (FDA CPG Sec.51.15) : 5µg/Kg Regulations for apple juice for infants and young children: Europe (EC 1881/26) : 1µg/Kg Patulin solution: 2.5mL apple juice and 2.5mL of water-2% acetic acid are mixed. Cleanup with a 3mL/1mg AFFINIMIP SPE Patulin cartridge 2mL Acetonitrile 1mL water 4mL of loading solution Washing of interferences (W1) 1mL NaHCO 3 2mL Water Drying by applying vacuum 1 seconds Washing of interferences (W2) 1mL Diethyl Ether 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of an apple juice spiked at 1µk/kg with Patulin (Green and blue) or not spiked (Red) Recovery of Patulin (n=9) at a contamination level of 1µg/kg in apple Juice after AFFINIMIP SPE Patulin Clean-up. Recoveries % (n=9) % RSD R 97.9 11 HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: Deionized water/acn (95/5, v/v) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. 3mL-1mg sorbent for apple juice and puree FS12-2 for 25 cartridges FS12-3 for 5 cartridges 6mL-2mg sorbent for a apple-based products, fruit juice and concentrate FS12-2B -2mg for 25 cartridges FS12-3B -2mg for 5 cartridges www.affinisep.com 1

2 18 16 14 12 1 8 6 4 2 7 6 5 4 3 2 1-1 -2 UV 6-276nm Brut carrefour.dat UV6-276nm C1 C5 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 Minutes 9 1 11 12 13 14 15 16 17 18 19 Minutes 7 6 5 4 3 2 1 2 18 16 14 12 1 8 6 4 2-1 -2 DETERMINATION OF PATULIN IN APPLE JUICE AFFINIMIP SPE Patulin Regulations for apple juice: Europe (EC 1881/26) : 5µg/Kg USA (FDA CPG Sec.51.15) : 5µg/Kg Regulations for apple juice for infants and young children: Europe (EC 1881/26) : 1µg/Kg Before clean-up solution: 2.5mL apple juice and 2.5mL of water-2% acetic acid are mixed. Clean-up with a 3mL/1mg AFFINIMIP SPE Patulin cartridge 2mL Acetonitrile 1mL water 4mL of loading solution Washing of interferences (W1) 1mL NaHCO 3 in Water 2mL Water Drying by applying vacuum 1 seconds Washing of interferences (W2) 1mL Diethyl Ether 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. Patulin Chromatograms of apple juice containing 25µg/kg of Patulin before (Red) and after (Blue) AFFINIMIP SPE Patulin Clean-up HMF Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of an apple juice spiked at 4µg/kg (tested twice, red) or at 1µg/kg (tested twice, blue) with Patulin or not spiked (orange) Recovery of Patulin in apple juice after AFFINIMIP SPE Patulin Clean-up and relative standard deviation calculated from results generated under reproducibility conditions. C of Patulin (ng/ml) Recoveries % % RSD R 1 97.9 11 (n=9) 4 9.6 11 (n=41) HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: Deionized water/acn (95/5, v/v) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. 3mL-1mg sorbent for apple juice and puree FS12-2 for 25 cartridges FS12-3 for 5 cartridges 6mL-2mg sorbent for a apple-based products, fruit juice and concentrate FS12-2B -2mg for 25 cartridges FS12-3B -2mg for 5 cartridges www.affinisep.com 11

AFFINIMIP SPE Patulin DETERMINATION OF PATULIN IN BABY FOOD APPLE PUREE Regulations for apple puree: Europe (EC 1881/26) : 25µg/Kg Regulations for apple puree for infants and young children: Europe (EC 1881/26) : 1µg/Kg Patulin 1g of apple puree, 15µL of a pectinase enzyme solution and 1mL water are mixed. Leave solution at room temperature overnight or for 2h at 4 C. Centrifuge at 45g for 5min and then filter the solution with a.2µm filter. This solution is used as the loading solution. Cleanup with a 3mL/1mg AFFINIMIP SPE Patulin cartridge 2mL Acetonitrile 1mL Water 5mL of loading solution Washing of interferences (W1) 4mL Water -1%Acetic acid 1mL NaHCO 3 1% solution 3mL Water Drying by applying vacuum 1 seconds Washing of interferences (W2) 5µL Diethyl Ether 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of different apple puree. In the lower part, clean-up of an apple puree from a well-known brand spiked at 25µg/kg (orange), 1µk/kg with Patulin (pink, tested twice) or not spiked (red). In the top part, clean-up of an apple puree second well known brand spiked at 25µg/kg (green), 1µk/kg with Patulin (blue, tested twice) or not spiked (turquoise). Recovery and repeatability of Patulin (n=4) at a contamination level of 1µg/kg in apple puree after AFFINIMIP SPE Patulin Clean-up. Recoveries % (n=4) % RSD R 81.2 2.1 HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: gradient Time (min) % water % ACN 98 2 2 98 2 21 5 5 25 5 5 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. 3mL-1mg sorbent FS12-2 for 25 cartridges FS12-3 for 5 cartridges FS12-2K for a kit of 25 cartridges + 5mL Pectinase FS12-3K for a kit of 5 cartridges + 5mL Pectinase REA-1-5mL for 5mL Pectinase solution www.affinisep.com 12

14 12 1 8 6 4 2 12 1 8 6 4 2 UV 6-276nm C7 UV 6-276nm C4-E-bis C21 UV 6-276nm C1 UV 6-276nm C6 UV 6-276nm C15 UV 6-276nm C11 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 Minutes 14 12 1 8 6 4 2 12 1 8 6 4 2 DETERMINATION OF PATULIN IN APPLE PUREE AFFINIMIP SPE Patulin Regulations for apple puree: Europe (EC 1881/26) : 25µg/Kg Regulations for apple juice for infants and young children: Europe (EC 1881/26) : 1µg/Kg Patulin 1g of apple puree, 15µL of a pectinase enzyme solution and 1mL water are mixed. Leave solution at room temperature overnight or for 2h at 4 C. Centrifuge at 45g for 5min and then filter the solution with a.2µm filter. This solution is used as the loading solution. Cleanup with a 3mL/1mg AFFINIMIP SPE Patulin cartridge 2mL Acetonitrile 1mL Water 5mL of loading solution Washing of interferences (W1) 4mL Water -1%Acetic acid 1mL NaHCO 3 1% solution 3mL Water Drying by applying vacuum 1 seconds Washing of interferences (W2) 5µL Diethyl Ether 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: gradient Time (min) % water % ACN 98 2 2 98 2 21 5 5 25 5 5 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 Minutes Chromatograms of apple puree containing 4µg/kg or 8µg/kg of Patulin before (Red) and after (Blue) AFFINIMIP SPE Patulin Clean-up Patulin Chromatograms of apple puree containing µg/kg (blue) or 2µg/kg (tested twice, green and red)) of Patulin after AFFINIMIP SPE Patulin Clean-up Recovery and repeatability of Patulin (n=3) at a contamination level of 2µg/kg in apple puree after AFFINIMIP SPE Patulin Clean-up. C of Patulin (µg/kg) Recoveries % (n=3) % RSDr 2 84 4.5 3mL-1mg sorbent FS12-2 for 25 cartridges FS12-3 for 5 cartridges FS12-2K for a kit of 25 cartridges + 5mL Pectinase FS12-3K for a kit of 5 cartridges + 5mL Pectinase REA-1-5mL for 5mL Pectinase solution www.affinisep.com 13

8 7 6 5 4 3 2 1 4 5 6 7 8 9 1 11 12 13 14 15 8 7 6 5 4 3 2 1 DETERMINATION OF PATULIN IN APPLE PUREE AFFINIMIP SPE Patulin A format tailored for the larger liquid volume required for apple puree protocol Regulations for apple puree: Europe (EC 1881/26) : 25µg/Kg Regulations for apple juice for infants and young children: Europe (EC 1881/26) : 1µg/Kg 1g of apple puree, 15µL of a pectinase enzyme solution and 1mL water are mixed. Leave solution at room temperature overnight or for 2h at 4 C. Centrifuge at 45g for 5min and then filter the solution with a.2µm filter. This solution is used as the loading solution. Cleanup with a 6mL/2mg AFFINIMIP SPE Patulin cartridge Patulin Minutes Chromatograms of apple puree spiked with 2µg/kg of Patulin (Red) and not spiked (blue) after AFFINIMIP SPE Patulin Clean-up 2mL Acetonitrile 1mL Water 5mL of loading solution Washing of interferences (W1) 4mL Water -1%Acetic acid 1mL NaHCO 3 1% solution 3mL Water Drying by applying vacuum 1 seconds Washing of interferences (W2) 5µL Diethyl Ether 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: gradient Time (min) % water % ACN 98 2 2 98 2 21 5 5 25 5 5 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. Recovery and repeatability of Patulin (n=6) at a contamination level of 1µg/kg in apple puree after AFFINIMIP SPE Patulin Clean-up. C of Patulin (µg/kg) Recoveries % % RSDr 1 (n=6) 9 9 2 (n=3) 92 11 6mL - 2mg sorbent FS12-2B-2mg for 25 cartridges FS12-3B-2mg for 5 cartridges FS12-2KB-2mg for a kit of 25 cartridges + 5mL Pectinase FS12-3KB-2mg for a kit of 5 cartridges + 5mL Pectinase REA-1-5mL for 5mL Pectinase solution www.affinisep.com 14

12 1 8 6 4 2 25 225 2 175 15 125 1 75 5 25-25 -5-75 UV 6-276nm C4-E-bis C21 UV 6-276nm C6 UV 6-276nm C11 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 Minutes 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 Minutes 12 1 8 6 4 2 25 225 2 175 15 125 1 75 5 25-25 -5-75 AFFINIMIP SPE Patulin DETERMINATION OF PATULIN IN APPLE FRUIT PUREE Regulations for apple puree: Europe (EC 1881/26) : 25µg/Kg Regulations for apple puree for infants and young children: Europe (EC 1881/26) : 1µg/Kg Patulin 1g of apple puree, 15µL of a pectinase enzyme solution and 1mL water are mixed. Leave solution at room temperature overnight or for 2h at 4 C. Centrifuge at 45g for 5min and then filter the solution with a.2µm filter. This solution is used as the loading solution. Cleanup with a 3mL/1mg AFFINIMIP SPE Patulin cartridge 2mL Acetonitrile 1mL Water 5mL of loading solution Washing of interferences (W1) 4mL Water -1%Acetic acid 1mL NaHCO 3 1% solution 3mL Water Drying by applying vacuum 1 seconds Washing of interferences (W2) 5µL Diethyl Ether 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: gradient Time (min) % water % ACN 98 2 2 98 2 21 5 5 25 5 5 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. Chromatograms of apple puree containing µg/kg (blue) or 2µg/kg (tested twice, green and red) of Patulin after AFFINIMIP SPE Patulin Clean-up. Apple-pear puree with Patulin (25µg/kg) Apple puree with Patulin (4µg/kg) Apple-strawberry puree with Patulin (4µg/kg) Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of different purees. Recovery and reproducibility of Patulin with different levels of contamination for all tested apple-fruit puree after AFFINIMIP SPE Patulin Clean-up. C of Patulin (µg/kg) Patulin Recoverie s % % RSD R 1 (n=9) 77.4 8.1 25 (n=8) 9.9 11.4 4 (n=6) 86. 11.9 3mL-1mg sorbent FS12-2 for 25 cartridges FS12-3 for 5 cartridges FS12-2K for a kit of 25 cartridges + 5mL Pectinase FS12-3K for a kit of 5 cartridges + 5mL Pectinase REA-1-5mL for 5mL Pectinase solution www.affinisep.com 15

DETERMINATION OF PATULIN IN WHOLE APPLE AFFINIMIP SPE Patulin Regulations for solid apple products: Europe (EC 1881/26) : 25µg/Kg Preparation with microwave Whole apple is cut into pieces and put in a microwave for 9s before crushing the pieces. 15g sample and 7.5mL water are mixed with 15µL pectinase solution and put overnight at room temperature or for 2h at 4 C before a filtration with filter 4-7µm to obtain the loading solution. Preparation with a blender Whole apple is cut into pieces, put in a blender with Water (2:1 Apple: Water) and mix for 1min. 15g sample and 3µL pectinase solution are put overnight at room temperature or for 2h at 4 C before a filtration with filter 4-7µm to obtain the loading solution. Cleanup with a 3mL/1mg AFFINIMIP SPE Patulin cartridge 2mL Acetonitrile 1mL Water 3mL of loading solution Washing of interferences (W1) 3mL Water-2% Acetic Acid Drying by applying vacuum 1 seconds Washing of interferences (W2) 25µL Diethyl Ether Drying by applying vacuum 1 seconds 1mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN 98 2 2 98 2 21 5 5 25 5 5 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. 9 8 7 6 5 4 3 2 1-1 -2-3 BLENDER PREPARATION MICROWAVE PREPARATION 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 Minutes Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of whole apple spiked at 4µg/kg with Patulin (dark colors) or not spiked (light colors). Recovery yields obtained after AFFINIMIP SPE Patulin Clean-up of spiked whole apple with 4µg/kg of Patulin. Whole apples are prepared according to 2 different methods Whole apple prepared with blender Patulin Whole apple prepared with microwave 96 96 95 88 3mL-1mg sorbent FS12-2 for 25 cartridges FS12-3 for 5 cartridges FS12-2K for a kit of 25 cartridges + 5mL Pectinase FS12-3K for a kit of 5 cartridges + 5mL Pectinase REA-1-5mL for 5mL Pectinase solution 9 8 7 6 5 4 3 2 1-1 -2-3 www.affinisep.com 16

DETERMINATION OF PATULIN IN CIDER AFFINIMIP SPE Patulin Regulations for cider: Europe (EC 1881/26) : 5µg/Kg Patulin The cider is degassed by sonicating sample for 1 hour. Then the degas cider is diluted by 2 with water containing 2% of acetic acid. This solution is mixed and used as the loading solution. Cleanup with a 3mL/1mg AFFINIMIP SPE Patulin cartridge 2mL Acetonitrile 1mL Water 4mL of loading solution Washing of interferences (W1) 1mL NaHCO 3 1% in Water 2mL Water Drying by applying vacuum 1 seconds Washing of interferences (W2) 5µL Diethyl Ether 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of a cider spiked at 4µg/kg (tested twice, pink) or at 1µg/kg (tested twice, blue) with Patulin or not spiked (red). Recovery of Patulin at a contamination level of 1µg/kg and 4µg/kg in cider after AFFINIMIP SPE Patulin Clean-up and relative standard deviation calculated from results generated under reproducibility conditions. C of Patulin (ng/ml) Recoveries % % RSD R 1 87.5 (n=2) - 4 8.5 (n=5) 7.5 HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: Deionized water/acn (95/5, v/v) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. 3mL-1mg sorbent FS12-2 for 25 cartridges FS12-3 for 5 cartridges FS12-2K for a kit of 25 cartridges + 5mL Pectinase FS12-3K for a kit of 5 cartridges + 5mL Pectinase REA-1-5mL for 5mL Pectinase solution www.affinisep.com 17

m AFFINIMIP SPE Patulin DETERMINATION OF PATULIN IN ALCOHOL POMMEAU AND LIQUOR Regulations for apple based beverage : Europe (EC 1881/26) : 5µg/Kg 3 3 275 275 Manzella liquor contains 2% alcohol and 2.1% of concentrated apple juice. Alcohol Pommeau is a mixture of Calvados and Apple Juice. It contains 17% Alcohol. 25 225 2 175 15 125 1 Patulin 25 225 2 175 15 125 1 To 1mL of Manzella Liquor or Alcohol Pommeau, add 2mL Water to obtain the loading solution. Cleanup with a 3mL/1mg AFFINIMIP SPE Patulin cartridge 2mL Acetonitrile 1mL Water 3mL of loading solution Washing of interferences (W1) 3mL Water (containing 2% Acetic Acid for AA W1 protocol) Drying by applying vacuum 1 seconds Washing of interferences (W2) 25µL Diethyl Ether Drying by applying vacuum 1 seconds 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. 75 5 25 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 Minutes Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of Manzella liquor spiked at 4µg/L with Patulin (dark blue for Water in W1 and red for Water AA in W1) or not spiked (light blue and pink). Washing with Acetic acid is more efficient. Recovery yields obtained for Pommeau and Manzella after AFFINIMIP SPE Patulin Cleanup. W1 with water or Water -2%Acetic acid Water for W1 Water-AA for W1 Pommeau 11 11 9 93 Manzella 12 16 87 9 75 5 25 HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN 98 2 2 98 2 21 5 5 25 5 5 26 98 2 ) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. 3mL-1mg sorbent FS12-2 for 25 cartridges FS12-3 for 5 cartridges FS12-2K for a kit of 25 cartridges + 5mL Pectinase FS12-3K for a kit of 5 cartridges + 5mL Pectinase REA-1-5mL for 5mL Pectinase solution www.affinisep.com 18

11 1 9 8 7 6 5 4 3 2 1 11 1 9 8 7 6 5 4 3 2 1-1 UV 6-276nm B14 UV 6-276nm B15 6 5 4 3 2 1 6 7 8 9 1 11 12 13 14 15 16 17 18 19 4 2 21 22 23 24 25 2 4 6 8 1 12 14 16 18 2 22 24 26 28 UV 6-276nm B16 11 1 UV 6-276nm B17 9 8 7 11 1 9 8 7 6 5 4 3 2 1 UV 6-276nm B14 UV 6-276nm B16 UV 6-276nm B15 UV 6-276nm B17 Minutes 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 4 26 27 28 29 3 Minutes 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 Minutes Minutes 11 1 9 8 7 6 5 3 2 1 11 1 9 8 7 6 5 3 2 1-1 11 1 9 8 7 6 5 4 3 2 1 11 1 9 8 7 6 5 4 3 2 1 AFFINIMIP SPE Patulin DETERMINATION OF PATULIN IN TOMATO KETCHUP AND TOMATO POWDER Preparation OF TOMATO KETCHUP 1g tomato ketchup and 1mL water are mixed with 15µL pectinase solution and left overnight at RT before a filtration with filter.2µm to obtain the loading solution. Preparation OF TOMATO POWDER 1g tomato ketchup and 2mL water are mixed. 1g of the mixture, 1mL water and 15µL pectinase solution are left overnight at RT before a centrifugation at 45rpm during 5 min. Then the mixture is filtered with filter.2µm to obtain the loading solution. Cleanup with a 3mL/1mg AFFINIMIP SPE Patulin cartridge 2mL Acetonitrile 1mL Water 5mL of loading solution from tomato ketchup or 2mL from tomato powder Washing of interferences (W1) 4mL Water-1% Acetic Acid 4mL Water Drying by applying vacuum 1 seconds Washing of interferences (W2) 5µL Diethyl Ether 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. Recovery yield 8% TOMATO KETCHUP Patuli n Patulin Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of TOMATO KETCHUP spiked at 4µg/kg with Patulin (red) or not spiked (light blue). Recovery yield 7% TOMATO POWDER Patulin Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of TOMATO POWDER spiked at 36µg/kg with Patulin (red) or not spiked (light blue). HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN 98 2 2 98 2 21 5 5 25 5 5 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. 3mL-1mg sorbent FS12-2 for 25 cartridges FS12-3 for 5 cartridges FS12-2K for a kit of 25 cartridges + 5mL Pectinase FS12-3K for a kit of 5 cartridges + 5mL Pectinase REA-1-5mL for 5mL Pectinase solution www.affinisep.com 19

m A U AFFINIMIP SPE Patulin DETERMINATION OF PATULIN IN BLUEBERRY JUICE 5mL Blueberry juice is diluted with 5mL water containing 2% of acetic acid to obtain the loading solution. 7 6 5 Recovery yields: 9 and 96% 7 6 5 Cleanup with a 3mL/1mg AFFINIMIP SPE Patulin cartridge 4 3 2 Patulin 4 3 2 2mL Acetonitrile 1mL Water 4mL of loading solution Washing of interferences (W1) 1mL NaHCO 3 1% in Water 2mL Water Drying by applying vacuum 1 seconds Washing of interferences (W2) 5µL Diethyl Ether 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. 1-1 -2 2 4 6 8 1 12 14 16 18 2 22 24 26 28 Minutes Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of Blueberry juice spiked at 4µg/L with Patulin (red) or not spiked (light blue). 1-1 -2 HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN 98 2 2 98 2 21 5 5 25 5 5 26 98 2 ) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. 3mL-1mg sorbent FS12-2 for 25 cartridges FS12-3 for 5 cartridges FS12-2K for a kit of 25 cartridges + 5mL Pectinase FS12-3K for a kit of 5 cartridges + 5mL Pectinase REA-1-5mL for 5mL Pectinase solution www.affinisep.com 2

16 14 12 1 8 6 4 2-2 2 4 6 8 1 12 14 16 18 2 22 24 26 28 Minutes 16 14 12 1 8 6 4 2-2 AFFINIMIP SPE Patulin DETERMINATION OF PATULIN IN CONCENTRATE JUICE AND THICK JUICE Preparation of fruit juice concentrate samples 2.5g of fruit juice concentrate are mixed with 1mL water and 1µL Pectinase. (REA-1-5mL). Leave the solution at room temperature overnight or for 2h at 4 C. Centrifuge at 4g for 1min and collect the supernatant. Dilute the supernatant by 2 with Acetic Acid 2% in water. This solution is used as the loading solution. 45 4 35 3 25 2 15 THICK JUICE Patulin Preparation of thick fruit juice samples 15mL of thick fruit juice are mixed with 12µL Pectinase (REA-1-5mL). Leave the solution at room temperature overnight or for 2h at 4 C. Centrifuge at 4g for 1min and collect the supernatant. Dilute the supernatant by 2 with acetic acid 2% in water. This solution is used as the loading solution. Cleanup with a 6mL/2mg AFFINIMIP SPE Patulin cartridge 4mL Acetonitrile 4mL Water 4 to 6mL of loading solution Washing of interferences (W1) 2mL NaHCO 3 1% in Water 4mL Water Drying by applying vacuum 3 seconds Washing of interferences (W2) 1mL Diethyl Ether 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN 98 2 2 98 2 21 5 5 25 5 5 26 98 2 ) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. 1 5 2 4 6 8 1 12 14 16 18 2 22 24 26 28 Minutes Chromatograms obtained after AFFINIMIP SPE Patulin clean-up of apple mango juice spiked at 2µg/kg (blue) with Patulin or not spiked (red). In green, Patulin solution at 5ng/mL. prepared by dilution of a 1µg/mL Patulin standard solution (REA-PAT-1mL) in mobile phase. CONCENTRATE JUICE Patulin Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of grapefruit juice concentrate spiked at 1µg/kg (blue) with Patulin or not spiked (red). 6mL-2mg sorbent FS12-2B-2mg for 25 cartridges FS12-3B-2mg for 5 cartridges FS12-2KB-2mg for a kit of 25 cartridges + 5mL Pectinase FS12-3KB-2mg for a kit of 5 cartridges + 5mL Pectinase REA-1-5mL for 5mL Pectinase solution www.affinisep.com 21

8 7 6 5 4 3 2 1-1 -2-3 -4-5 UV6-276nm B16 UV6-276nm B17 UV6-276nm B13 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 Minutes 8 7 6 5 4 3 2 1-1 -2-3 -4-5 DETERMINATION OF PATULIN IN DRIED APPLE AFFINIMIP SPE Patulin Regulations for solid apple products: Europe (EC 1881/26) : 25µg/Kg Patulin Recovery >9% 3g of dried apple dices, 3mL of water and 15µL of pectinase are mixed and left at room temperature overnight. Then, they are centrifuged at 45rpm during 5min and filtered with.2µm filter to obtain the loading solution. Cleanup with a 6mL/2mg AFFINIMIP SPE Patulin cartridge Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of dried apple dices spiked at 2µg/kg (red) or at 1µg/kg (blue) with Patulin or not spiked (green). 4mL Acetonitrile 2mL Water 1mL of loading solution Washing of interferences (W1) 5mL Water-2% Acetic Acid 5mL Water Drying by applying vacuum 3 seconds Washing of interferences (W2) 5µL Diethyl Ether 2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 15mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN 98 2 2 98 2 21 5 5 25 5 5 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: 1µL. 6mL - 2mg sorbent for apple-based products and fruit juice, concentrate FS12-2B-2mg for 25 cartridges FS12-3B-2mg for 5 cartridges FS12-2KB-2mg for a kit of 25 cartridges + 5mL Pectinase FS12-3KB-2mg for a kit of 5 cartridges + 5mL Pectinase REA-1-5mL for 5mL Pectinase solution www.affinisep.com 22

Intensity AFFINIMIP SPE Ochratoxin A DETERMINATION OF OCHRATOXIN A IN CEREALS Regulations for umprocessed cereals: Europe (EC 1881/26) : 5µg/Kg Codex Alimentarius Standard: 5µg/Kg for raw wheat 6 4 5g of finely ground wheat are mixed during 1 minute in a blender with 1mL of extraction solvent (6/4 Acetonitrile/deionized Water). The extract is filtered through a filter paper. Then, 5mL of the extract is diluted with 5mL of HCl solution ph=1,.1m. After a filtration through a filter paper, this solution is used as the loading solution. Cleanup with a 3mL/1mg AFFINIMIP SPE Ochratoxin A cartridge 3mL Acetonitrile 3mL Water 4mL of loading solution (eq. 1g wheat) Washing of interferences 6mL 6/4 HCl solution ph 1,.1M/ACN 2mL Methanol 2% Acetic acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. 2 Ochratoxin A, 2, 4, 6, 8, 1, 12, 14, 16, 18, Chromatogram obtained after Cleanup of wheat (spiked at 5µg / kg (pink) or not contaminated (orange)) with AFFINIMIP SPE Ochratoxin A Recoveries of Ochratoxin A after AFFINIMIP SPE Ochratoxin A Clean-up in wheat (n=6) C (µg/kg) Recoveries % % RSD 5 96.3 7.7 HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 2.1mm Mobile phase: water/acetic acid/meoh (39/1/6, v/v) Flow rate:.2ml/min Fluorescence detection: excitation/emission wavelengths: 333 / 46nm Injection volume: 2µL. FS11-2 for 25 cartridges FS11-3 for 5 cartridges www.affinisep.com 23

Intensity AFFINIMIP SPE Ochratoxin A DETERMINATION OF OCHRATOXIN A IN PAPRIKA Regulations for paprika: Europe (EC 594/212) : 3µg/Kg until 31.12.14 then 15µg/Kg 2 1g of paprika are shaken during 3 minutes with 1mL of NaHCO 3 1% in water. The extract is centrifuged for 3 minutes at 4 rpm at room temperature then filtered through a filter paper. 25mL of the extract is diluted with 25mL of HCl solution ph=1,.1m. After a filtration through a filter paper, this solution is used as the loading solution. Cleanup with a 3mL/1mg AFFINIMIP SPE Ochratoxin A cartridge 3mL Acetonitrile 3mL Water 4mL of loading solution (eq. 1g sample) Washing of interferences 6mL 6/4 HCl solution ph 1,.1M/ACN 2mL Methanol 2% Acetic acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. Ochratoxin A, 2, 4, 6, 8, 1, 12, 14, 16, 18, Chromatogram obtained after Cleanup of paprika (spiked at 3µg / kg (pink) or not contaminated (orange)) with AFFINIMIP SPE Ochratoxin A Recoveries of Ochratoxin A after AFFINIMIP SPE Ochratoxin A Clean-up in paprika (n=4). C (µg/kg) Recoveries % % RSD 3 93.3 3.4 HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 2.1mm Mobile phase: water/acetic acid/meoh (39/1/6, v/v) Flow rate:.2ml/min Fluorescence detection: excitation/emission wavelengths: 333 / 46nm Injection volume: 2µL. FS11-2 for 25 cartridges FS11-3 for 5 cartridges www.affinisep.com 24

Intensity Intensity Intensity AFFINIMIP SPE Ochratoxin A DETERMINATION OF OCHRATOXIN A IN RED AND WHITE WINE Regulations for wine: Europe (EC 1881/26) : 2µg/L 1 1mL of wine is diluted with 1mL of HCl solution ph=1,.1m. This solution is used as the loading solution. 5 Ochratoxin A Ochratoxin A, 2, 4, 6, 8, 1, 12, 14, 16, Cleanup with a 3mL/1mg AFFINIMIP SPE Ochratoxin A cartridge 3mL Acetonitrile 3mL Water 2 to 1mL of loading solution (eq. 1 to 5mL sample) Washing of interferences 6mL 6/4 HCl solution ph 1,.1M/ACN 2mL Methanol 2% Acetic acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. Chromatograms obtained after Cleanup of white wine spiked at 2µg/kg (loading with 5mL (blue); loading with 1mL (pink)) and after a loading of 5mL of not contaminated white wine (orange) with AFFINIMIP SPE Ochratoxin A 6 4 2 6 4 2, 2, 4, 6, 8, 1, 12, 14, 16, 18, Ochratoxin A HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 2.1mm Mobile phase: water/acetic acid/meoh (39/1/6, v/v) Flow rate:.2ml/min Fluorescence detection: excitation/emission wavelengths: 333 / 46nm Injection volume: 2µL., 2, 4, 6, 8, 1, 12, 14, 16, Chromatograms obtained after Cleanup of red wine spiked at 2µg / kg (loading with 2mL (orange); loading with 5mL (blue); loading with 1mL (pink)) and after a loading of 2mL of not contaminated red wine (grey) with AFFINIMIP SPE Ochratoxin A Recoveries of Ochratoxin A after AFFINIMIP SPE Ochratoxin A Clean-up in wine (white and red). Matrix White wine (n=1) Red wine (n=4) C (µg/kg) FS11-2 for 25 cartridges FS11-3 for 5 cartridges Recoveries % % RSD 2 91.3 6.2 2 78.8 2.8 www.affinisep.com 25

AFFINIMIP SPE Zearalenone DETERMINATION OF ZEARALENONE IN MAIZE AND RICE Regulations for unprocessed cereal except maize: Europe (EC 1126/27) : 1µg/Kg Regulations for maize: Europe (EC 1126/27) : 35µg/Kg Cleanup with a 3mL/1mg AFFINIMIP SPE Zearalenone cartridge 25g of ground cereal-based samples were extracted with 1 ml of acetonitrile/deionized water (75/25, v/v) for 3 min. The extract was filtered through a folded filter paper and 1 ml of the filtrate were diluted with 1 ml of deionized water. Then, this solution was filtered through a filter paper. This solution was used as the loading solution. 3mL Acetonitrile 3mL Water 12mL of loading solution (eq. 1.5g sample) Washing of interferences (W1) 3mL 58/2/4 Water/Acetic Acid/ACN 2mL Methanol 2% Acetic Acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. ZON Chromatogram obtained after Cleanup of Maize (contamined at 41 µg / kg) with AFFINIMIP SPE Zearalenone ZON Chromatogram obtained after Cleanup of Rice (contamined at 41 µg / kg) with AFFINIMIP SPE Zearalenone. Recoveries of Zearalenone at a contamination level of 41µg / kg after AFFINIMIP SPE Zearalenone. Clean-up in Maize (n=9) Recoveries % % RSD 86 8 HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 4.6mm Mobile phase: water/meoh (4/6, v/v) Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 275 / 45nm Injection volume: 1µL. 3mL-1mg sorbent FS1-2 for 25 cartridges FS1-3 for 5 cartridges www.affinisep.com 26

AFFINIMIP SPE Zearalenone DETERMINATION OF ZEARALENONE IN CEREAL-BASED BABY FOOD Regulations for processed cereal based food for baby food: Europe (EC 1126/27) : 2µg/Kg Cleanup with a 3mL/1mg AFFINIMIP SPE Zearalenone cartridge 25g of ground cereal-based samples were extracted with 1 ml of acetonitrile/deionized water (75/25, v/v) for 3 min. The extract was filtered through a folded filter paper and 1 ml of the filtrate were diluted with 1 ml of deionized water. Then, this solution was filtered through a filter paper. This solution was used as the loading solution. 3mL Acetonitrile 3mL Water 12mL of loading solution (eq. 1.5g sample) Washing of interferences (W1) 3mL 58/2/4 Water/Acetic Acid/ACN 2mL Methanol 2% Acetic Acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 4.6mm Mobile phase: water/meoh (4/6, v/v) Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 275 / 45nm Injection volume: 1µL. Recoveries of Zearalenone at a contamination level of 41µg / kg after AFFINIMIP SPE Zearalenone. Clean-up in Cereal based baby food (n=5) Recoveries % ZON Chromatogram obtained after Cleanup of Cereal-based babyfood (contamined at 41µg / kg) AFFINIMIP SPE Zearalenone (after dilution by 2 of the elution fraction with water). ZON Chromatograms obtained after Cleanup of Cereal-based babyfood (contamined at 1µg/kg (blue) or µg/kg (red)) with AFFINIMIP SPE Zearalenone (after evaporation of the elution fraction and dissolution in 1mL of the mobile phase). 3mL-1mg sorbent FS1-2 for 25 cartridges FS1-3 for 5 cartridges % RSD 8 3 www.affinisep.com 27

mvolt s 45 4 35 3 25 2 15 1 5 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 Minutes AFFINIMIP SPE Zearalenone DETERMINATION OF ZEARALENONE IN EDIBLE CORN OIL Regulations for processed cereal based food for baby food: Europe (EC 1126/27) : 2µg/Kg Zearalenone Cleanup with a 3mL/1mg AFFINIMIP SPE Zearalenone cartridge Corn oil is diluted 1/3 in Diethyl Ether to obtain the loading solution. 3mL Diethyl Ether 3mL of loading solution (eq. 1mL of corn oil) Washing of interferences (W1) 6mL Diethyl ether Drying 3 seconds Washing of interferences (W2) 6mL 58/2/4 Water/Acetic Acid/ACN 4mL Methanol 2% Acetic Acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. Chromatograms of Corn Oil spiked with Zearalenone at 4µg/L (blue) or not spiked (orange) obtained after cleanup by AFFINIMIP SPE Zearalenone. Zearalenone Chromatograms obtained after cleanup by AFFINIMIP SPE Zearalenone of Corn Oil spiked with Zearalenone at 2µg/L (red), 4µg/L (green), 6 µg/l (blue) or not spiked (purple). Recoveries of Zearalenone in Corn Oil at various contamination levels after AFFINIMIP SPE Zearalenone cleanup. HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 4.6mm Mobile phase: water/meoh (4/6, v/v) Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 275 / 45nm Injection volume: 1µL. C (µg/l) Mean C (µg/l) 3mL-1mg sorbent FS1-2 for 25 cartridges FS1-3 for 5 cartridges Recoveries % 2 23 115 4 44 11 6 678 113 www.affinisep.com 28

DETERMINATION OF ZEARALENONE IN MEAT AFFINIMIP SPE Zearalenone 25g of meat are mixed during 2 minutes with 1mL of a solution of deionized water/ acetonitrile (5/5). Then the mixture is filtered through filter paper (4-7µm). This solution was then diluted 1 times with deionized water to obtain the loading solution used for the clean-up protocol Purification with a 3mL/1mg AFFINIMIP SPE ZEARALENONE cartridge 2mL Acetonitrile 2mL Water solution Up to 6mL of loading solution Washing of interferences 6mL 4/6 Acetonitrile/deionized Water Drying 3-5min 2mL 2/98 Acetic Acid/Methanol The elution fraction was then concentrated and diluted to mobile phase before HPLC analysis. Recovery for ZON > 8%* * Tested at 96µg/kg Zearalenone SIM Chromatograms obtained after clean-up of MEAT with AFFINIMIP SPE ZEARALENONE. Blue trace for spiked with 96µg/kg of ZEARALENONE Red trace for the blank sample HPLC Method with LC-MS HPLC Column: Hypersil gold column (5mm x 2.1mm) Mobile phase: 73/27,1 Formic acid in water / Acetonitrile Flow rate:.2ml/min Injection volume: 2µL. 3mL-1mg sorbent FS1-2 for 25 cartridges FS1-3 for 5 cartridges www.affinisep.com 29

AFFINIMIP SPE Deoxynivalenol DETERMINATION OF DEOXYNIVALENOL IN CEREALS FOR FOOD (Water extraction) Regulations for unprocessed corn or durum wheat for food: Europe (EC 1126/27) : 175µg/Kg Deoxynivalenol (DON) with EXTRACTION WITH WATER 2g of cereals were ground in a blender for 1 minute. Then, 8 ml of deionized water were added. This mixture was then ground for 2 additional minutes. After grinding the mixture was placed in a beaker and left stirred under magnetic agitation for 3 minutes. Then the whole mixture was transferred in a centrifuge vial and centrifuged at 25 rpm for 15 minutes. After centrifugation the supernatant was filtered through filter paper. This solution was then diluted 5 times using deionized water. Cleanup with a 6mL/1mg AFFINIMIP SPE Deoxynivalenol cartridge. 2mL Acetonitrile 2mL Water 6mL of loading solution Washing of interferences (W1) 3mL NaHCO 3 1% in water Drying 3 seconds Washing of interferences (W2) 1mL Diethylether 4mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water -,1% HCOOH before HPLC analysis. HPLC Method with MS or UV detection Column: Hypersil Gold C18 column 5mm x 2,1mm Mobile phase: water with,1% formic acid/acn (95/5, v/v) Flow rate:,2ml/min MS detection: m/z 265 (ESI - ) UV detection: 22nm Injection volume: 2µL. UV chromatograms obtained after WATER extraction of DON from corn and clean-up with AFFINIMIP SPE Deoxynivalenol : black, red and green spiked with DON at 8µg/kg dark yellow not spiked blue, a standard solution of DON at 2ng/mL is prepared by dilution of a 1µg/mL Deoxynivalenol standard solution (reference : REA-DON-1mL) in mobile phase Recovery of Deoxynivalenol after AFFINIMIP SPE Deoxynivalenol clean-up and relative standard deviation (repeatability conditions). Matrix Corn (8µg/kg) Corn (8µg/kg) Wheat (n=3) (6µg/kg) Detec tion Mean µg/kg UV 623.4 78. MS 642.7 8.3 MS 54. 9. 6mL-1mg sorbent for (baby)food FS117-2B for 25 cartridges FS117-3B for 5 cartridges 6mL-2mg sorbent for feed FS117-2B-2mg for 25 cartridges FS117-3B-2mg for 5 cartridges R% %RSDr 1.4 (n=6) 3.4 (n=6) 9.8 (n=3) www.affinisep.com 3

AFFINIMIP SPE Deoxynivalenol DETERMINATION OF DEOXYNIVALENOL IN BABYFOOD CEREALS Regulations for cereal based food for baby food: Europe (EC 1126/27) : 2µg/Kg Deoxynivalenol (DON) 15 ml of deionized water were added to 2g of cereals - based babyfood. This mixture was then placed in a beaker and left stirring under magnetic agitation for 3 minutes. Then, the whole mixture was centrifuged at 25 g for 15 minutes. After centrifugation, the supernatant was filtered through filter paper. Cleanup with a 6mL/1mg AFFINIMIP SPE Deoxynivalenol cartridge. 2mL Acetonitrile 2mL Water 6mL of loading solution Washing of interferences (W1) 3mL NaHCO 3 1% in water Drying 3 seconds Washing of interferences (W2) 1mL Diethylether 4mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water -,1% HCOOH before HPLC analysis. MS chromatograms obtained after water extraction of Deoxynivalenol from cereals - based babyfoods and clean-up with AFFINIMIP SPE Deoxynivalenol: black, red and green spiked with Deoxynivalenol at 15µg/kg dark yellow not spiked blue, a standard solution of Deoxynivalenol at 2ng/mL is prepared by dilution of a 1µg/mL Deoxynivalenol standard solution (reference : REA-DON-1mL) in mobile phase Recovery of Deoxynivalenol after AFFINIMIP SPE Deoxynivalenol clean-up and relative standard deviation calculated from results generated under reproducibility conditions. Matrix Babyfoo d (n=3) C µg/kg Mean µg/kg R% %RSD R 15 136.5 91.4 HPLC Method with MS detection Column: Hypersil Gold C18 column 5mm x 2,1mm Mobile phase: water with,1% formic acid/acn (95/5, v/v) Flow rate:,2ml/min MS detection: m/z 265 (ESI - ) Injection volume: 2µL. 6mL-1mg sorbent for (baby)food FS117-2B for 25 cartridges FS117-3B for 5 cartridges 6mL-2mg sorbent for feed FS117-2B-2mg for 25 cartridges FS117-3B-2mg for 5 cartridges www.affinisep.com 31

AFFINIMIP SPE Deoxynivalenol DETERMINATION OF DEOXYNIVALENOL IN CEREALS FOR ANIMAL FEED Regulations for DON in animal feed: Europe (EC 576/26) : 8mg/Kg for cereals and cereals products 12mg/Kg for maize by-products Deoxynivalenol (DON) with EXTRACTION WITH WATER 2g of animal feed were ground in a blender for 1 minute. Then, 8 ml of deionized water were added. This mixture was then ground for 2 additional minutes. After grinding the mixture was placed in a beaker and left stirred under magnetic agitation for 3 minutes. Then, the whole mixture was centrifuged at 25 g for 15 minutes. After centrifugation the supernatant was filtered through filter paper. This solution was then diluted 5 times using deionized water. Cleanup with a 6mL/2mg AFFINIMIP SPE Deoxynivalenol cartridge. 2mL Acetonitrile 2mL Water 2mL of loading solution Washing of interferences (W1) 3mL NaHCO 3 1% in water Drying 3 seconds Washing of interferences (W2) 1mL Diethylether 4mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water -,1% HCOOH before HPLC analysis. HPLC Method with UV detection Column: Hypersil Gold C18 column 5mm x 2,1mm Mobile phase: water with,1% formic acid/acn (95/5, v/v) Flow rate:,2ml/min UV detection: 22nm Injection volume: 2µL. UV chromatograms obtained after WATER extraction of DON from wheat (animal feed) and clean-up with AFFINIMIP SPE Deoxynivalenol : black, red and green spiked with DON at 6mg/kg dark yellow not spiked blue, a standard solution of DON at 1µg/mL is prepared by dilution of a 1µg/mL Deoxynivalenol standard solution (reference : REA-DON-1mL) in mobile phase Analysis of Whiskas: a. Extraction solution with water b. solution c. Elution solution Recovery of Deoxynivalenol after AFFINIMIP SPE Deoxynivalenol clean-up and relative standard deviation - repeatability conditions (n=3). Feed Matrices C mg/k g a b c Mean mg/kg 6mL-1mg sorbent for (baby)food FS117-2B for 25 cartridges FS117-3B for 5 cartridges 6mL-2mg sorbent for feed FS117-2B-2mg for 25 cartridges FS117-3B-2mg for 5 cartridges R% %RSDr Wheat 6 5.7 94.1 Whiskas.8.73 91 2.4 www.affinisep.com 32

AFFINIMIP SPE Deoxynivalenol DETERMINATION OF DEOXYNIVALENOL IN MEAT 25g of meat are mixed during 2 minutes with 1mL of a solution of deionized water/ acetonitrile (5/5). Then the mixture is filtered through filter paper (4-7µm). This solution was then diluted 1 times with deionized water to obtain the loading solution used for the clean-up protocol Cleanup with a 6mL/1mg AFFINIMIP SPE DEOXYNIVALENOL cartridge 2mL Acetonitrile 2mL Water solution Up to 6mL of loading solution Washing of interferences 3mL NaHCO 3 1% solution Drying 3s Washing of interferences 1mL Diethyl Ether 4mL Ethyl Acetate The elution fraction was then concentrated and diluted to mobile phase before HPLC analysis. Elution Recovery for DON > 9%* *Tested at 8µg/kg Deoxynivalenol UV Chromatograms (22nm) obtained after clean-up of MEAT with AFFINIMIP SPE DEOXYNIVALENOL. Blue trace for spiked with 8µg/kg of DEOXYNIVALENOL Red trace for the blank sample HPLC Method with MS detection Column: Hypersil Gold C18 column 5mm x 2,1mm Mobile phase: water with,1% formic acid/acn (95/5, v/v) Flow rate:,2ml/min MS detection: m/z 265 (ESI - ) Injection volume: 2µL. Ordering information - FS117-2B for 25 cartridges, 6mL FS117-3B for 5 cartridges, 6mL www.affinisep.com 33

AFFINIMIP SPE Deoxynivalenol DETERMINATION OF DEOXYNIVALENOL, 3-AcetylDON AND 15-AcetylDON IN CEREALS (Hydro-organic extraction) Regulations for unprocessed corn or durum wheat for food: Europe (EC 1126/27) : 175µg/Kg 2 18 16 3-AcetylDeoxynivalenol (3-AcDON) 14 12 1 8 WITH HYDROORGANIC EXTRACTION 2g of cereals were ground in a blender for 1 minute. Then, a solution of deionized water: acetonitrile (5:5) was added. This mixture was then ground for 2 additional minutes. After grinding, the mixture was placed in a beaker and left stirred under magnetic agitation for 3 minutes. Then the mixture was centrifuged at 25 g for 15 minutes. After centrifugation, the supernatant was filtered through filter paper. This solution was then diluted 1 times using deionized water. Cleanup with a 6mL/1mg AFFINIMIP SPE Deoxynivalenol cartridge. 2mL Acetonitrile 2mL Water 6mL of loading solution Washing of interferences (W1) 3mL NaHCO 3 1% in water Drying 3 seconds Washing of interferences (W2) 1mL Diethylether 4mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water-.1% formic acid before HPLC analysis. HPLC Method with MS detection Column: Hypersil Gold C18 column 5mm x 2,1mm Mobile phase for DON analyses: water with,1% formic acid/acn (95/5, v/v) Mobile phase for 3-AcDON and 15-AcDON analyses: water with.1% formic acid/acn (9/1, v/v) Flow rate:.2ml/min MS detection: m/z 265 (ESI - ) Injection volume: 2µL. 6 4 2 5 1 15 2 25 3 Time (min) MS chromatograms obtained after hydroorganic extraction of 3-acetylDON from corn and clean-up with AFFINIMIP SPE Deoxynivalenol : -black, red and green: spiked with Deoxynivalenol at 8µg/kg -dark yellow: not spiked -blue: a standard solution of 3-AcetylDON at 2ng/mL is prepared by dilution of a 1µg/mL 3-AcetylDeoxynivalenol standard solution (reference : REA-3AcDON-1mL) in mobile phase Recovery obtained for DON, 3-acetylDON and 15-acetylDON after AFFINIMIP SPE Deoxynivalenol clean-up of Corn and relative standard deviation - repeatability conditions (n=3). Compound C µg/kg Mean µg/kg R% %RSDr DON 8 653.7 81.7.3 3- AcetylDON 8 61. 75.1 2.3 15- AcetylDON 8 641.8 8.2 3.4 6mL-1mg sorbent for (baby)food FS117-2B for 25 cartridges FS117-3B for 5 cartridges 6mL-2mg sorbent for feed FS117-2B-2mg for 25 cartridges FS117-3B-2mg for 5 cartridges www.affinisep.com 34

AFFINIMIP SPE VS IMMUNOAFFINITY COMPARATIVE STUDY Solid phase extraction is a simple tool to selectively extract analytes from complex matrices and quantify concentrations lower and lower. The major disadvantage of conventional SPE sorbents, such as C18 is a lack of selectivity and interference matrix components are co-extracted with the target analytes. To solve this problem, affinity-based SPE sorbents have been developed to be selective in extracting the target analytes like molecularly imprinted polymer (MIP) and immunoaffinity sorbent. Immunoaffinity columns (IAC) are biological sorbents based on the use of antibodies that are specific to the target analytes. Molecularly imprinted polymer is a synthetic material with artificially generated three-dimensional network able to specifically rebind a target molecule. Based on molecularly imprinted polymers, AFFINISEP s AFFINIMIP SPE cartridges have the advantages to be highly selective and specific. Contrary to IAC, AFFINIMIP SPE cartridges are chemically and thermally stable, compatible with all solvents as well as cost effective. PROPERTIES OF MIP AND IAC Feature IAC AFFINIMIP SPE Selectivity High High Capacity Analyte recognition in water Analyte recognition in Organics 6µmol/g Good Poor 1-1µmol/g Variable Good Stability Poor Very High Reproducibility Variable Good Cost Expensive Inexpensive PROTOCOL: Zearalenone (ZON) from maize flour Step Extraction of target analyte Preparatio n loading solution Washing Elution Protocol time Vicam IAC 25g sample in 1mL 9/1 Methanol/water Blender 3 minutes + filtration 4mL extract + 96mL water 1mL solution 2mL Water 1.5mL Methanol 55min AFFINIMIP SPE ZON 25g sample in 1mL 75/25 ACN/water Blender 3 minutes + filtration 1mL extract + 1mL Water 8mL solution 4mL 2/58/4 Acetic acid / water / ACN 2mL 98/2 Methanol/Aceti c acid 3min Compared to IAC, AFFINIMIP SPE provides: Easier and faster protocol Lower dilution Easier automatisation (Cf. Automated method for the selective SPE of Ochratoxin A from wheat Using Molecularly Imprinted Polymer; Gilson Application Notes Handbook 211; volume 1 Issue 4) PROTOCOL: Ochratoxin A (OTA) from wheat flour Step Extraction of target analyte Preparation loading solution Washing Elution Protocol time Vicam IAC AFFINIMIP SPE OTA 5g sample in 1mL 6/4 ACN/water Blender 1 minute + filtration 1mL extract + 4mL PBS 1mL solution 1mL PBS 1mL Water 1.5mL Methanol 3min 1mL extract + 1mL HCl.1M ph=1 4mL solution 7mL 6/4 HCl.1M ph=1/acn 2mL 98/2 Methanol/Aceti c acid 2min www.affinisep.com 35

Are a mvolts 52 51 5 49 48 47 46 45 44 43 42 41 Fluo elution B6 Fluo elution immuno B2 2 4 6 8 1 12 14 16 18 2 22 24 26 Minutes 52 51 5 49 48 47 mvolts 46 45 44 43 42 41 Level found ng/g AFFINIMIP SPE CARTRIDGE VS IMMUNOAFFINITY COLUMN CHROMATOGRAM ASPECT Equivalent chromatograms IA C AFFINIMIP SPE Figure 1. Chromatogram of Maize sample spiked with Zearalenone at 85 µg/kg obtained after cleanup by AFFINIMIP SPE Zearalenone (red) or Vicam IAC (blue). Higher Recoveries RECOVERIES obtained with AFFINIMIP SPE 1ng/g OTA 85% Recovery 1 9 8 Recovery % 98 97 ZON 85 OTA 79 7 6 AFFINIMIP SPE IAC 6ng/g OTA 79% Recovery 5 4 3 2 1 Figure 2. Chromatogram of wheat sample spiked with Ochratoxin A obtained after cleanup by AFFINIMIP SPE Zearalenone (red, spiked at 1ng/g) or Vicam IAC (blue, spiked at 6ng/g). MIP IAC MIP IAC Figure 3. Recovery of Ochratoxin A or Zearalenone obtained after cleanup by AFFINIMIP SPE or Vicam IAC. CAPACITY Capacity MIP > Capacity IAC 3,5E+8 3,E+8 2,5E+8 2,E+8 1,5E+8 1,E+8 5,E+7 AFFINIMIP SPE IAC,E+ 2 4 6 8 1 Spiking level ng/g Figure 4. Comparison of capacity between AFFINIMIP SPE Zearalenone (red) and Vicam IAC (blue). 5 45 4 35 3 25 2 15 1 5 AFFINIMIP SPE IAC 1 2 3 4 5 6 Spiking level ng/g Figure 5. Comparison of capacity between AFFINIMIP SPE OTA (red) and Vicam IAC (blue). www.affinisep.com 36

Analysis of ENDOCRINE DISRUPTING COMPOUNDS www.affinisep.com 37 37

DETERMINATION OF ESTROGENS IN PLASMA AFFINIMIP SPE Estrogens Regulations for Estrogens: Europe (EC directive) : 4pg/mL of plasma or serum of bovine animals 17β-Estradiol-d 3 419>285 Blank plasma 1ppt 4ppt 1ppt 17 α/β-estradiol 416>129 17 α/β-estradiol 416>285 2mL serum samples spiked with 4pg 17β- Estradiol-d3. Then 2mL of Acetate buffer (.8M, ph 6.8) and 1µL β-glucuronidase were added. Hydrolysis performed overnight at 37 C and samples centrifuged at 4 rpm for 1min. Upper layer was used as loading solution. Cleanup with a 3mL/1mg AFFINIMIP SPE Estrogens cartridge 3mL Methanol 3mL Acetonitrile 3mL Water solution from sample preparation Washing of interferences 3mL Water 3mL Water/Acetonitrile (6/4) 3mL Methanol The elution fraction was then evaporated and estrogens were derivatised 4min at 6 C with BSTFA before GC-MS/MS analysis. GC-MS/MS Analysis Column: RTX-1614 Resteck 15m x.25mm x.1µm Gradient temperature: 8 to 32 C (15 C/min) MRM chromatograms from GC-MS/MS analysis of fortified calves plasma samples at, 1, 4 and 1 pg.ml -1 with 17α-estradiol, 17β-estradiol and estrone. Chromatograms obtained after a clean-up with AFFINIMIP SPE Estrogens (Courtesy of Emmanuelle Bichon - LABERCA) Data extracted from Quantification of estrogens at ppt levels in bovine plasma by Molecularly Imprinted Solid Phase Extraction and GC-MS/MS analysis, Emmanuelle Bichon et al. (LABERCA) Poster session, HTSP-2 and HTC 212 FS14-2 for 25 cartridges, 3mL FS14-3 for 5 cartridges, 3mL www.affinisep.com 38

AFFINIMIP SPE Estrogens PROTOCOL COMPARISON AFFINIMIP SPE ESTROGENS vs usual protocol AFFINISEP method Usual method 2mL Bovine plasma Enzymatic hydrolysis Liquid - Liquid Extraction with Ether Copolymeric SPE AFFINIMIP SPE Estrogens 3cc Liquid - Liquid Extraction with Pentane Silica SPE Preparative dimethylaminopropyle HPLC Derivatisation PFB/TMS HPLC analysis : GC-MS/MS Performance. Save your time. Data extracted from Quantification of estrogens at ppt levels in bovine plasma by Molecularly Imprinted Solid Phase Extraction and GC-MS/MS analysis, Emmanuelle Bichon et al. (LABERCA) Poster session, HTSP-2 and HTC 212 FS14-2 for 25 cartridges, 3mL FS14-3 for 5 cartridges, 3mL www.affinisep.com 39

mvolts mvolts 11 1 9 8 7 6 5 4 3 2 1 9 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 Minutes 9, 9,2 9,4 9,6 9,8 1, 1,2 1,4 1,6 1,8 11, 11,2 11,4 11,6 11,8 12, 12,2 12,4 12,6 12,8 13, Minutes AFFINIMIP SPE Bisphenol A DETERMINATION OF BISPHENOL A IN LIQUID INFANT FORMULA Regulations for Bisphenol A: Europe (directive 211/8/EU) : forbiden in infant feeding bottles Injection 5µL of Infant Formula before treatment Bisphenol A after treatment of 15mL of Infant Formula Cleanup with a 3mLor 6mL/1mg AFFINIMIP SPE Bisphenol A cartridge 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water Up to 15mL of infant formula Washing of interferences 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 seconds 3mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 5 5 2 5 5 2.5 65 35 35 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: 5µL. Chromatograms of Infant Formula containing 1µg/L of Bisphenol A before clean-up (Red) and after clean-up (Blue) with AFFINIMIP SPE Bisphenol A. Bisphenol A Chromatograms obtained after clean-up with AFFINIMIP SPE Bisphenol A of 15mL of Infant Formula spiked with Bisphenol A at 2µg/L (tested twice, blue) or at 1µg/L (tested twice, red) or not spiked (pink). Recovery of Bisphenol A in 15mL of infant formula after AFFINIMIP SPE Bisphenol A clean-up and relative standard deviation calculated from results generated - under reproducibility conditions % RSDR ). C (µg/l) Mean (µg/l) Recoveries % % RSD R 1..8 84.4 7.4 2. 1.7 85.8 5.3 3mL-1mg sorbent in a PP cartridge FS16-2 for 25 cartridges FS16-3 for 5 cartridges 6mL-1mg sorbent in a PP cartridge FS16-2B for 25 cartridges FS16-3B for 5 cartridges 6mL-1mg sorbent in a glass cartridge FS16-2G for 25 cartridges FS16-3G for 5 cartridges www.affinisep.com 4

mvolts 14 Fluo 2-MD1.dat 13 12 11 1 9 8 7 6 5 4 3 2 1-1 Fluo 2-md2.dat Fluo 4-md1.dat Fluo 4-md2.dat Fluo mnd.dat 6,5 7, 7,5 8, 8,5 9, 9,5 1, 1,5 11, 11,5 12, 12,5 13, 13,5 14, Minutes AFFINIMIP SPE Bisphenol A DETERMINATION OF BISPHENOL A IN POWDERED INFANT FORMULA Regulations for Bisphenol A: Europe (directive 211/8/EU) : forbiden in infant feeding bottles Bisphenol A 4.4g powdered infant milk was reconstituted in 3 ml of water and warmed up at ~ 5 C during 2 seconds using microwaves. Then 2 ml of acetonitrile were added to 2 ml of warm milk and centrifuged at 4 rpm during 1 minutes. The supernatant was collected and filtered on filter paper (4-7µm). This extract was diluted 1:1 with water to form the loading solution. Cleanup with a 3mL or 6mL/1mg AFFINIMIP SPE Bisphenol A cartridge 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water Up to 4mL of infant formula Washing of interferences 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 seconds 3mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 5 5 2 5 5 2.5 65 35 35 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: 5µL. Chromatograms obtained after clean-up with AFFINIMIP SPE Bisphenol A of equivalent at 1mL of Infant Formula spiked with Bisphenol A at 4.3µg/L (tested twice, red) or at 2.1µg/L (tested twice, blue) or not spiked (pink). Recovery of Bisphenol A spiked at different concentrations after 3mL/1mg AFFINIMIP SPE Bisphenol A clean-up of 4mL of loading solution (equivalent to 1mL of reconstituted Infant milk) and relative standard deviation calculated from results generated under repeatability conditions C of BPA in reconstituted milk (µg/l) Mean concentration (µg/l) Recoveries % RSD r % 2.1 2.3 (n=5) 18 8.7 4.3 4. (n=4) 95 3.7 3mL-1mg sorbent in a PP cartridge FS16-2 for 25 cartridges FS16-3 for 5 cartridges 6mL-1mg sorbent in a PP cartridge FS16-2B for 25 cartridges FS16-3B for 5 cartridges 6mL-1mg sorbent in a glass cartridge FS16-2G for 25 cartridges FS16-3G for 5 cartridges www.affinisep.com 41

AFFINIMIP SPE Bisphenol FS16 A PROTOCOL COMPARISON AFFINIMIP SPE Bisphenol A vs competitor POWDERED INFANT FORMULA ANALYSIS AFFINISEP method Precipitation (15g sample + 15mL ACN) Centrifuge and collect supernatant WATERS method* Precipitation (1g sample + 1mL ACN) Centrifuge and collect supernatant Quecher 1 Add contents from DisQue tube 1. Shake. Centrifuge and collect 1mL Quecher 2 Add contents from DisQue tube 2. Shake. Centrifuge and collect supernatant Dilute 2mL supernatant with 2mL H 2 Load on AFFINIMIP SPE Bisphenol A 3cc (4mL) Wash 1mL H 2 and 6mL 4% ACN ; Elute 3mL 1% MeOH Dilute supernatant with 7mL H2 Load on SPE cartridge OASIS HLB 3cc (7mL) Wash 2mL 4% Methanol ; Elute 1mL 1% MeOH HPLC analysis : LC Fluorescence detector or GC-MS/MS or LC-MS/MS Performance. Save your time. HPLC analysis : LC-MS/MS Extract from Waters application note, published 212 : Rapid analysis of Bisphenol A 3mL-1mg sorbent in a PP cartridge FS16-2 for 25 cartridges FS16-3 for 5 cartridges 6mL-1mg sorbent in a PP cartridge FS16-2B for 25 cartridges FS16-3B for 5 cartridges 6mL-1mg sorbent in a glass cartridge FS16-2G for 25 cartridges FS16-3G for 5 cartridges www.affinisep.com 42

mvolts 22 2 18 16 14 12 1 8 6 4 2-2 -4-6 -8 4,5 5, 5,5 6, 6,5 7, 7,5 8, 8,5 9, 9,5 1, 1,5 11, Minutes AFFINIMIP SPE Bisphenol A DETERMINATION OF BISPHENOL A IN CANNED FOOD (Liquid form) Regulations for Bisphenol A: Europe (directive 211/8/EU) : Specific migration limit in food from packaging of.6mg/kg Bisphenol A Cleanup with a 3mL or 6mL/1mg AFFINIMIP SPE Bisphenol A cartridge 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water 1mL liquid from canned food after filter paper filtration Washing of interferences 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 seconds 3mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. Chromatograms after clean-up with AFFINIMIP SPE Bisphenol A of 1mL liquid form of canned Peas and carrots spiked with Bisphenol A at 1µg/L (tested twice, blue) or not spiked (green). Recovery of Bisphenol A after AFFINIMIP SPE Bisphenol A clean-up of 1mL of canned peas and carrots (liquid) spiked at 1µg/L and relative standard deviation calculated from results generated - under repetability conditions (n=4). C (µg/l) Mean (µg/l) Recoveries % % RSD R 1. 1.5 15.1 5 - under reproducibility conditions (n=4). HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 5 5 2 5 5 2.5 65 35 35 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: 5µL. C (µg/l) Mean (µg/l) Recoveries 3mL-1mg sorbent in a PP cartridge FS16-2 for 25 cartridges FS16-3 for 5 cartridges 6mL-1mg sorbent in a glass cartridge FS16-2G for 25 cartridges FS16-3G for 5 cartridges % % RSD R 1. 1.4 14.3 1 More information in the application note on our website www.affinisep.com 43

mvolts 55 Fluo MD1.dat 5 45 4 35 3 25 2 15 1 5-5 Fluo md2.dat Fluo mnd.dat 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 mvolts 65 6 55 5 45 4 35 3 25 2 15 1 5-5 Fluo MD1.dat Fluo md2.dat Fluo mnd.dat 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 Minutes Minutes AFFINIMIP SPE Bisphenol A DETERMINATION OF BISPHENOL A IN CANNED FOOD (Vegetable) Regulations for Bisphenol A: Europe (directive 211/8/EU) : Specific migration limit in food from packaging of.6mg/kg 15g of drained canned peas - carrots and 2mL of Water /ACN (5/5) are blended during 2 min and centrifuged during 1min at 4rpm. The supernatant solution is collected, filtered (4-7µm) and diluted ½ with water to give the loading solution ZOOM Bisphenol A Cleanup with a 3mL or 6mL/1mg AFFINIMIP SPE Bisphenol A cartridge 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water 2mL loading solution Washing of interferences 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 seconds 3mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. Bisphenol A Recovery yield : 97-99% Chromatograms after clean-up with AFFINIMIP SPE Bisphenol A of 2mL loading solution of extract of canned Peas- carrots spiked with Bisphenol A at 2µg/L (tested twice, blue and red) or not spiked (green). HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 5 5 2 5 5 2.5 65 35 35 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: 5µL. 3mL-1mg sorbent in a PP cartridge FS16-2 for 25 cartridges FS16-3 for 5 cartridges 6mL-1mg sorbent in a PP cartridge FS16-2B for 25 cartridges FS16-3B for 5 cartridges 6mL-1mg sorbent in a glass cartridge FS16-2G for 25 cartridges FS16-3G for 5 cartridges www.affinisep.com 44

DETERMINATION OF BISPHENOL A IN BEER AFFINIMIP SPE Bisphenol A Regulations for Bisphenol A: Europe (directive 211/8/EU) : Specific migration limit in food from packaging of.6mg/kg The beer is degassed by sonication for 1 hour. Cleanup with a 3mL or 6mL/1mg AFFINIMIP SPE Bisphenol A cartridge 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water 1mL of degassed beer Washing of interferences 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 seconds 3mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 5 5 12.5 65 35 22 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: 5µL. Injection 5µL of beer before treatment Bisphenol A after treatment of 1mL of Beer Chromatograms of beer containing 1µg/L of Bisphenol A before (Red) and after (Blue) AFFINIMIP SPE Bisphenol A Clean-up. Bisphenol A Chromatograms obtained after AFFINIMIP SPE Bisphenol A Clean-up of 1mL of beer spiked at 2µg/L (tested 3 times, orange) or at 1µg/L (tested 3 times, blue) with Bisphenol A or not spiked (red) Recovery of Bisphenol A in spiked beer after AFFINIMIP SPE Bisphenol A clean-up and relative standard deviation calculated from results generated under reproducibility conditions ( % RSDR). C (µg/l) Mean µg/l Recoveries % 3mL-1mg sorbent in a PP cartridge FS16-2 for 25 cartridges FS16-3 for 5 cartridges 6mL-1mg sorbent in a glass cartridge FS16-2G for 25 cartridges FS16-3G for 5 cartridges % RSD R 1. 1. 99.3 8.9 2. 1.8 9.6 6. www.affinisep.com 45

mvolts 11 1 9 8 7 6 5 4 3 2 1-1 Fluo MND.dat Fluo md1.dat Fluo md2.dat Fluo md3.dat 5, 5,5 6, 6,5 7, 7,5 8, 8,5 9, 9,5 1, 1,5 11, 11,5 12, 12,5 13, Minutes AFFINIMIP SPE Bisphenol A DETERMINATION OF BISPHENOL A IN RED/WHITE WINES Regulations for Bisphenol A: Europe (directive 211/8/EU) : Specific migration limit in food from packaging of.6mg/kg Bisphenol A Cleanup with a 3mL or 6mL/1mg AFFINIMIP SPE Bisphenol A cartridge 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water Up to 1mL of wine Washing of interferences 9mL Water 6mL Water/Acetonitrile (6/4) Drying 1 minute 3mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. Chromatograms obtained after clean-up with AFFINIMIP SPE Bisphenol A of 1mL of white wine spiked with Bisphenol A at 2µg/kg (tested three times, blue) or not spiked (red). The white wine naturally contained 2µg/kg of BPA Recovery of Bisphenol A spiked at 2µg/kg after AFFINIMIP SPE Bisphenol A clean-up of 6mL of red wine or 1mL of white wine. Matrice Spiked at 2µg/kg Mean C (µg/kg) Recoveries % Red wine 1 1.93 (n=2) 96.6 Red wine 2 2.13 (n=2) 16.5 HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 5 5 12.5 65 35 22 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: 5µL. Red wine 3 1.66 (n=2) 83. White wine 1.6 (n=3) 8. 3mL-1mg sorbent in a PP cartridge FS16-2 for 25 cartridges FS16-3 for 5 cartridges 6mL-1mg sorbent in a PP cartridge FS16-2B for 25 cartridges FS16-3B for 5 cartridges 6mL-1mg sorbent in a glass cartridge FS16-2G for 25 cartridges FS16-3G for 5 cartridges www.affinisep.com 46

AFFINIMIP SPE Bisphenol A DETERMINATION OF BISPHENOL A IN COLA DRINKS PROTOCOL OF CLEAUNP Cleanup with a 3mL or 6mL/1mg AFFINIMIP SPE Bisphenol A cartridge 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water 6mL of Cola drinks after 3min degassing with ultrasounds Washing of interferences 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 minute 3mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. Bisphenol A Chromatograms obtained after clean-up with AFFINIMIP SPE Bisphenol A of 1mL of white wine spiked with Bisphenol A at 2µg/kg (tested three times, blue) or not spiked (red). The white wine naturally contained 2µg/kg of BPA Recovery of Bisphenol A spiked at 5µg/kg after AFFINIMIP SPE Bisphenol A clean-up of 6mL of Cola drinks Mean concentration (µg/kg) Recoverie s % RSDr % 1.93 (n=2) 96.6 1. HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 5 5 2 5 5 2.5 65 35 4 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: 5µL. 3mL-1mg sorbent in a PP cartridge FS16-2 for 25 cartridges FS16-3 for 5 cartridges 6mL-1mg sorbent in a PP cartridge FS16-2B for 25 cartridges FS16-3B for 5 cartridges 6mL-1mg sorbent in a glass cartridge FS16-2G for 25 cartridges FS16-3G for 5 cartridges www.affinisep.com 47

mvolts 5 45 4 35 3 25 2 15 1 5-5 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 Minutes 5 45 4 35 3 25 2 15 1 5-5 mvolts AFFINIMIP SPE Bisphenol A DETERMINATION OF BISPHENOL A AND BADGE IN MILK Bisphenol A BADGE Bisphenol A Diglycidyl Ether (BADGE) Bisphenol A Cleanup with a 3mL or 6mL/1mg AFFINIMIP SPE Bisphenol A cartridge 3mL Methanol -2% Formic Acid 3mL Acetonitrile 3mL Water 9mL of Milk Washing of interferences 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 minute 3mL Methanol (E1) 3mL Acetonitrile (E2) The elution fractions E1 and E2 were gathered, evaporated and dissolved in the mobile phase before HPLC analysis. HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 15mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 5 5 2 2 8 25 2 8 3 65 35 4 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: 5µL. Fluorescence chromatograms obtained after clean-up with AFFINIMIP SPE Bisphenol A of 9mL of milk spiked with 1µg/kg Bisphenol A and 1µg/kg BADGE (tested twice, blue) or not spiked (red). Recovery of Bisphenol A and BADGE spiked at 1ng/mL after AFFINIMIP SPE Bisphenol A clean-up of 9mL of milk. Matrice Spiked at 1ng/mL Mean concentration (µg/kg) Recoveries % BPA 1.85 18.5 BADGE 7.5 75 3mL-1mg sorbent in a PP cartridge FS16-2 for 25 cartridges FS16-3 for 5 cartridges 6mL-1mg sorbent in a PP cartridge FS16-2B for 25 cartridges FS16-3B for 5 cartridges 6mL-1mg sorbent in a glass cartridge FS16-2G for 25 cartridges FS16-3G for 5 cartridges www.affinisep.com 48

AFFINIMIP SPE Bisphenol A DETERMINATION OF TOTAL BISPHENOL A IN HUMAN URINE 3mL urine sample, 1mL of sodium acetate buffer.1m at ph 5. and 2µL of β- glucuronidase/sulfatase Helix pomatia enzyme solution at 1.mg/mL in the same buffer were mixed thoroughly by vortex. The enzymatic reaction was carried out for 2h at 37 C to obtain the loading solution. Mean percentage recoveries of Bisphenol A spiked at different concentrations in 3mL of urine after AFFINIMIP SPE Bisphenol A cleanup: C (ng/ml) 1 1 1 Recoveries % 12.6 94.7 97.6 Cleanup with a 6mL/1mg AFFINIMIP SPE Bisphenol A glass cartridge 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water solution Up to 12mL of loading solution (Equivalent to around 9mL of urine) Washing of interferences 4mL Water 4mL Water/Acetonitrile (6/4) 3mL Methanol The elution fraction was then concentrated and diluted to 1mL before HPLC analysis. HPLC Method with LC-MS/MS HPLC Column: Kinetex 2.6µm PFP 1mm x 4.6mm Mobile phase: gradient profile Time (min) % water % Methanol 7 3 1 7 3 2 5 95 5 5 95 6 7 3 9 7 3 Flow rate:.5ml/min Injection volume: 2µL. Detector: ESI-MS/MS By courtesy of Nadia Diano, Dept. of Experimental Medicine, Second University of Naples (Italy) More details in the following article C. Nicolucci, S. Rossi, C. Menale, E. Giudice, P. Miraglia del Giudice, L. Perrone, P. Gallo, D. Mita, N. Diano, Analytical and Bioanalytical Chemistry, 1618-2642, 213. 6mL-1mg sorbent in a PP cartridge FS16-2B for 25 cartridges FS16-3B for 5 cartridges 6mL-1mg sorbent in a glass cartridge FS16-2G for 25 cartridges FS16-3G for 5 cartridges www.affinisep.com 49

12 11 1 9 8 7 6 5 4 3 2 1 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 Minutes Minutes 12 11 1 9 8 7 6 5 4 3 2 1 AFFINIMIP SPE Phenolics DETERMINATION OF PARABENS IN COSMETIC PRODUCTS 1g of Lotion was mixed 1minute with 1mL of H2SO4 2M and 5mL of 9/1 Ethanol/Water. The mixture was heated during 5min at 6 C. Then the solution is filtered on filter paper (4-7µm). This extract was diluted by 3 with water. The solution was spiked with methylparaben to simulate a concentration of paraben in the lotion at.2%,.4% and.8%. Cleanup with a 3mL/1mg AFFINIMIP SPE Phenolics cartridge 3mL Acetonitrile 3mL Water Up to 5mL of loading solution Washing of interferences 3mL Water / Acetonitrile (75/25 v/v) 3mL Methanol The elution fraction was diluted by 2 with water prior to analysis. Chromatograms of a cream containing.2% of methylparaben before clean-up (blue) and after clean-up (Red) with AFFINIMIP SPE Phenolics. 18 16 14 12 1 8 6 4 2 Before After Cream spiked at.8%, yield 91% (n=2) Cream spiked at.4%, yield 16 (n=2) Cream spiked at.2%, yield 16 (n=2) 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 Chromatograms obtained after clean-up with AFFINIMIP SPE Phenolics of a cream (without parabens) spiked with different concentrations of methylparaben 18 16 14 12 1 8 6 4 2 Recovery yields and reproducibility after AFFINIMIP SPE Phenolics Clean-up. Recoveries % (n=6) RSD R % 11.1 8 HPLC-UV Method Column: Thermo Hypersil gold, 15mm x 2.1mm Mobile phase: 6/4 (v/v) Water/Methanol Flow rate:.2ml/min Detection: UV - 254nm Injection volume: 2µL. 3mL-1mg sorbent FS13-2 for 25 cartridges FS13-3 for 5 cartridges www.affinisep.com 5

Analysis of ANTIBIOTICS AND DRUG RESIDUES www.affinisep.com 51

Intensity AFFINIMIP SPE Chloramphenicol DETERMINATION OF CHLORAMPHENICOL IN HONEY Regulations for Chloramphenicol in residues in food of animal origin: Europe 23/181/EC prohibited with a minimum required performance limits of.3µg/kg 1g of honey and 1mL Water were mixed under magnetic stirring during 1 minutes and used as the loading solution. Cleanup with a 1mL/5mg AFFINIMIP SPE Chloramphenicol cartridge 2mL Acetonitrile 2mL Water 1mL of loading solution for 15µg/kg (or 1mL for.3µg/kg) Washing of interferences (W1) 1mL Water 1mL (Water -.5% AA)/ACN (95/5) 2mL of Ammonia (1%) in Water 2mL (Water-1% Ammonia)/ACN (8/2) Drying 1 min Washing of interferences (W2).25mL Diethyl ether 2mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis..3µg/kg Chloramphenicol Chloramphenicol SIM Chromatogram obtained after cleanup with AFFINIMIP SPE Chloramphenicol of 1g of Honey spiked with Chloramphenicol at.3µg/kg. HPLC Method with MS detection Column: Thermo Accucore C18 column 5mm x 2.1mm Mobile phase: Ammonium acetate (1mM) in water /Methanol (75/25) flow rate:.2ml/min MS detection: m/z 322 (ESI - ) Injection volume: 2µL. 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 UV Chromatograms obtained after clean-up with AFFINIMIP SPE Chloramphenicol of 1g of Honey spiked with Chloramphenicol at 15.7µg/kg (red) or not spiked (blue). 2 3 4 5 6 7 8 9 1 Time (min) 1mL-5mg sorbent FS11-2A for 25 cartridges FS11-3A for 5 cartridges Honey spiked at 15.7µg/kg Chloramphenicol Not spiked SIM Chromatograms obtained after clean-up with AFFINIMIP SPE Chloramphenicol of 1g of Honey spiked with Chloramphenicol at 15.7µg/kg (red) or not spiked (blue). Recovery of Chloramphenicol spiked at 16µg/kg after AFFINIMIP SPE Chloramphenicol clean-up of 1g of Honey and relative standard deviation calculated from results generated under reproducibility conditions ( % RSD R ). C (µg/kg) Mean (µg/kg) Recoverie s % % RSD R 15.7 16.9 18.1 6.5 (n=6) 18.2 16.6 91.4 11.4 (n=12) www.affinisep.com 52

uau AFFINIMIP SPE Chloramphenicol DETERMINATION OF CHLORAMPHENICOL IN BOVINE URINE Regulations for Chloramphenicol in residues in food of animal origin: Europe (23/181/EC) : prohibited with a Minimum Required Performance Limits of.3µg/kg USA FDA: prohibited Chloramphenico l 1 ml of urine were adjusted at ph 7 with Ammonia 1%. This solution was mixed and used as the loading solution. Cleanup with a 1mL/5mg Chloramphenicol cartridge AFFINIMIP SPE 2mL Acetonitrile 2mL Water 1mL of loading solution Washing of interferences (W1) 1mL (Water -.5% Acetic Acid)/Acetonitrile (95/5) 2mL of Ammonia (1%) in Water 2mL (Water-1% Ammonia)/Acetonitrile (8/2)) Drying 1 min Washing of interferences (W2).25mL Diethyl ether 2mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. SIM Chromatograms obtained after clean-up with AFFINIMIP SPE Chloramphenicol of 1 ml of Urine spiked with Chloramphenicol at 17.6µg/kg (red and blue) or not spiked (green). RT:. - 3. 14 13 12 11 1 9 8 7 6 5 4 3 2 1 UV: VERY LOW BACKGROUND 2 4 6 8 1 12 14 16 18 2 22 24 26 28 Time (min) UV Chromatograms of Urine spiked with Chloramphenicol at 17.6 µg/kg (red and black) or not spiked (green) after clean-up with AFFINIMIP SPE Chloramphenicol Recovery of Chloramphenicol spiked at 17.6µg/kg after AFFINIMIP SPE Chloramphenicol clean-up of 1 ml of Urine. C (µg/kg) Mean (µg/kg) Recovery % NL: 1.32E5 Channel A UV UrineD1-E NL: 1.32E5 Channel A UV urined2-e NL: 1.32E5 Channel A UV UrineND-E 17.6 16.7 9 HPLC Method with MS detection Column: Thermo Accucore C18 column 5mm x 2.1mm Mobile phase: Ammonium acetate (1mM) in water /Methanol (75/25) flow rate:.2ml/min MS detection: m/z 321 (ESI - ) Injection volume: 2µL. 1mL-5mg sorbent FS11-2A for 25 cartridges FS11-3A for 5 cartridges www.affinisep.com 53

uau Intensity AFFINIMIP SPE Chloramphenicol DETERMINATION OF CHLORAMPHENICOL IN SHRIMP Regulations for Chloramphenicol in residues in food of animal origin: Europe (23/181/EC) : prohibited with a Minimum Required Performance Limits of.3µg/kg USA FDA: prohibited 5g peeled shrimp were homogenized 2min with a vortex in 2mL of ethyl acetate. Then the solution was filtered on filter paper (25µm). The supernatant was evaporated to dryness and reconstituted in 1mL of Water to obtain the loading solution. Cleanup with a 1mL/5mg Chloramphenicol cartridge AFFINIMIP SPE 2mL Acetonitrile 2mL Water 1 or 2mL of loading solution Washing of interferences (W1) 1mL Water 1mL (Water -.5% Acetic Acid)/Acetonitrile (95/5) 2mL of Ammonia (1%) in Water 2mL (Water-1% Ammonia)/Acetonitrile (8/2)) Drying 1 min Washing of interferences (W2).25mL Diethyl ether 2mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. HPLC Method with MS detection Column: Thermo Accucore C18 column 5mm x 2.1mm Mobile phase: Ammonium acetate (1mM) in water /Methanol (75/25) flow rate:.2ml/min MS detection: m/z 321 (ESI - ) Injection volume: 2µL. 65 6 55 5 45 4 35 3 25 2 15 1 5 2 4 6 8 1 12 14 16 Time (min) 1mL-5mg sorbent FS11-2A for 25 cartridges FS11-3A for 5 cartridges Chloramphenicol SIM Chromatograms obtained after clean-up with AFFINIMIP SPE Chloramphenicol of Shrimp spiked with Chloramphenicol at 38µg/kg. of 1mL (spiked in green and not spiked in black) and of 2mL (spiked in red and not spiked in blue) 9 8 7 6 5 4 3 2 1-1 UV: VERY LOW BACKGROUND UV Chromatograms of the same solutions Recovery of Chloramphenicol spiked at 38µg/kg after AFFINIMIP SPE Chloramphenicol clean-up of Shrimp. C (µg/kg) Chloramphenicol 1 2 3 4 5 6 7 8 9 1 11 12 13 Time (min) volume Mean (µg/kg) Recovery % 38 1mL 38.7 11.7 38 2mL 36.4 95.8 www.affinisep.com 54

AFFINIMIP SPE Amphetamines DETERMINATION OF AMPHETAMINES IN HUMAN URINE Example of Regulations: France : prohibited cut-off limit of 1µg/mL in urine and 5ng/mL of blood Virginia (USA): 1ng/mL of blood 85 8 75 7 65 6 55 5 Amphetamine MDA Human urine is diluted by 2 with an ammonium acetate buffer (13mM, ph 8.5). The ph of the diluted urine is adjusted with NH 3 or CH 3 COOH at ph 8.5. Cleanup with a 3mL AFFINIMIP SPE Amphetamines cartridge 1mL Acetonitrile 2mL Water 5mL of diluted urine Washing of interferences (W1) 3mL Water 3mL Water/Acetonitrile (6/4) Drying 3 seconds 1.5mL Methanol 2% Formic acid The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. HPLC Method with MS detection Column: Syncronis Aq column 15mm x 2.1mm Mobile phase: gradient profile with A (Water Ammonium Acetate 1mM) and B (Acetonitrile Ammonium Acetate 1mM) Time (min) % A % B 95 5 2 95 5 12 6 4 12.1 95 5 flow rate:.4ml/min MS detection (ESI + ) : m/z 136 (Amphetamine) ; 18 (MDA); 15 (Methamphetamine); 194 (MDMA); 28 (MDEA) Injection volume: 2µL. 45 4 35 3 25 2 15 1 5 Methamphetamine MDMA MDEA 2 4 6 8 1 12 14 16 18 2 22 24 Recovery of Amphetamines in human urine spiked at 2ng/mL after AFFINIMIP SPE Amphetamines clean-up and relative standard deviation calculated from results generated under reproducibility conditions. Sample Mean ng/ml 3mL-5mg cartridge DG12-2 for 25 cartridges DG12-3 for 5 cartridges Recoveri es % % RSD R Amphetamine 17.5 87.5 8.9 (n=8) MDA 18.6 93.1 9.6 (n=8) Methampheta mine 18.6 93.2 9.2 (n=8) MDMA 21.1 15.4 1.5 (n=4) MDEA 2.3 11.7 12.4 (n=8) Capacity: different concentrations of Amphetamine in urine were applied on AFFINIMIP SPE Amphetamines cartridge (25mg) to measure the capacity of the product. Quantity loaded µg Time (min) Mass Chromatogram (SIM) obtained after AFFINIMIP SPE Amphetamines clean-up of a human urine sample spiked at 2ng/mL with Amphetamine and its derivatives. Quantity obtained in the elution fraction µg 1..9 2.5 2.41 5. 3.51 www.affinisep.com 55

AFFINIMIP SPE Amphetamines DETERMINATION OF AMPHETAMINES IN HUMAN SERUM Example of Regulations: France : prohibited cut-off limit of 1µg/mL in urine and 5ng/mL of blood Virginia (USA): 1ng/mL of blood Human serum is diluted by 5 with an ammonium acetate buffer (13mM, ph 8.5). The ph of the diluted urine is adjusted with NH 3 or CH 3 COOH at ph 8.5. 9 8 7 6 5 4 3 2 1 Amphetamine MDA Methamphetamine MDMA MDEA 2 4 6 8 1 12 14 16 18 2 22 24 Time (min) Cleanup with a 3mL AFFINIMIP SPE Amphetamines cartridge 1mL Acetonitrile 2mL Water 2.5mL of diluted serum Washing of interferences (W1) 3mL Water 3mL Water/Acetonitrile (6/4) Drying 3 seconds 1.5mL Methanol 2% Formic acid The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. HPLC Method with MS detection Column: Syncronis Aq column 15mm x 2.1mm Mobile phase: gradient profile with A (Water Ammonium Acetate 1mM) and B (Acetonitrile Ammonium Acetate 1mM) Time (min) % A % B 95 5 2 95 5 12 6 4 12.1 95 5 flow rate:.4ml/min MS detection (ESI + ) : m/z 136 (Amphetamine) ; 18 (MDA); 15 (Methamphetamine); 194 (MDMA); 28 (MDEA) Injection volume: 2µL. Mass Chromatogram (SIM) obtained after AFFINIMIP SPE Amphetamines clean-up of a human serum sample spiked at 1ng/mL with Amphetamine and its derivatives. Recovery of Amphetamines in human serum spiked at 1ng/mL after AFFINIMIP SPE Amphetamines clean-up and relative standard deviation calculated from results generated under reproducibility conditions (n=4). Sample Mean ng/ml 3mL-5mg cartridge DG12-2 for 25 cartridges DG12-3 for 5 cartridges Recoveri es % % RSD R Amphetamine 87.9 87.9 5. MDA 94.4 94.4 3.7 Methamphetam ine 9.7 9.7 2.2 MDMA 16.2 16.2 2.5 MDEA 111. 111. 4.9 www.affinisep.com 56

8 7 6 5 4 3 2 1-1 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48 5 Minutes 8 7 6 5 4 3 2 1-1 AFFINIMIP SPE Tetracyclines DETERMINATION OF TETRACYCLINES, THEIR EPIMERS AND DOXYCYCLINE IN MILK AND SALMON for Milk Mix 1.5mL of Milk with 6mL of EDTA/Mc Ilvaine s Buffer and centrifuge at 4rpm for 1 min at a temperature below 15 C. Collect the supernatant and add 75µL 1N NaOH solution. Adjust to ph 1 with a NaOH solution (this mixture was the loading solution). Sample Preparation for Salmon based on AOAC 995.9 method Blend 1g Salmon with 4mL of EDTA/Mc Ilvaine s Buffer during 3 s and stir during 1min with a magnetic stirrer. Centrifuge the mixture at 25g for 1 min at a temperature < 15 C. Collect the supernatant Repeat this operation with 4mL buffer and again with 2mL of buffer. Then, gather all the supernatants and centrifuge during 2min at 25g, filter on Buchner. Add 75µL 1N NaOH solution to the filtrate and adjust to ph 1 with a NaOH solution (this mixture was the loading solution). Cleanup with a 1mL/1mg AFFINIMIP SPE Tetracyclines cartridge 1mL Acetonitrile 1mL Water solution (7.5mL) Washing of interferences 1mL Water 2mL Water/Acetonitrile (6/4) Drying 3 minutes 2mL Methanol with 2% Formic acid The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. HPLC Method with UV detection Column: Hypersil Gold C18 column 15mm x 2.1mm, 3µm Mobile phase: gradient profile Time min % 1mM Oxalic Acid Water Flow rate:.2ml/min UV detection: 355nm Injection volume: 1µL. % 1mM Oxalic Acid ACN % MeOH 9 5 5 2 9 5 5 21 8 1 1 4 8 1 1 41 9 5 5 4-epiOTC TC UV Chromatograms (355nm) obtained after clean-up with AFFINIMIP SPE Tetracyclines of 1.5mL of Milk spiked with Tetracycline, Chlortetracycline and 4-epioxytetracycline (4- epiotc) at 5µg/L (blue) or not spiked (red) or of 1.5mL of water spiked with Tetracycline, Chlortetracycline and 4-epioxytetracycline at 5µg/L (pink) Recovery of Tetracyclines after AFFINIMIP SPE Tetracyclines clean-up of Salmon or milk spiked at 5 or 1µg/L and relative standard deviation calculated from results generated under repeatability conditions (n=3). Molecules Mean (µg/l) Milk Salmon R % % RSDr R% Tetracycline 49.6 99.4 4.9 113 Oxytetracycline 45.6 91.3 7.1 - Chlortetracycline 37.2 74.4 6.3 74 4-epitetracycline (4- epitc) 47.9 95.9 5.1-4- 18.4 epichlortetracycline 18.4 15. 97 4- epioxytetracycline CTC 43.7 87.4 9.1 71 Doxycycline (DOX) 43.8 88. 2.9 89 1mL-1mg sorbent FS112-2A for 25 cartridges 1mL FS112-3A for 5 cartridges 1mL kit of 12 reservoirs 15ml and adapters for use with 1, 3 & 6 ml columns ACC-AR2 www.affinisep.com 57

4 35 3 25 2 15 1 5-5 -1. 2.5 5. 7.5 1. 12.5 15. 17.5 2. 22.5 25. 27.5 3. 32.5 35. 37.5 4. 42.5 45. 47.5 5. Minutes 4 35 3 25 2 15 1 5-5 -1 AFFINIMIP SPE Tetracyclines DETERMINATION OF TETRACYCLINES, THEIR EPIMERS IN MEAT Preparation of loading solution for Meat based on AOAC 995.9 method 1g meat were blend during 3 seconds with 4mL of EDTA/Mc Ilvaine s Buffer and stirred during 1min with a magnetic stirrer. The mixture was centrifuged at 25g for 1 minutes at a temperature below 15 C. The supernatant was collected. This operation was repeated with 4mL of buffer and again with 2mL of buffer. Then, all the supernatants were gathered and centrifuged during 2min at 25g, filtered on Buchner. 75µL 1N NaOH solution were added to the filtrate and adjusted to ph 1 with a NaOH solution (this mixture was the loading solution). Cleanup with a 1mL/1mg AFFINIMIP SPE Tetracyclines cartridge 1mL Acetonitrile 1mL Water 5mL solution Washing of interferences 1mL Water 2mL Water/Acetonitrile (6/4) Drying 1 minute (only if elution is evaporated) 2mL Methanol with 2% Formic acid The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. HPLC Method with UV detection Column: Hypersil Gold C18 column 15mm x 2.1mm, 3µm Mobile phase: gradient profile Time min % 1mM Oxalic Acid Water Flow rate:.2ml/min UV detection: 355nm Injection volume: 1µL. % 1mM Oxalic Acid ACN % MeOH 9 5 5 2 9 5 5 21 8 1 1 4 8 1 1 41 9 5 5 4-epiOTC TC CTC 4-epiCTC UV Chromatograms (355nm) obtained after clean-up with AFFINIMIP SPE Tetracyclines of meat spiked with Tetracycline, Chlortetracycline, 4-epichlortetracycline (4- epictc) and 4-epioxytetracycline (4-epiOTC) at 5µg/L (red), not spiked (pink) or of water spiked (green) Recovery of Tetracyclines after AFFINIMIP SPE Tetracyclines clean-up of Meat spiked at 2µg/kg (4-epiCTC at 4µg/kg) Molecules R% (n=2) Tetracycline 98 Chlortetracycline 7 4-epichlortetracycline 74 4-epioxytetracycline 91 1mL-1mg sorbent FS112-2A for 25 cartridges 1mL FS112-3A for 5 cartridges 1mL kit of 12 reservoirs 15ml and adapters for use with 1, 3 & 6 ml columns ACC-AR2 www.affinisep.com 58

AttractSPE TM W/O-HLB DETERMINATION OF ANTIBACTERIAL AMINOGLYCOSIDES ON MILK OR MEAT PROTOCOL OF PURIFICATION FOR MEAT and MILK for meat 2g meat are mixed during 1min with 1mL extraction buffer (1 mm KH2PO4,.4 mm EDTA, 2% trichloroacetic acid). Then centrifuge during 1 min at 4 rpm, and collect the supernatant. Repeat two times. Adjust the ph 7.5 ~ 8. with 5 M NaOH (.3 ~.4 ml) to obtain the loading solution. for Milk Mix 5mL milk in 3µL 5% trichloroacetic acid during 1min. Then centrifuge during 15min at 5rpm and collect the supernatant. Add 3µL 5% trichloroacetic acid to the supernatant and centrifuge again 15sec. Adjust the ph 7.5 ~ 8. with 1M NaOH to obtain the loading solution. streptomycin (STR) Dihydrostreptomycin (DHS) hygromycin B (HB) kanamycin (KM) apramycin (APM) destomycin A (DA) amikacin (AK) Paromomycin (PM) Tobramycin Purification with a 6mL/2mg AttractSPE TM W/O - HLB cartridge 5mL Methanol 5mL Water solution Washing 5mL water Drying 5 min 2x 3mL 1mM Heptafluorobutyric acid (HFBA) in Acetonitrile Methanol (2+1, v/v) Evaporate under nitroger at 5 C Reconstitute with 1mL 2mM HFBA solution before analysis. Detection LC-MS/MS 6mL-2mg sorbent HLB-5.S.6.2 for 5 cartridges www.affinisep.com 59

AttractSPE TM W/O-HLB MULTI-RESIDUE DETERMINATION OF SEVERAL FAMILIES OF ANTIBIOTICS IN KIDNEY PROTOCOL OF PURIFICATION Vortex 1 g of kidney with 1mL of McIlvaine/EDTA buffer during 1min. Shake for 15min and ultrasonic for 5 min. Centrifuge at 38g for 1min at 5 C. Filter and collect supernatant. Repeat extraction with 3mL buffer solution. Combine the supernatants to obtain the loading solution. Purification with a 6mL/2mg AttractSPE TM W/O - HLB cartridge 5mL Methanol 5mL Water solution Washing 6mL water/methanol (95/5, v /v) Drying 1 min 6mL Methanol Evaporate under nitroger at 4 C and reconstituted with 1mL water/acetonitrile (9/1, v/v). Vortex, centrifuge and filter Analysis: LC-MS/MS 73 substances measured from drug families: Quinolones Macrolides Lincosamides Sulfonamides Penicillins Cephalosporine Pleuromutilins Diamino pyrimidine derivatives 6mL-2mg sorbent HLB-5.S.6.2 for 5 cartridges AttractSPE TM W/O-HLB MULTI-RESIDUE DETERMINATION OF NSAID IN MUSCLE TISSUE PROTOCOL OF PURIFICATION Shake 2g of homogenized meat with 1mL Acetonitrile for 2min. Centrifuge during 5min at 55rpm and evaporate the supernatant at 6 C under nitrogen. Reconstitute with 1µL Methanol 9µL Water to obtain the loading solution. Purification with a 3mL/6mg AttractSPE TM W/O - HLB cartridge 1mL Methanol 1mL Water All the loading solution Washing of interferences 1mL Methanol/Water (5/95) Drying under vaccuum 1mL Methanol 1mL Hexane with 1% Acetic acid The elution fraction was then evaporated at 6 C under nitrogen and reconstituted before HPLC analysis. Analyse: LC/MS-MS Regulations - MRL for NSAID in bovive muscle: Carprofen 5µg/kg Flunixin 2µg/kg Tolfenamic acid 5µg/kg Meloxicam 2µg/kg Salicylic acid Phenylbutazone Flunixin Tolfenamic acid Meloxicam Desoximethasome (IS) Ketoprofen 3mL-6mg sorbent HLB-5.S.3.6 for 5 cartridges www.affinisep.com 6

AttractSPE TM W/O-HLB DETERMINATION OF PENICILLIN BASED ANTIBACTERIALS ON MUSCLES PROTOCOL OF PURIFICATION FOR MUSCLES for muscle 2g muscle samples are mixed in a 5mL centrifuge tube with 1mL.1 M sodium phosphate buffer (ph 4.5) and then homogenized. Add 2.5 ml.17 M sulfuric acid, 2.5 ml 5% sodium tungstate and mix it well,. Centrifuge at 5 rpm during 15 min. The supernatant was adjusted at ph 8.1 ~ 8.5 with 5M NaOH, centrifuged at 5 rpm during 15 min and the supernatant is collected and mix with 1mL NaCl (2%) to obtain the loading solution. Ampicillin, Amoxicillin Penicillin G or benzypenicillin Penicillin V Oxacillin Nafcillin, Cloxacillin Dicloxacillin Purification with a 6mL/2mg AttractSPE TM W/O HLB cartridge 5mL Methanol 5mL Water 5mL 2% NaCl solution Washing 5mL 2% NaCl 5mL 25mM PBS (ph 9,) Drying 5 min 2x 3mL Acetonitrile Evaporate under nitroger at 4 C and reconstituted before analysis. Detection LC-MS/MS 6mL-2mg sorbent HLB-5.S.6.2 for 5 cartridges AttractSPE TM W/O-HLB DETERMINATION OF ERYTHROMYCIN AND CLINDAMYCIN PROTOCOL OF PURIFICATION Mix 2g of crushed samples with 6mL of 2% acetic acid solution in centrifuge tube and then centrifuge at 12g for 1 min to form the loading solution. Purification with a 6mL/2mg AttractSPE TM W/O HLB cartridge 5mL Methanol 5mL Water solution Washing 5mL distilled water 6mL Methanol Detection LC-MS Regulation EC 37/21 Erythromycin 2kg/kg muscle 6mL-2mg sorbent HLB-5.S.6.2 for 5 cartridges www.affinisep.com 61

AttractSPE TM SAX DETERMINATION OF QUINOXALINE-2 -CARBOXYLIC ACID AND 3- METHYL QUINOXALINE-2-CARBOXYLIC ACID IN MUSCLE, LIVER, KIDNEYS PROTOCOL OF PURIFICATION Step 1: Mix 5 g homogenized sample with 1µL of internal standard (Quinoxaline-2-carboxylic acid-d4) and 1mL solution of 1% Methanol in water containing 5% metaphosphoric acid in a 5 ml centrifuge tube and shake, then centrifuge at 45rpm for 1 min at 3 C. Step 2: Collect the supernatant in a 5mL centrifuge tube. Repeat the step 1 with 1mL solution of 1% Methanol in water containing 5% metaphosphoric acid and centrifuge at 5rpm for 2 min at 3 C. Sept 3: Combine the supernatants (~2mL) Step 4: Mix vigorously with 1mL Ethyl Acetate for 15min then centrifuge at 5rpm for 1 min at 3 C and collect the supernatant. Step 5: repeat step 4, combine the supernatants, concentrate them at 6 C under nitrogen. The residue is dissolved in 5mL HCl.1M. Purification with a 3mL/6mg AttractSPE TM SAX cartridge 3mL Methanol 3mL Water solution Washing 3mL water Drying for 5min 3mL Methanol -.1M HCl (9-1 v/v) This eluate was dried at 6 under nitrogen Analysis: LC-MS/MS 3mL-6mg sorbent SAX-5.S.3.6 for 5 cartridges www.affinisep.com 62

MULTI-CLASS METHOD OF ANTIBIOTICS IN MILK AttractSPE TM W/O - HLB PROTOCOL OF PURIFICATION Mix 1mL Milk and 1mL Acetonitrile in a PP centrifuge tube. Voxtex for 1-15s and centrifuge at 4rpm (4 C) for 1min. Collect the supernatant (avoiding any visible fat layer) and add to a glass test tube containing 9mL,1% Formic acid. Vortex the tube during 1 s to obtain the loading solution. 25 substances measured from Fluoroquinolones Beta lactam Sulfonamide Macrolides Purification with a 3mL/6mg AttractSPE TM W/O HLB cartridge 3mL Acetonitrile 3mL water with,1% formic acid/acetonitrile (95/5, v /v) solution Washing 2x2mL,1% formic acid Drying 3s 2.5mL Acetonitrile/Methanol 7/3 Analysis: LC-MS/MS DETERMINATION OF VANCOMYCIN IN FISH 6mL-2mg sorbent HLB-5.S.3.6 for 5 cartridges Same method as FDA Lab information bulletin LIB# 4443, Susan B. Clark, Joseph M. Storey, Sherri B. Turnipseed AttractSPE TM SCX PROTOCOL OF PURIFICATION Step 1: Mix 5 g homogenized sample with a 15mL solution of 2% ACN in water in a 5 ml centrifuge tube and shake for 2 min, then centrifuge at 7 6rpm for 1 min. Step 2: Collect the supernatant in a 5mL centrifuge tube. Repeat the step 1 with 1mL solution of 2% ACN in water. Combine the supernatants (~25mL) and mix vigorously with 1mL Hexane for 1min then centrifuge at 7 6rpm for 1 min and remove Hexane to obtain the loading solution. Purification with a 3mL/6mg AttractSPE TM SCX cartridge 3mL Methanol 3mL Water-.1% Formic acid 3mL solution Washing 3mL water 3mL Methanol with 3% Ammonium hydroxide This eluate was dried at 5 under nitrogen and reconstituted in 1mL water and filtered at.2µm. Analysis: LC-MS/MS 3mL-6mg sorbent SCX-5.S.3.6 for 5 cartridges www.affinisep.com 63

DETERMINATION OF GLUCOCORTICOIDS AttractSPE TM W/O-HLB PROTOCOL OF PURIFICATION Mix 2 g of the sample and 1mL of acetate buffer solution (3M, ph 4.6) in a 5 ml centrifuge tubes and homogenize for about 2 minutes. Make an enzymatic hydrolysis by adding 5µL Helix pomatia β- Glucuronidase/Arylsulfatase for 1h in an oven at 6. After cooling at RT, add 8 ml CAN and centrifuge at 45rpm for 1 min. Repeat the above steps. Collect the supernatants and concentrate under nitrogen at 5. Dissolve the residue in 1mL Ethanol and add 5mL of distilled water to obtain the loading solution. Purification with a 6mL/5mg AttractSPE TM W/O HLB cartridge 5mL Methanol 5mL Water solution Washing 5mL acetone/distilled water (2/8, v / v) 5mL n-hexane Drying 2 min 6mL Ethyl Acetate Evaporate under nitrogen at 5 C and reconstituted before analysis with 1mL mobile phase. Centrifuge at -4, 15rpm during 15 min. The clear supernatant is filterer.2 μm nylon filter and analyzed by LC-MS/MS after the filtration Detection LC-MS/MS Cortisone, Corticosterone, Aldosterone, Betamethasone, Dexamethasone, Flumethasone, Prednisone, Prednisolone, Methylprednisolone 6mL-5mg sorbent HLB-5.S.6.5 for 5 cartridges AttractSPE TM SCX DETERMINATION OF SULFONAMIDES IN MILK PROTOCOL OF PURIFICATION Purification with a 3mL/6mg AttractSPE TM SCX cartridge 2mL Methanol 2mL Water 5mL Milk Washing of interferences 2mL Methanol/Water (5/95) 1mL.5M HCl 2mL Methanol/Water (2/8) 2.5mL Ammonium bicarbonate/methanol (1/9) The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. Analyse LC/MS-MS Regulations for Sulfonamides: Sulfadimethoxine.1ppm in milk (U.S. FDA 21 CFR 556.64) Sulfaethoxypyridazine in milk (U.S. FDA 21 CFR 556.65) EC 37/21 1µg/kg Milk 6mL-15mg sorbent SCX-25.S.3.6 for 25 cartridges SCX-5.S.3.6 for 5 cartridges www.affinisep.com 64

AttractSPE TM W/O-HLB MULTI-CLASS METHOD OF ANTIBIOTICS IN DISTILLER GRAINS PROTOCOL OF PURIFICATION Mix 5mL Distillers grains with 2mL 1,5mM EDTA and 2mL 1% Trichloroacetic acid in water in a 5mL PP centrifuge tube. Shake for 15min and centrifuge at 4rpm for 1min. Collect the supernatant and dilute it with 15mL water. Repeat extraction with 3mL methanol and centrifuge and combine supernatants. Dilute the supernatants to 2mL with water to obtain the loading solution. Purification with a 6mL/15mg AttractSPE TM W/O HLB cartridge 3mL Methanol 3mL water with Trichloroacetic acid (ph~4) 1mL solution Drying under vacuum for 5min Washing 5mL water Drying under vacuum for 5min 2.5mL Methanol Evaporate eluate to about 1mL under nitrogen at 35 C. Analysis: LC-MS/MS Analyses of 13 antibiotics Ampicillin, bacitracin A, erythromycin, tylosin, clarithromycin, penicillin G, virginiamycin M1 and monensin 6mL-15mg sorbent HLB-5.S.6.15 for 5 cartridges Same method as FDA Lab information bulletin LIB# 4438 David N. Heller G.K. Hemakanthi de Alwis AttractSPE TM W/O-HLB DETERMINATION OF PRAZIQUANTEL AND TIAMULIN PROTOCOL OF PURIFICATION Mix 2g of pulverized samples with 6mL of 2% ammonium hydroxide solution in centrifuge tube and then centrifuge at 12g for 1 min to form the loading solution. Purification with a 6mL/2mg AttractSPE TM W/O HLB cartridge 5mL Methanol 5mL Water solution Washing 5mL distilled water 6mL Methanol Analysis: LC-MS 6mL-2mg sorbent HLB-5.S.6.2 for 5 cartridges www.affinisep.com 65

DETERMINATION OF CEPHALEXIN IN FISH AttractSPE TM SCX PROTOCOL OF PURIFICATION Mix 2g of homogenized samples with 5mL of 1% trichloroacetic acid solution in a 5mL centrifuge tube and then centrifuge at 3g for 1 min. Collect the supernatant and make a 1:1 mix with a 4% phosphoric acid to form the loading solution. Purification with a 3mL/6mg AttractSPE TM SCX cartridge 2mL Methanol 2mL Water solution Washing 2mL 2% Formic acid 2mL Methanol 4mL Methanol with 5% Ammonium hydroxide This eluate was concentrated at 5 under nitrogen and reconstituted with 2µL.1% formic acid containing 1% acetonitrile. The solution was centrifuged at 12 G for 1 minutes and the supernatant was filtered with a membrane filter. Analysis: LC-UV (26nm) 3mL-6mg sorbent SCX-5.S.3.6 for 5 cartridges AttractSPE TM SCX DETERMINATION OF VALNEMULIN AND TIAMULIN IN FISH PROTOCOL OF PURIFICATION Step 1: Mix 1 g homogenized sample with a 1mL solution of 4-6 (v:v) ACN-,1M HCl in a 5 ml centrifuge tube and shake at 3rpm for 15 min, then centrifuge at 1 rpm for 1 min. Step 2: Collect the supernatant in a 5mL centrifuge tube. Repeat the step 1 with the lower layer of the first centrifuge tube. Combine the supernatants (~2mL) and mix vigorously with 2mL Hexane and remove Hexane to obtain the loading solution. Purification with a 3mL/6mg AttractSPE TM SCX cartridge 3mL Methanol 3mL Water 3mL solution Washing 3mL 4-6 (v:v) ACN-,1M HCl Drying 1min 3mL Methanol with 5% Ammonium hydroxide This eluate was dried at 4 under nitrogen and reconstituted in the mobile phase. B. Assay conditions Analysis: LC-MS/MS 3mL-6mg sorbent SCX-5.S.3.6 for 5 cartridges www.affinisep.com 66

AttractSPE TM W/O-HLB DETERMINATION OF PHARMACEUTICALS AND PERSONAL CARE PRODUCTS (GROUP 1 COMPOUNDS) OF EPA METHOD 1694 PROTOCOL OF PURIFICATION Filtrate 1L solution and adjust the ph to 2 while stirring the water. Add 5mg Na 4 EDTA and mix. Equilibrate during 1-2h to obtain the loading solution. Purification with a 6mL/2mg AttractSPE TM W/O - HLB cartridge 2mL Methanol 6mL Water 6mL Water ph 2 1L of loading solution, ph 2 Washing 1mL water Drying 5 min 6mL Methanol or 6mL Methanol Acetonitrile (5/5) The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. EPA methods 1694 Acetaminophen Ormetoprim Caffeine Sarafloxacin Carbadox Sulfachloropyrida Cefotaxime zine Ciprofloxacin Sulfadiazine Clinafloxacin Sulfamerazine Codeine Sulfamethazine Cotinine Sulfamethizole 1,7- Sulfamethoxazole Dimethylxanthine Sulfanilamide Enrofloxacin Sulfathiazole Lincomycin Thiabendazole Lomefloxacin Trimethoprim Norfloxacin Ofloxacin 6mL-2mg sorbent HLB-5.S.6.2 for 5 cartridges AttractSPE TM W/O-HLB DETERMINATION OF MISCELLANEOUS DRUGS IN WASTEWATER PROTOCOL OF PURIFICATION Filtrate 5mL to 1L of wastewater with,45µm glass fiber to form the loading solution. Purification with a 3mL/6mg AttractSPE TM W/O - HLB cartridge 3mL Ethyl Acetate 3mL Methanol 3mL Water solution (15mL/min) Washing 3mL Methanol/ water (5/95, v/v) 3mL n-hexane 3x1mL Ethyl Acetate Evaporate under nitroger and reconstituted with.5ml Methanol. Analysis: LC-DAD-Fluorescence Caffeine Acetaminophen Diclofenac Ibuprofen Ketoprofen Naproxen Carbamazepine 3mL-6mg sorbent HLB-5.S.3.6 for 5 cartridges www.affinisep.com 67

Analysis of PESTICIDES www.affinisep.com 68 68

uau AFFINIMIP SPE Picolinic herbicides DETERMINATION OF AMINOPYRALID, CLOPYRALID AND PICLORAM IN COMPOST AND WATER Efficient clean-up and enrichment AFFINIMIP SPE 5 45 4 35 3 25 2 15 1 Amino pyralid Clopyralid Picloram Spiked Water solution Elution solution 5-5 -1-15 -2 Spiked Compost Not spiked Compost 2 4 6 8 1 12 14 16 18 2 Time (min) PROTOCOL OF PURIFICATION for compost 5g of compost sample and 1mL water are shaked during 6minutes. Centrifuge at 3g for 1min and then filter the solution with a 4-7µm filter. This solution is used as the loading solution. Purification with a 3mL/6mg AFFINIMIP SPE Picolinic Herbicides cartridge 2mL Acetonitrile 1mL Water 3mL of loading solution Washing of interferences (W1) 1mL Water Drying by applying vacuum 1 min Washing of interferences (W2) 1mL Acetonitrile 3mL 98/2 Ethyl acetate / Trifluoroacetic acid The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. UV chromatogram of compost or water spiked with Aminopyralid, Picloram and Clopyralid after AFFINIMIP SPE Picolinic Herbicides clean-up Recovery and repeatability of Picloram, Aminopyralid and Clopyralid in compost (n=3) and after AFFINIMIP SPE Picolinic Herbicides Clean-up. Analytes Recoveries % for Water 3mL-6mg sorbent FS115-2 for 25 cartridges FS115-3 for 5 cartridges 6mL-1mg sorbent FS115-2B for 25 cartridges FS115-3B for 5 cartridges Recoveries % for Compost % RSDr compos t Aminopyralid 95 84 3 Clopyralid 19 12 4 Picloram 88 89 3 www.affinisep.com 69

AFFINIMIP SPE Glysophate - AMPA DETERMINATION OF GLYPHOSATE AND AMPA IN WATER Efficient clean-up and enrichment Also tested with up to 2% Methanol for percolation and washing PROTOCOL OF PURIFICATION Purification with a 3mL AFFINIMIP SPE Glyphosate AMPA cartridge 6mL Water Water Washing of interferences (W1) 3mL Water 3mL HCl solution (1mM) The elution fraction was then derivatized with FMOC and dissolved in the mobile phase before HPLC analysis. Method UPLC MS/MS Column: Acquity UPLC HSS T3 (2.1mm x 1mm, 1,8µm) Mobile phase: % Time (min) Water/Ammo nium Acetate % ACN 5mM 9 1 2 9 1 7 5 5 7.5 1 11 1 flow rate:.2ml/min MS detection: m/z 321 (ESI - ) Injection volume: 2µL. Recovery Yields of AMPA and Glyphosate in water for a range of concentration of 5 to 45ng/L of Glyphosate and of 5 to 55ng/L of AMPA after AFFINIMIP SPE Glyphosate - AMPA clean-up Sample Recoveries % GLYPHOSATE >8% AMPA >75% Poster: Extraction SPE basée sur un polymère à empreintes moléculaires pour l extraction du glyphosate et de son métabolite (AMPA) dans des eaux souterraines, by BRGM and ICOA (ANR project Origami), AFSEP 214 Paris. New Selective SPE Clean-Up Method Based on Molecularly Imprinted Polymers for Glyphosate and AMPA Analysis with and without Derivatization for Water, Food and Feed, Pittcon 215 New Orleans. 3mL cartridge FS113-2 for 25 cartridges FS113-3 for 5 cartridges www.affinisep.com 7

AttractSPE TM W/O-HLB DETERMINATION OF 16 PESTICIDES FROM GROUNDWATER PROTOCOL OF PURIFICATION Purification with a 6mL/2mg W/O -HLB cartridge 1mL Dichloromethane 1mL Acetonitrile 1mL Water 1L Water sample Washing of interferences 5mL Water/Methanol 95/5 5mL Acetonitrile 5mL Methanol Analyse HPLC AttractSPE TM Desysopropylatrazine, Desethylatrazine, Aldocarb, Simazine, Carbofuran, Metalaxyl, Atrazine, 2, 4-D, Metazachlor, Dicloran, Phenmedipham, Linuron, Iprodione, Procymidone, Fenitrothion, Vinclozolin 6mL-2mg sorbent HLB-5.S.6.2 for 5 cartridges AttractSPE TM W/O-HLB DETERMINATION OF PESTICIDES FROM SURFACE WATER PROTOCOL OF PURIFICATION Purification with a 6mL/2mg AttractSPE TM W/O - HLB cartridge 3mL Methanol/Acetonitrile 5/5 6mL Water 1L Water sample (+.5g NaCl) Drying 3mL Acetonitrile/Methanol 5/5 Analyse HPLC Desethylsimazin, 2, 6- Dichlorbenzamid, Ethidimuron, Chloridazon, Desethylatrazin, Desethylsebuthylazin, Bromacil, Simazin, Metribuzin, Desethylterbuthylazin, Metabenzthiazuron, Chlortoluron, Atrazin, Diuron, Isoproturon, Matazaclor, Terbumeton, Sebuthylazin, Propazin, Dimefuron, Terbuthylazin, Triadimenol, Epoxiconazol, Terbutryn, Metolachlor, propiconazol, Kresoxim- methyl 6mL-2mg sorbent HLB-5.S.6.2 for 5 cartridges www.affinisep.com 71

AttractSPE TM W/O-HLB DETERMINATION OF ACETAMIDE HERBICIDES IN DRINKING WATER PROTOCOL OF PURIFICATION Purification with a 3mL/6mg cartridge 3mL Methanol 2mL Water 15mL Water sample Washing of interferences 1mL Water 1mL Methanol Analyse HPLC AttractSPE TM HLB Metolachlor metabolite, 2- Chloro 2, 6 diethylacetanilide, 2,6 Diethylaniline, Alachlor, Metolachlor 3mL-6mg sorbent HLB-5.S.3.6 for 5 cartridges AttractSPE TM W/O-HLB DETERMINATION OF HERBICIDES IN DRINKING WATER PROTOCOL OF PURIFICATION Purification with a 3mL/6mg AttractSPE TM HLB cartridge 3mL Methanol 6mL Water 75mL Water sample Washing of interferences 1mL Water 1mL Methanol Analyse HPLC Desispropylatrazine, Hydroxyatrazine, Desethylatrazine, Simazine, Cyanazine, Atrazine 6mL-2mg sorbent HLB-5.S.3.6 for 5 cartridges www.affinisep.com 72

Analyses of OTHER RESIDUES AND MISCELLANEOUS www.affinisep.com 73

AFFINIMIP SPE Metanephrines DETERMINATION OF METANEFHRINES IN PLASMA COMPARISON WITH WCX CARTRIDGES The plasma or serum is diluted by 5 with water. This solution is used as the loading solution. WCX AFFINIMIP SPE Cleanup with a 1mL AFFINIMIP SPE Metanephrines cartridge 1mL of phosphate buffer ph 7 2mL Water 1.5mL of loading solution Washing of interferences (W1) 1mL Water 5µL Water/Methanol (6/4) Drying 1 seconds Washing of interferences (W2) 5µL Methanol 1mL Methanol 5% Acetic acid The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. Analysis by LC-MS/MS: Total Ion Current of a calf serum after Cleanup by AFFINIMIP SPE Metanephrines. The sample naturally contained Metanephrine. Concentration of MN found: 3nM. In parallel, a SPE was performed on a protocol developed for the analysis of MN using WCX cartridges: the concentration obtained was 7nM for the same sample. WCX Recovery : 33% Recoveries of MN and NMN at a contamination level of 5nM in rabbit plasma after AFFINIMIP SPE Metanephrines Clean-up and relative standard deviation calculated from results generated under reproducibility conditions (Analysis by LC-MS). Analytes Recoveries % % RSD R Metanephrine 79.4 6.3 Normetanephrine 19 11 HPLC Method with LC-MS/MS detection Column: Syncronis aq column 15mm x 2.1mm Mobile phase: Water.1% Formic Acid flow rate:.2ml/min MS detection: m/z 322 (ESI + ) Injection volume: 2µL. NMN 1mL cartridge DG11-2A for 25 cartridges DG11-3A for 5 cartridges 3mL cartridge DG11-2 for 25 cartridges DG11-3 for 5 cartridges AFFINIMIP SPE Recovery: 11% Analysis by LC-MS/MS: Selected ion monitoring of Normetanephrine (m/z 18). Chromatograms obtained after Cleanup by AFFINIMIP SPE Metanephrines or by WCX of a calf serum spiked at 27nM with Normetanephrine. www.affinisep.com 74

35 3 25 2 15 1 5-5 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 26 24 22 2 18 16 14 12 1 8 6 4 2-2 Minutes 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 Minutes 2 35 3 25 2 15 1 5-5 AFFINIMIP SPE Phenolics DETERMINATION OF GUAIACOL General structure of Guaîacol Guaïacol Cleanup with a 3mL/1mg AFFINIMIP SPE Phenolics cartridge 3mL Acetonitrile 3mL Water Up to 2mL of red or white wine Washing of interferences 3mL Water / Acetonitrile (8/2 v/v) 2mL Methanol Chromatograms obtained after clean-up with AFFINIMIP SPE Phenolics of red wine spiked with Guaïacol (.1µM) (red) or not spiked (blue). Recovery yields and reproducibility evaluated with 3 cartridges and 3 different batches of AFFINIMIP SPE Phenolics by matrix (n=9) Guaïacol C (µm) Recoveries % RSD R % Red wine 1.1 88.1 3.9 Red wine 2.1 93.1 3.7 White wine 1.2 96.8 1.7 Chromatograms obtained before (red) and after (blue) clean-up with AFFINIMIP SPE Phenolics of red wine spiked with Guaïacol (.1µM) White wine 2.2 93.5 2.6 HPLC-UV Method Column: Thermo Hypersil gold, 15mm x 4.6mm Mobile phase: 15/85 (v/v) Acetonitrile Water Flow rate: 1mL/min Detection: UV - 272nm Injection volume: 1µL. 3mL-1mg sorbent FS13-2 for 25 cartridges FS13-3 for 5 cartridges www.affinisep.com 75

45 4 35 3 25 2 15 1 5 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 5 45 4 35 3 25 2 15 1 5 UV 6-23nm E ZON ethanol.dat UV 6-23nm E ZON 75-25.dat Minutes 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 Minutes 5 45 4 35 3 25 2 15 1 5 45 4 35 3 25 2 15 1 5 DETERMINATION OF CARNOSIC ACID IN MEAT AFFINIMIP SPE Phenolics 25g of turkey was mixed with 2mL of 74.5/25/.5 ACN/H 2 O/H 3 PO 4 or Ethanol-.5% H 3 PO 4 using a blender during 3 minutes. After, the mixture was mixed during 3 minutes with magnetic stirrer. The mixture was filtered on filter paper (4-7µm). Then the mixture was diluted by 2 with water. Carnosic acid Cleanup with a 3mL/1mg AFFINIMIP SPE Phenolics cartridge 3mL Acetonitrile 3mL Water Up to 8mL of loading solution Washing of interferences 3mL Water / Acetonitrile (6/4 v/v) 2mL Methanol -1% H 3 PO 4 Chromatogram of a turkey containing 5ppm of Carnosic acid after clean-up with AFFINIMIP SPE Phenolics. Extraction of the turkey with Ethanol-.5% H3PO4 Carnosic acid Chromatogram of a turkey containing 5ppm of Carnosic acid after clean-up with AFFINIMIP SPE Phenolics. Extraction of the turkey with 74.5/25/.5 ACN/H2O/H3PO4 Recovery yields obtained by both extraction solvent after AFFINIMIP SPE Phenolics Cleanup. Extraction solvent Recoveries % HPLC-UV Method Column: Thermo Hypersil gold, 15mm x 4.6mm Mobile phase: 65/35 (v/v) ACN/Water-.5% H 3 PO 4 Flow rate: 1mL/min Detection: UV - 23nm Injection volume: 5µL. 74.5/25/.5 ACN/H 2 O/H 3 PO 4 >85% Ethanol-.5% H 3 PO 4 >8% 3mL-1mg sorbent FS13-2 for 25 cartridges FS13-3 for 5 cartridges www.affinisep.com 76

AttractSPE TM W/O-HLB DETERMINATION OF ARTIFICIAL SWEETENERS IN WATER PROTOCOL OF PURIFICATION Purification with a 6mL/2mg AttractSPE TM W/O HLB cartridge 5mL Methanol 5mL Water ph~2 5mL of water (adjusted to ph ~2) Washing 1mL distilled water ph ~2 Drying 3min 2mL Methanol Acesulfame, Aspartame, Cyclamate, Neohespiridine dihydrochalcone, Saccharin, Sucralose 6mL-2mg sorbent HLB-5.S.6.2 for 5 cartridges Analysis: LC-MS/MS AttractSPE TM W/O-HLB DETERMINATION OF COCAINE AND MAIN METABOLITES IN WASTE WATER PROTOCOL OF PURIFICATION Waste water was adjusted to ph 2 with 37%HCl and filtered to form the loading solution. Purification with a 6mL/5mg AttractSPE TM W/O HLB cartridge 3mL Methanol 3mL Water ph~2 1mL Waste water ph ~6 Washing 3mL Methanol/Water (5/95) Drying 15min 2x4mL Methanol Analysis: LC-MS/MS Cocaine, benzoylecgonine and ecgonine methyl ester 6mL-5mg sorbent HLB-5.S.6.5 for 5 cartridges www.affinisep.com 77

DETERMINATION OF MELAMINE IN MILK AttractSPE TM SCX PROTOCOL OF PURIFICATION Add 4mL water to 5g liquid infant formula or 1g dry infant formula. Shake during 1-2min with 2mL 5/5 ACN/Water and centrifuge for 1minutes at 34 rpm. The supernatant is the loading solution. Purification with a 6mL/15mg AttractSPE TM SCX cartridge 5mL.1M NaOH in Acetonitrile 5mL.1M HCl in Acetonitrile 5mL Acetonitrile 5mL 4% Formic acid in Water 3mL 4% Formic acid in Water 2mL of loading solution Washing of interferences 5mL Acetonitrile 5mL.2% diethyamine in Acetonitrile 4mL 2% diethylamine in Acetonitrile The elution fraction was filtered and then evaporated under nitrogen and dissolved in the mobile phase before HPLC analysis. Analyse LC-MS/MS Regulations for Melamine: Codex alimentarius 35 th CAC session (july 212): Maximum limit.15mg/kg for liquid infant milk 6mL-15mg sorbent SCX-25.S.6.15 for 25 cartridges SCX-5.S.6.15 for 5 cartridges AttractSPE TM SAX DETERMINATION OF CYANURIC ACID IN MILK PROTOCOL OF PURIFICATION Add 4mL water to 5g liquid infant formula or 1g dry infant formula. Shake during 1-2min with 2mL 5/5 ACN/Water and centrifuge for 1minutes at 34 rpm. The supernatant is the loading solution Purification with a 6mL/15mg AttractSPE TM SAX cartridge 5mL.1M HCl in Acetonitrile 5mL.1M NaOH in Acetonitrile 5mL Acetonitrile 5mL 5% NH 4 OH in Water 3mL 5% NH 4 OH in Water 2mL of loading solution Washing of interferences 5mL Acetonitrile 2mL 4% Formic acid in Acetonitrile The elution fraction was filtered and then evaporated under nitrogen and dissolved in the mobile phase before HPLC analysis. Analyse LC-MS/MS 6mL-15mg sorbent SAX-25.S.6.15 for 25 cartridges SAX-5.S.6.15 for 5 cartridges www.affinisep.com 78

DETERMINATION OF MELAMINE IN FOOD AttractSPE TM SCX PROTOCOL OF PURIFICATION Add 5mL water and 5mL Acetonitrile to 1g pulverized sample. Shake thoroughly for 3 min and centrifuge for 1minutes at 26 rpm. The supernatant is filtered through,45µm membrane filter to obtain the loading solution. Purification with a 6mL/15mg AttractSPE TM SCX cartridge 5mL Acetonitrile 5mL 4% Formic acid in Water 3mL 4% Formic acid in Water 2mL of loading solution Washing of interferences 5mL Acetonitrile 5mL.2% diethyamine in Acetonitrile 4mL 2% diethylamine in Acetonitrile The elution fraction was filtered and then evaporated under nitrogen at 5 C and dissolved in the mobile phase before HPLC analysis. Analyse LC-MS/MS 6mL-15mg sorbent SCX-25.S.6.15 for 25 cartridges SCX-5.S.6.15 for 5 cartridges AttractSPE TM W/O-HLB DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS FROM DRINKING WATER PROTOCOL OF PURIFICATION Purification with a 6mL/2mg AttractSPE TM W/O - HLB cartridge 5mL Dichloromethane 5mL Methanol 5mL Water 5mL Water sample Washing of interferences 6mL Water 8mL Dichloromethane Analyse HPLC Naphtalene, Acenaphthylene, Acenaphthene, Fluorene, Phenanthrene, Anthracene, Fluoranthene, Pyrene, Benzo(a)anthracene, Chrysene, Benzo(b)fluoranthene, Benzo(k)fluoranthene, Benzo(a)pyrene, Dibenzo(g, h, l)perylene, Benzo(a)pyrene, Benzoperylene, Indenopyrene 6mL-2mg sorbent HLB-5.S.6.2 for 5 cartridges www.affinisep.com 79

PRODUCT LIST www.affinisep.com 8

AFFINIMIP SPE PRODUCT LIST FOR MYCOTOXINS ANALYSES Products Designation Definition Reference Multimycotoxins Zearalenone and Fumonisins Patulin Ochratoxin A Deoxynivalenol Zearalenone AFFINIMIP SPE MultimycoLCMSMS AFFINIMIP SPE FumoZON AFFINIMIP SPE Patulin AFFINIMIP SPE Patulin & Pectinase kit AFFINIMIP SPE Ochratoxin A AFFINIMIP SPE Deoxynivalenol AFFINIMIP SPE Zearalenone 3mL Selective SPE cartridges for Multimycotoxin analyses 6mL Selective SPE cartridges for Multimycotoxin analyses 3mL Selective SPE cartridges for Zearalenone and Fumonisins 3mL 1mg Selective SPE cartridges for Patulin 6mL 2mg Selective SPE cartridges for Patulin Kit of 3mL selective SPE cartridges for Patulin + 5mL Pectinase enzyme solution Kit of 6mL - 2mg selective SPE cartridges for Patulin in dried apple + 5mL Pectinase enzyme solution 3mL Selective SPE cartridges for Ochratoxin A 6mL Selective SPE cartridges for Ochratoxin A 6mL -1mg selective SPE cartridges for Deoxynivalenol mycotoxin in food and babyfood 6mL 2mg selective SPE cartridges for Deoxynivalenol mycotoxin in feed 3mL Selective SPE cartridges for ZON Nber of cartridges FS118-2 25 FS118-3 5 FS118-2B 25 FS118-3B 5 FS19-2 25 FS19-3 5 FS12-2 25 FS12-3 5 FS12-2B-2mg 25 FS12-3B-2mg 5 FS12-2K 25 FS12-3K 5 FS12-2KB- 2mg FS12-3KB- 2mg 25 5 FS11-2 25 FS11-3 5 FS11-2B 25 FS11-3B 5 FS117-2B 25 FS117-3B 5 FS117-2B-2mg 25 FS117-3B-2mg 5 FS1-2 25 FS1-3 5 Pectinase 5 ml Pectinase enzyme solution REA-1-5mL For other formats, please contact us AFFINISEP can provide you with other formats than the one described in the product list. Other formats available on demand and with an adapted protocol can be : different volumes of SPE cartridges (1mL, 3mL, LRC, 6mL, etc ) 96 well plates, HPLC columns, Preparative HPLC columns the format adapted to your application and your automate www.affinisep.com 81

AFFINIMIP SPE PRODUCT LIST (MISCELLANEOUS) Products Designation Definition Reference Bisphenol A Estrogens Catecholamines Metanephrines Picloram, Aminopyralid, Clopyralid Glyphosate, AMPA Amphetamines Chloramphenicol Tamoxifen Tetracyclines Zeranol Residues Phenolics AFFINIMIP SPE Bisphenol A AFFINIMIP SPE Estrogens AFFINIMIP SPE Catecholamines AFFINIMIP SPE Metanephrines AFFINIMIP SPE Picolinic Herbicides AFFINIMIP SPE Glyphosate - AMPA AFFINIMIP SPE Amphetamines AFFINIMIP SPE Chloramphenicol AFFINIMIP SPE Tamoxifen AFFINIMIP SPE Tetracyclines AFFINIMIP SPE Zeranol Residues AFFINIMIP SPE Phenolics www.affinisep.com 82 3mL Selective SPE cartridges for Bisphenol A (PP) 6mL Selective SPE cartridges for Bisphenol A (PP) 6mL Selective SPE cartridges for Bisphenol A (Glass) 1mL Selective SPE cartridges for Estrogens 3mL Selective SPE cartridges for Estrogens 3mL Selective SPE cartridges for Catecholamines 1mL Selective SPE cartridges for Catecholamines 3mL Selective SPE cartridges for Metanephrines 1mL Selective SPE cartridges for Metanephrines 3mL Selective SPE cartridges for Picolinic acid based herbicides 3mL Selective SPE cartridges for Glyphosate and AMPA 3mL Selective SPE cartridges for Amphetamines derivatives 1mL Selective SPE cartridges for Chloramphenicol Nber of cartridges FS16-2 25 FS16-3 5 FS16-2B 25 FS16-3B 5 FS16-2G 25 FS16-3G 5 FS14-2A 25 FS14-3A 5 FS14-2 25 FS14-3 5 DG1-2 25 DG1-3 5 DG1-2A 25 DG1-3A 5 DG11-2 25 DG11-3 5 DG11-2A 25 DG11-3A 5 FS115-2 25 FS115-3 5 FS113-2 25 FS113-3 5 DG12-2 25 DG12-3 5 FS11-2A 25 FS11-3A 5 3mL Selective SPE cartridges for Tamoxifen PH11-2 25 1mL Selective SPE cartridges for Tetracyclines 3mL Selective SPE cartridges for Zeranol Residues 3mL Selective SPE cartridges for Phenolic compounds FS112-2A 25 FS112-3A 5 FS15-2 25 FS15-3 5 FS13-3 5

POLYMERIC AttractSPE TM PRODUCT LIST Cartridges format, Sorbent amount Nber /box AttractSPETM W/O - HLB AttractSPE TM SCX AttractSPE TM WCX AttractSPE TM SAX AttractSPE TM WAX AttractSPE TM DVB 1mL, 3mg 1 HLB-1.S.1.3 SCX-1.S.1.3 WCX-1.S.1.3 SAX-1.S.1.3 WAX- 1.S.1.3 DVB-1.S.1.3 3mL, 6mg 5 HLB-5.S.3.6 SCX-5.S.3.6 WCX-5.S.3.6 SAX-5.S.3.6 WAX-5.S.3.6 DVB-5.S.3.6 1 HLB-1.S.3.6 SCX-1.S.3.6 WCX-1.S.3.6 SAX-1.S.3.6 WAX- 1.S.3.6 DVB-1.S.3.6 25 HLB-25.S.6.2 SCX-25.S.6.2 WCX-25.S.6.2 SAX-25.S.6.2 WAX- 25.S.6.2 DVB-25.S.6.2 6mL, 2mg 5 HLB-5.S.6.2 SCX-5.S.6.2 WCX-5.S.6.2 SAX-5.S.6.2 WAX- 5.S.6.2 DVB-5.S.6.2 1 HLB- 1.S.6.2 SCX-1.S.6.2 WCX- 1.S.6.2 SAX- 1.S.6.2 WAX- 1.S.6.2 DVB- 1.S.6.2 25 HLB-25.S.6.5 SCX-25.S.6.5 WCX-25.S.6.5 SAX-25.S.6.5 WAX- 25.S.6.5 DVB-25.S.6.5 6mL, 5mg 5 HLB-5.S.6.5 SCX-5.S.6.5 WCX-5.S.6.5 SAX-5.S.6.5 WAX- 5.S.6.5 DVB-5.S.6.5 1 HLB- 1.S.6.5 SCX-1.S.6.5 WCX- 1.S.6.5 SAX- 1.S.6.5 WAX- 1.S.6.5 DVB- 1.S.6.5 96 wells Plate, 3mg 1 HLB-1.96W.3 SCX-1.96W.3 WCX-1.96W.3 SAX-1.96W.3 WAX-1.96W.3 DVB-1.96W.3 Reversible.7mL, 3mg 25 5 HLB- 25.REV.1.N1 HLB- 5.REV.1.N1 SCX- 25.REV.1.N1 SCX- 5.REV.1.N1 WCX- 25.REV.1.N1 WCX- 5.REV.1.N1 SAX- 25.REV.1.N1 SAX- 5.REV.1.N1 WAX- 25.REV.1.N1 WAX- 5.REV.1.N1 DVB- 25.REV.1.N1 DVB- 5.REV.1.N1 Reversible.7mL, 1mg (2mg for DVB) 25 HLB-25.REV.1.F SCX-25.REV.1.F WCX-25.REV.1.F SAX-25.REV.1.F 5 HLB-5.REV.1.F SCX-5.REV.1.F WCX-5.REV.1.F SAX-5.REV.1.F WAX- 25.REV.1.F WAX- 5.REV.1.F DVB- 25.REV.1.F DVB- 5.REV.1.F Reversible 2mL, 225mg 25 5 HLB- 25.REV.2.N1 SCX- 25.REV.2.N1 For other formats, please contact us AFFINISEP can provide you with other formats than the one described in the product list. Other formats available on demand and with an adapted protocol can be : different volumes of SPE cartridges (1mL, 3mL, LRC, 6mL, etc ) 96 well plates, HPLC columns, Preparative HPLC columns the format adapted to your application and your automate www.affinisep.com 83 WCX- 25.REV.2.N1 SAX- 25.REV.2.N1 WAX- 25.REV.2.N1 HLB- 5.REV.2.N1 SCX- 5.REV.2.N1 WCX- 5.REV.2.N1 SAX- 5.REV.2.N1 WAX- 5.REV.2.N1

MYCOTOXINS STANDARDS & SPE ACCESSORIES- Product List MYCOTOXINS STANDARDS Product List Mycotoxines Aflatoxins Product Concentration (µg/ml) Volum e Référence Aflatoxin B1 2 1mL REA-AFLB1-1ML Aflatoxin B2.5 1mL REA-AFLB2-1ML Aflatoxin G1 2 1mL REA-AFLG1-1ML Aflatoxin G2.5 1mL REA-AFLG2-1ML Mixture Aflatoxins: B1-B2-G1-G2 2-.5-2-.5 1mL Aflatoxin M1.5 1mL REA-MIXAFL- 1ML REA-AFLM1-1ML Ochratoxin Ochratoxin A 1 1mL REA-OTA-1ML Patulin Patulin 1 1mL REA-PAT-1ML Trichothecenes A T2 Toxin 1 1mL REA-T2-1ML HT-2 Toxin 1 1mL REA-HT2-1ML Trichothecenes B Deoxynivalenol (DON) 1 1mL REA-DON-1ML Zearalenone Zearalenone 1 1mL REA-ZON-1ML Fumonisine B1 5 1mL REA-FB1-1ML Fumonisines Fumonisine B2 5 1mL REA-FB2-1ML Mixture Fumonisine: B1-B2 5 1mL REA-MIXFB-1ML SPE ACCESSORIES Product list SPE Accessories Designation Definition Reference Manifold SPE Vaccum Manifold 12-port model ACC-MAN1 SPE Adapter & Reservoir kit Mini-Vap Mini PUMP SPE Adapter & Reservoir kit Mini Evaporator/Concentrator Mini vacuum pump Kit of 12 reservoirs 6ml and adapters for use with 1,3 & 6 ml cartridges ACC-AR1 6 port Mini-Vap Evaporator/Concentrator for use with 1 ACC-VAP1 to 25mL containers Laboport diaphragm vacuum mini pump, 5.5L/min ACC-PUMP Vacuum pump trap SPE Vacuum pump trap kit 1L trap kit ACC-TRAP www.affinisep.com 84

NONE EXHAUSTIVE LIST OF PUBLICATIONS AND POSTERS Analysis of Mycotoxins Solid-phase extraction using molecularly imprinted polymers for selective extraction of a mycotoxin in cereals, J. Chrom. A., 1217, 6668-6673, 21. Molecularly imprinted polymer solid-phase extraction for detection of zearalenone in cereal sample extracts detection, Analytica Chimica Acta, 672, 15 19, 21. Molecularly imprinted polymer as sorbent in micro-solid phase extraction of ochratoxin A in coffee, grape juice and urine, Tien Ping Lee, Bahruddin Saad, Wejdan Shakir Khayoon, Baharuddin Salleh, Talanta, 88, 129 135, 212. Molecularly imprinted polymer-based solid phase clean-up for analysis of ochratoxin A in ginger and LC-MS/MS confirmation, J. Cao, S. Zhou, W. Kong, M. Yang, L. Wan, S. Yang, Food control, 33(2), 337-343, 213. Molecularly imprinted polymer-based solid phase clean-up for analysis of ochratoxin A in beer, red wine, and grape juice, J. Cao, W. Kong, S. Zhou, L. Yin, L. Wan, M. Yang, J. Sep. Sci., 36(7), 1291-1297, 213. Application of molecularly imprinted polymers to determine B1, B2, and B3 fumonisins in cereal products, M. Bryła, R. Jedrzejczak, M. Roszko, K. Szymczyk, M. W. Obiedziński, J. Sękul, M. Rzepkowska, J. Sep. Sci., 36(3), 578-584, 213. Breakthrough innovation in rapid detection kits using MOLECULARLY IMPRINTED POLYMERS SPE for an early quantification of PATULIN from apple-based food matrices, D. Derrien, C. Pérollier, O. Lépine, K. Naraghi, S. Bayoudh, poster at 7th World Mycotoxin Forum, Rotterdam, The Netherlands, November 5-9, 212. Molecularly Imprinted Polymer for Solid Phase Extraction of Patulin mycotoxin, D. Derrien, M. Mulet, F. Alix, C. Pérollier, O. Lépine, K. Naraghi, S. Bayoudh, 33rd Mycotoxin Workshop, Freising, Germany, 3 May - 1 June 211 Analysis of Endocrine Disrupting Compounds Solid-phase extraction using molecularly imprinted polymer for selective extraction of natural and synthetic estrogens from aqueous samples, P. Lucci, O. Núñez, M.T. Galceran, J. Chrom. A, 1218, 4828-4833, 211. On-line molecularly imprinted solid-phase extraction coupled to liquid chromatography-tandem mass spectrometry for the determination of hormones in water and sediment samples, D. Matějíček, J. Vlček, A. Burešová, P. Pelcová, J. Sep. Sci., 36(9-1), 159-1515, 213. Quantification of estrogens at ppt levels in bovine plasma by Molecularly Imprinted Solid Phase Extraction and GC-MS/MS analysis, S. Rochereau, E. Bichon, F. Courant, F. Monteau, S. Prévost, F. Hanganu, N. Cesbron, G. Dervilly-Pinel, B. Le Bizec (LABERCA), Poster Euroresidues VIIth conference, 212. The use of molecularly imprinted polymers for the multicomponent determination of endocrinedisrupting compounds in water and sediment, D. Matějíček, A. Grycová, J. Vlček, J. Sep. Sci., 36(6), 197-113, 213. Utilisation de la spectrométrie de masse pour le dosage du Bisphénol A dans les matrices alimentaires, Y. DECEUNINCK, Z. ZENDONG, E. BICHON, J.-P. ANTIGNAC, B. LE BIZEC (LABERCA) poster SMAP 211, Avignon, France, 19-22 sept. 211. www.affinisep.com 85

NONE EXHAUSTIVE LIST OF PUBLICATIONS AND POSTERS Quantitative analysis of Bisphenol A in all liquid or solid food matrices were carried out by using AFFINIMIP SPE Bisphenol A (p132, Annex 12 of Annexes to the report on the assessment of the risks associated with bisphenol A (BPA) for human health, and on toxicological data and data on the use of bisphenols S, F, M, B, AP, AF, and BADGE (In French)), ANSES April 213. A high selective and sensitive liquid chromatography tandem mass spectrometry method for quantization of BPA urinary levels in children, C. Nicolucci, S. Rossi, C. Menale, E. Giudice, P. Miraglia del Giudice, L. Perrone, P. Gallo, D. Mita, N. Diano, Analytical and Bioanalytical Chemistry, 1618-2642, 213. A developmental hepatotoxicity study of dietary bisphenol A in Sparus aurata juveniles, F. Maradonna, V. Nozzi, L. Dalla Valle, I. Traversi, G. Gioacchini, F. Benato, E. Colletti, P. Gallo, I. Di Marco Pisciottano, D. G. Mita, G. Hardiman, A. Mandich, O. Carnevali, Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 214, 166, 1 13. Molecularly imprinted solid phase extraction for the selective extraction of bisphenol analogues in beverages and canned food, Y. Yang, J. Yu, J. Yin, B. Shao, J. Zhang, J. Agric. Food Chem., 214, 62 (46), pp 1113 11137 Determination of bisphenol A and related substitutes/analogues in human breast milk using gas chromatography-tandem mass spectrometry, Y. Deceuninck, E. Bichon, P. Marchand, C-Y Boquien, A.Legrand, C. Boscher, J. P. Antignac, B. Le Bizec, Analytical and Bioanalytical Chemistry, 215, DOI 1.17/s216-15-8469-9 Analysis of Antibiotics and Drug residues Interest of molecularly imprinted polymers in the fight against doping. Extraction of tamoxifen and its main metabolite from urine followed by high-performance liquid chromatography with UV detection. J. Chrom. A, 1196 1197, 81 88, 28. Molecularly imprinted polymer applied to the selective isolation of urinary steroid hormones: An efficient tool in the control of natural steroid hormones abuse in cattle, M. Doué, E. Bichon, G. Dervilly-Pinel, V. Pichon, F. Chapuis-Hugon, E. Lesellier, C. West, F. Monteau, B. Le Bizec, J. Chrom A, 127, 51-56, 212. High efficiency of semi-preparative Supercritical Fluid Chromatography with Molecularly Imprinted Polymer as stationary phase (SFC-MIP). Application on urinary steroids purification for IRMS analysis, M. Doué, E. Bichon, F. Monteau, B. Le Bizec (LABERCA), poster 2nd International Symposium on HTSP 212. New technological tools for isolating and measuring growth promoting agents in edible tissues and biological fluids, E. Bichon, S. Rochereau, L. Seree, S. Prevost, F. Monteau, B. Le Bizec (LABERCA) Poster session RAFA 211. Analysis of other residues and miscellaneous Analysis of urinary neurotransmitters by capillary electrophoresis: Sensitivity enhancement using field-amplified sample injection and molecular imprinted polymer solid phase extraction, B. Claude, R. Nehmé, P. Morin, Analytica Chimica Acta, 699 (2), 242 248, 211. Molecularly imprinted polymers-liquid chromatography/fluorescence for the selective clean-up of hydroxylated polycyclic aromatic hydrocarbons in soils, O. Baltrons, M. Lopez-Mesas, C. Palet, F. Lederf and F. Portet-Koltalo, Anal. Methods, 213,5, 6297-635 Selective solid phase extraction of catecholamines and metanephrines from serum using a new molecularly imprinted polymer, B. Claude, P. Morin and L. Denoroy, J. Liquid Chromatography & Related Technologies, 214, 37 (18), 2624-2638. www.affinisep.com 86

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About AFFINISEP AFFINISEP is a worldwide expert in purification and sample preparation applications as well as for the design and the development of intelligent polymers with Molecularly Imprinted Polymers (MIP). AFFINISEP is dedicated to the development of analytical applications in various fields such as water, biological fluids, food and feed analysis with a complete set of products and services for sample preparation. Our mission is to develop and market innovative products of high value to customers by a practical contribution to their work. By offering you a most comprehensive range of solid phase extraction products: - AFFINIMIP SPE products based on molecularly imprinted polymers, - AttractSPE TM a range of polymeric phases - SilactSPE TM Silica based products, associated reagents, - QuEChERS - small equipment, the analytical chemists can find any solution for sample preparation, selective extraction and sample clean-up needs in various sectors: food and feed safety and quality, pharmaceutical R&D and quality control, clinical diagnosis, environment and doping. Furthermore, by exploiting our library of innovative polymers and our know-how in chromatography and solid phase extraction, we have a strong capacity to adapt these polymers to meet any specific requirements and to solve unsatisfied purification and extraction needs. Numerous documents related to our products (Application notebooks, publication references, posters, catalog for different applications ) can be found on our website www.affinisep.com. ORDERING INFORMATION For any order, please, choose one of the following ways: By mail: AFFINISEP Pharma Parc II Voie de l innovation 271 Val de Reuil, FRANCE By fax: +33 ()2 32 59 61 1 By email: contact@affinisep.com On-line by E-commerce: To save your time and a quicker delivery, order via our web site www.affinisep.com Columnex LLC represents all Affinisep products in America. Information about new products or new matrices: Newsletters Columnex LLC Stay up-to-date with the news on AFFINISEP s latest innovations by subscribing to our newsletter, 2181 N Tracy Blvd Ste 22, Tracy, CA 95376 USA delivering new information straight to your inbox. phone: 1-347-688-4752 support@columnex.com www.columnex.com