APPLICATION NOTEBOOK FOR AFFINIMIP SPE

APPLICATION NOTEBOOK FOR AFFINIMIP SPE Selective Solid Phase Extraction Molecularly Imprinted Polymers for the Selective Extraction of Trace Analytes from Complex Matrices Q3 214

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. To make you enjoy of this background and for an wealthy, very instructive and easily-accessed overview of AFFINIMIP SPE performances, we are pleased to introduce you this Application Notebook which collects a good abstract of AFFINISEP s experience on AFFINIMIP SPE. This Application notebook will be an essential tool to address your technical issues. QUALITY POLICY To develop a long term and durable partnership with its customers, AFFINISEP ensures the best quality of its products and services. As an ISO91:28 certified company, AFFINISEP has implemented Quality management system requirements to show its commitment to quality, customers, and a willingness to work towards improving efficiency. In addition, to ensure the best quality of its products, the performance is checked by following several QC tests according to each product s quality control procedure. After passing all these tests, the products receive a certificate of analysis which proved the compliance with the defined criterion. 2

TABLE OF CONTENTS AFFINIMIP SPE VS IMMUNOAFFINITY COMPARATIVE STUDY 5 AFFINIMIP SPE FOR MYCOTOXINS ANALYSES 7 AFFINIMIP SPE Multimyco1 Simultaneous determination of Aflatoxins, Fumonisins, Ochratoxin A, HT-2, T-2, Zearalenone in Wheat 8 AFFINIMIP SPE Patulin Patulin in Baby food apple juice 9 Patulin in Apple juice 1 Patulin in Baby food apple puree 11 Patulin in Apple puree 12 Patulin in Apple Fruit puree 13 Patulin in WHOLE apple 14 Patulin in Cider 15 Patulin in Alcohol Pommeau and Liquor 16 Patulin in Tomato Ketchup and Tomato Powder 17 Patulin in Blueberry juice 18 Patulin in Thick Juice and Concentrate Juice 19 Patulin in Apple puree 2 Patulin in Dried apple 21 AFFINIMIP SPE Ochratoxin A Ochratoxin A in Cereals 22 Ochratoxin A in Paprika 23 Ochratoxin A in Red and White wine 24 AFFINIMIP SPE Zearalenone Zearalenone in Maize and Rice 25 Zearalenone in Cereal-based Baby food 26 Zearalenone in Edible corn oil 27 AFFINIMIP SPE Deoxynivalenol Deoxynivalenol in cereals for food (Water extraction) 28 Deoxynivalenol in babyfood cereals 29 Deoxynivalenol, 3-AcetylDON and 15-AcetylDON in cereals (Hydro-organic extraction) 3 Deoxynivalenol in cereals for animal feeds 31 AFFINIMIP SPE FumoZON Fumonisins B1 + B2 and Zearalenone in Maize Flour 32 Fumonisins B1 + B2 and Zearalenone in Maize-based baby food 33 AFFINIMIP SPE Estrogens 34 Determination of estrogens in plasma 35 Protocol comparative - AFFINIMIP SPE Estrogens vs usual protocol 36 3

TABLE OF CONTENTS AFFINIMIP SPE Bisphenol A 37 Bisphenol A in Liquid infant formula 38 Bisphenol A in powdered infant formula 39 PROTOCOL COMPARATIVE WITH A COMPETITOR POWDERED INFANT FORMULA 4 Bisphenol A in canned food (liquid form) 41 Bisphenol A in canned food (Vegetable) 42 Bisphenol A in Beer 43 Bisphenol A in White/Red wines 44 Bisphenol A in Cola drinks 45 Bisphenol A and BADGE in Milk 46 Total Bisphenol A in Human Urine 47 AFFINIMIP SPE Chloramphenicol 48 Chloramphenicol in Honey 49 Chloramphenicol in Bovine urine Chloramphenicol in Shrimp 51 AFFINIMIP SPE Amphetamines 52 Amphetamines in Human urine 53 Amphetamines in Human serum 54 AFFINIMIP SPE Tetracyclines 55 Tetracyclines (Oxytetracycline, Chlortetracycline, Tetracycline), theirs epimers and Doxycycline in Milk and Salmon 56 AFFINIMIP SPE Metanephrines 57 Methanephrines in Plasma comparison with WCX cartridges 58 AFFINIMIP SPE Phenolics 59 Parabens in cosmetic products 6 Guaïacol 61 Carnosic acid in meat 62 AFFINIMIP SPE PRODUCT LIST FOR MYCOTOXIN ANALYSES 63 AFFINIMIP SPE PRODUCT LIST (MISCELLANEOUS) 64 LIST OF PUBLICATIONS AND POSTERS 66 4

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- µmol/g Variable Good Stability Poor Very High Reproducibility Variable Good Cost Expensive Inexpensive PROTOCOL: Zearalenone (ZON) from maize flour Step Extraction of target analyte Preparation loading solution Washing Elution Protocol time Vicam IAC 25g sample in ml 9/1 Methanol/water Blender 3 minutes + filtration 4mL extract + 96mL water ml solution 2mL Water 1.5mL Methanol 55min AFFINIMIP SPE ZON 25g sample in ml 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/Acetic 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 Vicam IAC AFFINIMIP SPE OTA g sample in ml 6/4 ACN/water Blender 1 minute + filtration 1mL extract + 4mL PBS 1mL solution 1mL PBS 1mL Water 1.5mL Methanol 1mL extract + 1mL HCl.1M ph=1 4mL solution 7mL 6/4 HCl.1M ph=1/acn 2mL 98/2 Methanol/Acetic acid Protocol time 3min 2min

52 51 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 52 51 49 48 47 46 45 44 43 42 41 AFFINIMIP SPE CARTRIDGE VS IMMUNOAFFINITY COLUMN CHROMATOGRAM ASPECT Equivalent chromatograms IAC 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). RECOVERIES Higher Recoveries obtained with AFFINIMIP SPE 1ng/g OTA 85% Recovery 9 8 Recovery % 98 97 ZON 85 OTA 79 7 6 mvolts AFFINIMIP SPE IAC 6ng/g OTA 79% Recovery mvolts 4 3 2 1 MIP IAC MIP IAC 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). Figure 3. Recovery of Ochratoxin A or Zearalenone obtained after cleanup by AFFINIMIP SPE or Vicam IAC. CAPACITY Capacity MIP > Capacity IAC Area 3,E+8 3,E+8 2,E+8 2,E+8 1,E+8 1,E+8 5,E+7 AFFINIMIP SPE IAC,E+ 2 4 6 8 Spiking level ng/g Figure 4. Comparison of capacity between AFFINIMIP SPE Zearalenone (red) and Vicam IAC (blue). Level found ng/g 4 4 3 3 2 2 1 AFFINIMIP SPE IAC 2 3 4 6 Spiking level ng/g Figure 5. Comparison of capacity between AFFINIMIP SPE OTA (red) and Vicam IAC (blue).

AFFINIMIP SPE for mycotoxins analyses Mycotoxins are toxic secondary metabolites produced by different fungi present in agricultural commodities. They are regulated in food and feed due to nephrotoxic, neurotoxic, carcinogenic, estrogenics, and immunosuppressive effects. AFFINISEP has developed two sets of product for mycotoxins analyses: For single mycotoxin extraction Designed for the analysis of one specific mycotoxin or for mycotoxins analyzed by fluorescence detection AFFINISEP has developed the following products AFFINIMIP SPE Patulin (PI-FS12) AFFINIMIP SPE Ochratoxin A (PI-FS11) AFFINIMIP SPE Zearalenone (PI-FS) AFFINIMIP SPE Deoxynivalenol (PI-FS117) For multimycotoxins extraction Designed for the simultaneous extraction of several mycotoxins which are present in a same matrix prior to LC-MS/MS analyses These mycotoxins are all present in the same matrix to be analyzed. Their extraction is done all at once by SPE. Then the elution solution containing all these mycotoxins is evaporated, reconstituted and analyzed by LC-MS/MS. The protocol is short and efficient AFFINISEP has developed the following products AFFINIMIP SPE FumoZON (PI-FS19) for the analyses of Fumonisins Zearalenone AFFINIMIP SPE Multimyco1 (PI-FS114) for the analyses of Fumonisins Aflatoxins Ochratoxin A T-2 and HT-2 Zearalenone 7

AFFINIMIP SPE Multimyco1 SIMULTANEOUS DETERMINATION OF MULTIMYCOTOXINS IN WHEAT FS114 AFFINIMIP SPE Multimyco1 are Multimycotoxins solid-phase extraction cartridges that selectively and SIMULTANEOUSLY clean-up and concentrate Fumonisins, Aflatoxins, Ochratoxin A, T-2, HT-2 and Zearalenone prior to analysis by LC-MS/MS from complex matrices such as cereals. 25g of ground wheat were extracted with ml of Acetonitrile/Water (/, v/v/v) for 2 min using a blender. The extract was filtered through a folded filter paper and 4mL of the filtrate were diluted with 16mL of water. Then, this solution was filtered through a filter paper. Cleanup with a 3mL/6mg Multimyco1 cartridge AFFINIMIP SPE 2mL MeOH -2% Formic acid 3mL Acetonitrile 2mL water 3mL of loading solution Washing of interferents (W1) 3mL Water 3mL Water/Acetonitrile (85/15 v/v) Drying by applying vacuum 3 minutes 3mL Methanol/ACN/Formic acid (48.5/48.5/3, v/v/v) dissolved in water containing.1% acetic acid before HPLC analysis. WHEAT MRM parameters for mycotoxins analyses Compound name RT MRM transition Aflatoxin B1 Positive ion mode Fumonisin B1 Positive ion mode HT-2 Positive ion mode T-2 Positive ion mode Zearalenone Negative ion mode Ochratoxin A Positive ion mode Recovery yield Recovery of multimycotoxins extracted from wheat and analyzed after AFFINIMIP SPE Multimyco1 cleanup Compound name C µg/k g 1.4 2 1.7 1 1.7 6 1.8 9 2. 5 2.1 3 Mean µg/kg 312.8 > 285. 722.4 > 141.1 722.4 > 334.5 722.4 > 352.3 441.9 > 14.9 441.9 > 214.7 441.9 > 263.2 484. > 15. 484. > 185.1 484. > 35.1 316.8 > 131.1 316.8 > 174.9 316.8 > 273.1 43.8 > 221.1 43.8 > 239.2 43.8 > 356.1 R% Aflatoxin B1 2 1.6 85 Fumonisin B1 937 94 HT-2 119 119 T-2 56.5 113 Zearalenone 54 18 UFLC Method Column: Phenomenex Kinetix XB-C18 Detection: LC-MS/MS with ESI source - MRM mode Injection volume: 2µL. Ochratoxin A 3 2.6 88 PI-FS114-3 for cartridges, 3mL PI-FS114-4 for cartridges, 3mL 8

AFFINIMIP SPE Patulin DETERMINATION OF PATULIN IN BABY FOOD APPLE JUICE Regulations for apple juice: Europe (EC 1881/26) : µg/kg USA (FDA CPG Sec.51.1) : µg/kg Regulations for apple juice for infants and young children: Europe (EC 1881/26) : 1µg/Kg 4 35 3 25 2 Patulin 4 35 3 25 2 15 15 1 1 solution: 2.5mL apple juice and 2.5mL of water-2% acetic acid are mixed. Cleanup with a 3mL/mg Patulin cartridge AFFINIMIP SPE 2mL Acetonitrile 1mL water 4mL of loading solution Washing of interferents (W1) 1mL NaHCO 3 2mL Water Drying by applying vacuum 1 seconds Washing of interferents (W2) 1mL Diethyl Ether 2mL Ethyl Acetate dissolved in water containing.1% acetic acid before HPLC analysis. 5-5 -1-15 -2-25 9 1 11 12 13 14 15 16 17 18 19 2 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 5-5 -1-15 -2-25 HPLC Method Column: Atlantis T3 column, 1mm x 2.1mm Mobile phase: Deionized water/acn (95/5, v/v) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. 3mL-mg sorbent for apple juice and puree PI-FS12-2 for 25 cartridges PI-FS12-3 for cartridges 6mL-2mg sorbent for a apple-based products, fruit juice and concentrate PI-FS12-2B -2mg for 25 cartridges PI-FS12-3B -2mg for cartridges 9

DETERMINATION OF PATULIN IN APPLE JUICE AFFINIMIP SPE Patulin Regulations for apple juice: Europe (EC 1881/26) : µg/kg USA (FDA CPG Sec.51.1) : µg/kg Regulations for apple juice for infants and young children: Europe (EC 1881/26) : 1µg/Kg 2 18 16 14 12 UV 6-276nm Brut carrefour.dat UV6-276nm C1 C5 Before cleanup 2 18 16 14 12 solution: 2.5mL apple juice and 2.5mL of water-2% acetic acid are mixed. Clean-up with a 3mL/mg Patulin cartridge AFFINIMIP SPE 2mL Acetonitrile 1mL water 4mL of loading solution Washing of interferents (W1) 1mL NaHCO 3 in Water 2mL Water Drying by applying vacuum 1 seconds Washing of interferents (W2) 1mL Diethyl Ether 2mL Ethyl Acetate dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 1mm x 2.1mm Mobile phase: Deionized water/acn (95/5, v/v) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. 8 6 4 2 7 6 4 3 2 1-1 -2 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 Patulin Chromatograms of apple juice containing 25µg/kg of Patulin before (Red) and after (Blue) AFFINIMIP SPE Patulin Clean-up HMF 9 1 11 12 13 14 15 16 17 18 19 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. Concentration of Patulin (ng/ml) Recoveries % % RSD R 1 97.9 11 (n=9) 4 9.6 11 (n=41) 3mL-mg sorbent for apple juice and puree PI-FS12-2 for 25 cartridges PI-FS12-3 for cartridges 6mL-2mg sorbent for a apple-based products, fruit juice and concentrate PI-FS12-2B -2mg for 25 cartridges PI--FS12-3B -2mg for cartridges 7 6 4 3 2 1 8 6 4 2-1 -2 1

12 8 6 4 2 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 12 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 14 12 UV 6-276nm C7 UV 6-276nm C1 UV 6-276nm C15 Patulin 14 12 8 8 1g of apple puree, 1µL of a pectinase enzyme solution and 1mL water are mixed. Leave solution at room temperature overnight or for 2h at 4 C. Centrifuge at 4g for 5min and then filter the solution with a.2µm filter. This solution is used as the loading solution. Cleanup with a 3mL/mg Patulin cartridge AFFINIMIP SPE 2mL Acetonitrile 1mL Water 5mL of loading solution Washing of interferents (W1) 4mL Water -1%Acetic acid 1mL NaHCO 3 1% solution 3mL Water Drying by applying vacuum 1 seconds Washing of interferents (W2) µl Diethyl Ether 2mL Ethyl Acetate dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 1mm x 2.1mm Mobile phase: gradient Time (min) % water % ACN 98 2 2 98 2 21 25 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. 6 4 2 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 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. Concentration of Patulin (µg/kg) Recoveries % (n=3) % RSDr 2 84 4.5 3mL-mg sorbent PI-FS12-2 for 25 cartridges PI-FS12-3 for cartridges PI-FS12-2K for a kit of 25 cartridges + ml Pectinase PI-FS12-3K for a kit of cartridges + ml Pectinase PI-REA-1-mL for ml Pectinase solution 6 4 2 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, 1µL of a pectinase enzyme solution and 1mL water are mixed. Leave solution at room temperature overnight or for 2h at 4 C. Centrifuge at 4g for 5min and then filter the solution with a.2µm filter. This solution is used as the loading solution. Cleanup with a 3mL/mg Patulin cartridge AFFINIMIP SPE 2mL Acetonitrile 1mL Water 5mL of loading solution Washing of interferents (W1) 4mL Water -1%Acetic acid 1mL NaHCO 3 1% solution 3mL Water Drying by applying vacuum 1 seconds Washing of interferents (W2) µl Diethyl Ether 2mL Ethyl Acetate 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, 1mm x 2.1mm Mobile phase: gradient Time (min) % water % ACN 98 2 2 98 2 21 25 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. 3mL-mg sorbent PI-FS12-2 for 25 cartridges PI-FS12-3 for cartridges PI-FS12-2K for a kit of 25 cartridges + ml Pectinase PI-FS12-3K for a kit of cartridges + ml Pectinase PI-REA-1-mL for ml Pectinase solution 12

12 8 6 4 2 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 12 8 6 4 2 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, 1µL of a pectinase enzyme solution and 1mL water are mixed. Leave solution at room temperature overnight or for 2h at 4 C. Centrifuge at 4g for 5min and then filter the solution with a.2µm filter. This solution is used as the loading solution. Cleanup with a 3mL/mg Patulin cartridge AFFINIMIP SPE 2mL Acetonitrile 1mL Water 5mL of loading solution Washing of interferents (W1) 4mL Water -1%Acetic acid 1mL NaHCO 3 1% solution 3mL Water Drying by applying vacuum 1 seconds Washing of interferents (W2) µl Diethyl Ether 2mL Ethyl Acetate dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 1mm x 2.1mm Mobile phase: gradient Time (min) % water % ACN 98 2 2 98 2 21 25 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. Chromatograms of apple puree containing µg/kg (blue) or 2µg/kg (tested twice, green and red) of Patulin after AFFINIMIP SPE Patulin Clean-up. 2 225 2 175 1 125 75 25-25 - -75 Apple-pear puree with Patulin (25µg/kg) Apple puree with Patulin (4µg/kg) Apple-strawberry puree with Patulin (4µg/kg) 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 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. Concentration of Patulin (µg/kg) Patulin Recoveries % % RSD R 1 (n=9) 77.4 8.1 25 (n=8) 9.9 11.4 4 (n=6) 86. 11.9 3mL-mg sorbent PI-FS12-2 for 25 cartridges PI-FS12-3 for cartridges PI-FS12-2K for a kit of 25 cartridges + ml Pectinase PI-FS12-3K for a kit of cartridges + ml Pectinase PI-REA-1-mL for ml Pectinase solution 2 225 2 175 1 125 75 25-25 - -75 13

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 1µL 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/mg AFFINIMIP SPE Patulin cartridge 2mL Acetonitrile 1mL Water 3mL of loading solution Washing of interferents (W1) 3mL Water-2% Acetic Acid Drying by applying vacuum 1 seconds Washing of interferents (W2) 2µL Diethyl Ether Drying by applying vacuum 1 seconds 1mL Ethyl Acetate dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 1mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN 98 2 2 98 2 21 25 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. 9 8 7 6 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 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-mg sorbent PI-FS12-2 for 25 cartridges PI-FS12-3 for cartridges PI-FS12-2K for a kit of 25 cartridges + ml Pectinase PI-FS12-3K for a kit of cartridges + ml Pectinase PI-REA-1-mL for ml Pectinase solution 9 8 7 6 4 3 2 1-1 -2-3 14

DETERMINATION OF PATULIN IN CIDER AFFINIMIP SPE Patulin Regulations for cider: Europe (EC 1881/26) : µg/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/mg Patulin cartridge AFFINIMIP SPE 2mL Acetonitrile 1mL Water 4mL of loading solution Washing of interferents (W1) 1mL NaHCO 3 1% in Water 2mL Water Drying by applying vacuum 1 seconds Washing of interferents (W2) µl Diethyl Ether 2mL Ethyl Acetate 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. Concentration 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, 1mm x 2.1mm Mobile phase: Deionized water/acn (95/5, v/v) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. 3mL-mg sorbent PI-FS12-2 for 25 cartridges PI-FS12-3 for cartridges PI-FS12-2K for a kit of 25 cartridges + ml Pectinase PI-FS12-3K for a kit of cartridges + ml Pectinase PI-REA-1-mL for ml Pectinase solution 15

AFFINIMIP SPE Patulin DETERMINATION OF PATULIN IN ALCOHOL POMMEAU AND LIQUOR Regulations for apple based beverage : Europe (EC 1881/26) : µg/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. 2 225 2 175 1 125 Patulin 2 225 2 175 1 125 m To 1mL of Manzella Liquor or Alcohol Pommeau, add 2mL Water to obtain the loading solution. Cleanup with a 3mL/mg AFFINIMIP SPE Patulin cartridge 2mL Acetonitrile 1mL Water 3mL of loading solution Washing of interferents (W1) 3mL Water (containing 2% Acetic Acid for AA W1 protocol) Drying by applying vacuum 1 seconds Washing of interferents (W2) 2µL Diethyl Ether Drying by applying vacuum 1 seconds 2mL Ethyl Acetate dissolved in water containing.1% acetic acid before HPLC analysis. 75 25 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 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. 16 14 12 8 6 4 2 UV C1 6-276nm UV C12 6-276nm UV C11 6-276nm UV C13 6-276nm UV C15 6-276nm UV C14 6-276nm Patulin 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 Chromatograms obtained after AFFINIMIP SPE Patulin Clean-up of Pommeau 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 Clean-up. W1 with water or Water -2%Acetic acid 75 25 16 14 12 8 6 4 2 Water for W1 Water-AA for W1 HPLC Method Column: Atlantis T3 column, 1mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN 98 2 2 98 2 21 25 26 98 2 ) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. Pommeau 11 11 9 93 Manzella 12 16 87 9 3mL-mg sorbent PI-FS12-2 for 25 cartridges PI-FS12-3 for cartridges PI-FS12-2K for a kit of 25 cartridges + ml Pectinase PI-FS12-3K for a kit of cartridges + ml Pectinase PI-REA-1-mL for ml Pectinase solution 16

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

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 Recovery yields: 9 and 96% 7 6 Cleanup with a 3mL/mg Patulin cartridge AFFINIMIP SPE 4 3 m A U 2 Patulin 4 3 2 2mL Acetonitrile 1mL Water 4mL of loading solution Washing of interferents (W1) 1mL NaHCO 3 1% in Water 2mL Water Drying by applying vacuum 1 seconds Washing of interferents (W2) µl Diethyl Ether 2mL Ethyl Acetate 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 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, 1mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN 98 2 2 98 2 21 25 26 98 2 ) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. 3mL-mg sorbent PI-FS12-2 for 25 cartridges PI-FS12-3 for cartridges PI-FS12-2K for a kit of 25 cartridges + ml Pectinase PI-FS12-3K for a kit of cartridges + ml Pectinase PI-REA-1-mL for ml Pectinase solution 18

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 µl Pectinase. (REA-1- ml). 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. 4 4 3 3 2 2 THICK JUICE Patulin Preparation of thick fruit juice samples 15mL of thick fruit juice are mixed with 12µL Pectinase (REA-1-mL). 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 interferents (W1) 2mL NaHCO 3 1% in Water 4mL Water Drying by applying vacuum 3 seconds Washing of interferents (W2) 1mL Diethyl Ether 2mL Ethyl Acetate dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 1mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN 98 2 2 98 2 21 25 26 98 2 ) Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. 1 2 4 6 8 1 12 14 16 18 2 22 24 26 28 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 ng/ml. prepared by dilution of a µg/ml Patulin standard solution (REA-PAT-1mL) in mobile phase. 16 14 12 8 6 4 2-2 CONCENTRATE JUICE 2 4 6 8 1 12 14 16 18 2 22 24 26 28 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 PI-FS12-2B-2mg for 25 cartridges PI-FS12-3B-2mg for cartridges PI-FS12-2KB-2mg for a kit of 25 cartridges + ml Pectinase PI-FS12-3KB-2mg for a kit of cartridges + ml Pectinase PI-REA-1-mL for ml Pectinase solution 16 14 12 8 6 4 2-2 19

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 8 7 6 8 7 6 1g of apple puree, 1µL of a pectinase enzyme solution and 1mL water are mixed. Leave solution at room temperature overnight or for 2h at 4 C. Centrifuge at 4g for 5min and then filter the solution with a.2µm filter. This solution is used as the loading solution. Cleanup with a 6mL/2mg Patulin cartridge AFFINIMIP SPE 2mL Acetonitrile 1mL Water 5mL of loading solution Washing of interferents (W1) 4mL Water -1%Acetic acid 1mL NaHCO 3 1% solution 3mL Water Drying by applying vacuum 1 seconds Washing of interferents (W2) µl Diethyl Ether 2mL Ethyl Acetate dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 1mm x 2.1mm Mobile phase: gradient Time (min) % water % ACN 98 2 2 98 2 21 25 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. 4 3 2 4 5 6 7 8 9 1 11 12 13 14 15 Chromatograms of apple puree spiked with 2µg/kg of Patulin (Red) and not spiked (blue) after AFFINIMIP SPE Patulin Clean-up Recovery and repeatability of Patulin (n=6) at a contamination level of 1µg/kg in apple puree after AFFINIMIP SPE Patulin Clean-up. Concentration of Patulin (µg/kg) Patulin Recoveries % % RSDr 1 (n=6) 9 9 2 (n=3) 92 11 6mL - 2mg sorbent PI-FS12-2B-2mg for 25 cartridges PI-FS12-3B-2mg for cartridges PI-FS12-2KB-2mg for a kit of 25 cartridges + ml Pectinase PI-FS12-3KB-2mg for a kit of cartridges + ml Pectinase PI-REA-1-mL for ml Pectinase solution 4 3 2 2

UV6-276nm B16 UV6-276nm B17 UV6-276nm B13 DETERMINATION OF PATULIN IN DRIED APPLE AFFINIMIP SPE Patulin Regulations for solid apple products: Europe (EC 1881/26) : 25µg/Kg 8 8 7 7 6 Patulin 6 4 Recovery >9% 4 3 3 2 2 3g of dried apple dices, 3mL of water and 1µL of pectinase are mixed and left at room temperature overnight. Then, they are centrifuged at 4rpm during 5min and filtered with.2µm filter to obtain the loading solution. Cleanup with a 6mL/2mg AFFINIMIP SPE Patulin cartridge 1-1 -2-3 -4-9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 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). 1-1 -2-3 -4-4mL Acetonitrile 2mL Water 1mL of loading solution Washing of interferents (W1) 5mL Water-2% Acetic Acid 5mL Water Drying by applying vacuum 3 seconds Washing of interferents (W2) µl Diethyl Ether 2mL Ethyl Acetate dissolved in water containing.1% acetic acid before HPLC analysis. HPLC Method Column: Atlantis T3 column, 1mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN 98 2 2 98 2 21 25 26 98 2 Flow rate:.2ml/min Detection: UV - 276nm Injection volume: µl. 6mL - 2mg sorbent for apple-based products and fruit juice, concentrate PI-FS12-2B-2mg for 25 cartridges PI-FS12-3B-2mg for cartridges PI-FS12-2KB-2mg for a kit of 25 cartridges + ml Pectinase PI-FS12-3KB-2mg for a kit of cartridges + ml Pectinase PI-REA-1-mL for ml Pectinase solution 21

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 g of finely ground wheat are mixed during 1 minute in a blender with ml 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/mg AFFINIMIP SPE Ochratoxin A cartridge 3mL Acetonitrile 3mL Water 4mL of loading solution (eq. 1g wheat) Washing of interferents 6mL 6/4 HCl solution ph 1,.1M/ACN 2mL Methanol 2% Acetic acid dissolved in water before HPLC analysis. Intensity 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 1mm 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. PI-FS11-2 for 25 cartridges PI-FS11-3 for cartridges 22

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 ml 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. Intensity 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 Cleanup with a 3mL/mg AFFINIMIP SPE Ochratoxin A cartridge 3mL Acetonitrile 3mL Water 4mL of loading solution (eq. 1g sample) Washing of interferents 6mL 6/4 HCl solution ph 1,.1M/ACN 2mL Methanol 2% Acetic acid dissolved in water before HPLC analysis. 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 1mm 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. PI-FS11-2 for 25 cartridges PI-FS11-3 for cartridges 23

AFFINIMIP SPE Ochratoxin A DETERMINATION OF OCHRATOXIN A IN RED AND WHITE WINE Regulations for wine: Europe (EC 1881/26) : 2µg/L 1mL of wine is diluted with 1mL of HCl solution ph=1,.1m. This solution is used as the loading solution. Intensity Ochratoxin A Ochratoxin A, 2, 4, 6, 8, 1, 12, 14, 16, Cleanup with a 3mL/mg AFFINIMIP SPE Ochratoxin A cartridge 3mL Acetonitrile 3mL Water 2 to 1mL of loading solution (eq. 1 to 5mL sample) Washing of interferents 6mL 6/4 HCl solution ph 1,.1M/ACN 2mL Methanol 2% Acetic acid dissolved in water before HPLC analysis. Intensity 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 Intensity 6 4 2, 2, 4, 6, 8, 1, 12, 14, 16, 18, Ochratoxin A, 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). HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 1mm 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. Matrix White wine (n=1) C (µg/kg ) PI-FS11-2 for 25 cartridges PI-FS11-3 for cartridges Recoveries % % RSD 2 91.3 6.2 Red wine (n=4) 2 78.8 2.8 24

AFFINIMIP SPE Zearalenone DETERMINATION OF ZEARALENONE IN MAIZE AND RICE Regulations for unprocessed cereal except maize: Europe (EC 1126/27) : µg/kg Regulations for maize: Europe (EC 1126/27) : 3µg/Kg Cleanup with a 3mL/mg AFFINIMIP SPE Zearalenone cartridge 25g of ground cereal-based samples were extracted with 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 interferents (W1) 3mL 58/2/4 Water/Acetic Acid/ACN 2mL Methanol 2% Acetic Acid 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 1mm x 4.6mm Mobile phase: water/meoh (4/6, v/v) Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 275 / 4nm Injection volume: µl. 3mL-mg sorbent PI-FS-2 for 25 cartridges PI-FS-3 for cartridges 25

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/mg AFFINIMIP SPE Zearalenone cartridge 25g of ground cereal-based samples were extracted with 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 interferents (W1) 3mL 58/2/4 Water/Acetic Acid/ACN 2mL Methanol 2% Acetic Acid dissolved in water before HPLC analysis. 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 Cerealbased 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). Recoveries of Zearalenone at a contamination level of 41µg / kg after AFFINIMIP SPE Zearalenone. Clean-up in Cereal based baby food (n=5) Recoveries % % RSD HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 1mm x 4.6mm Mobile phase: water/meoh (4/6, v/v) Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 275 / 4nm Injection volume: µl. 8 3 3mL-mg sorbent PI-FS-2 for 25 cartridges PI-FS-3 for cartridges 26

s 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 4 5 mvolt 4 3 5 3 2 5 2 1 5 Zearalenone Cleanup with a 3mL/mg 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 interferents (W1) 6mL Diethyl ether Drying 3 seconds Washing of interferents (W2) 6mL 58/2/4 Water/Acetic Acid/ACN 4mL Methanol 2% Acetic Acid dissolved in water before HPLC analysis. 1 5 2 4 6 8 1 1 2 1 4 Chromatograms of Corn Oil spiked with Zearalenone at 4µg/L (blue) or not spiked (orange) obtained after cleanup by AFFINIMIP SPE 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). 1 6 Zearalenone 1 8 2 2 2 2 4 2 6 2 8 3 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 1mm x 4.6mm Mobile phase: water/meoh (4/6, v/v) Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 275 / 4nm Injection volume: µl. C (µg/l) Mean C (µg/l) Recoveries % 2 23 115 4 44 11 6 678 113 3mL-mg sorbent PI-FS-2 for 25 cartridges PI-FS-3 for cartridges 27

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) : 17µg/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 2 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/mg AFFINIMIP SPE Deoxynivalenol cartridge. 2mL Acetonitrile 2mL Water 6mL of loading solution Washing of interferents (W1) 3mL NaHCO 3 1% in water Drying 3 seconds Washing of interferents (W2) 1mL Diethylether 4mL Ethyl Acetate dissolved in water -,1% HCOOH before HPLC analysis. HPLC Method with MS or UV detection Column: Hypersil Gold C18 column mm 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 µg/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) Recovery of Deoxynivalenol after AFFINIMIP SPE Deoxynivalenol clean-up and relative standard deviation (reproducibility conditions). Matrix Detec tion Detect ion Mean µg/kg UV 623.4 78. MS 642.7 8.3 MS 54. 9. Mean µg/kg R% %RSDr 1.4 (n=6) 3.4 (n=6) 9.8 (n=3) R% %RSD R Corn (n=6) UV 658.7 82.3 7.5 Corn (n=6) MS 659.5 82.4 7.8 6mL-mg sorbent for (baby)food PI-FS117-2B for 25 cartridges PI-FS117-3B for cartridges 6mL-2mg sorbent for feed PI-FS117-2B-2mg for 25 cartridges PI-FS117-3B-2mg for cartridges 28

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) 1 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 2 g for 15 minutes. After centrifugation, the supernatant was filtered through filter paper. Cleanup with a 6mL/mg AFFINIMIP SPE Deoxynivalenol cartridge. 2mL Acetonitrile 2mL Water 6mL of loading solution Washing of interferents (W1) 3mL NaHCO 3 1% in water Drying 3 seconds Washing of interferents (W2) 1mL Diethylether 4mL Ethyl Acetate dissolved in water -,1% HCOOH before HPLC analysis. HPLC Method with MS detection Column: Hypersil Gold C18 column mm 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. 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 1µg/kg dark yellow not spiked blue, a standard solution of Deoxynivalenol at 2ng/mL is prepared by dilution of a µg/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 repeatability conditions. Matrix Babyfood (n=3) C µg/kg Mean µg/kg R% %RSDr 1 136.5 91.4 Recovery of Deoxynivalenol after AFFINIMIP SPE Deoxynivalenol clean-up and relative standard deviation calculated from results generated under reproducibility conditions. Matrix Babyfood (n=3) C µg/kg Mean µg/kg R% %RSDR 1 136.5 91.4 6mL-mg sorbent for (baby)food PI-FS117-2B for 25 cartridges PI-FS117-3B for cartridges 6mL-2mg sorbent for feed PI-FS117-2B-2mg for 25 cartridges PI-FS117-3B-2mg for cartridges 29

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) : 17µg/Kg 2 18 16 3-AcetylDeoxynivalenol (3-AcDON) 14 12 8 WITH HYDROORGANIC EXTRACTION 2g of cereals were ground in a blender for 1 minute. Then, a solution of deionized water: acetonitrile (:) 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 2 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/mg AFFINIMIP SPE Deoxynivalenol cartridge. 2mL Acetonitrile 2mL Water 6mL of loading solution Washing of interferents (W1) 3mL NaHCO 3 1% in water Drying 3 seconds Washing of interferents (W2) 1mL Diethylether 4mL Ethyl Acetate dissolved in water-.1% formic acid before HPLC analysis. HPLC Method with MS detection Column: Hypersil Gold C18 column mm 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 MS chromatograms obtained after hydro-organic 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 µg/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 Time (min) 8 641.8 8.2 3.4 6mL-mg sorbent for (baby)food PI-FS117-2B for 25 cartridges PI-FS117-3B for cartridges 6mL-2mg sorbent for feed PI-FS117-2B-2mg for 25 cartridges PI-FS117-3B-2mg for cartridges 3

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 2 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 interferents (W1) 3mL NaHCO 3 1% in water Drying 3 seconds Washing of interferents (W2) 1mL Diethylether 4mL Ethyl Acetate dissolved in water -,1% HCOOH before HPLC analysis. HPLC Method with UV detection Column: Hypersil Gold C18 column mm 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 µg/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 (n=3). Feed Matrices a b c Analysis of Whiskas: a. Extraction solution with water b. solution c. Elution solution C mg/kg Mean mg/kg R% %RSDr Wheat 6 5.7 94.1 Whiskas.8.73 91 2.4 6mL-mg sorbent for (baby)food PI-FS117-2B for 25 cartridges PI-FS117-3B for cartridges 6mL-2mg sorbent for feed PI-FS117-2B-2mg for 25 cartridges PI-FS117-3B-2mg for cartridges 31

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) : µg/kg for maize flour USA: FDA advisory 2µg/Kg RT:.8 RT:.8-2. 22 2 2 19 18 17 16 Intensity 14 13 12 1 9 8 7 6 4 3 2 RT:.8-3. 4 3 Fumonisin B2 2 4 6 8 1 12 14 16 18 2 Time (min) 6.19 Intensity 3 2 2 Fumonisin B1 12 1 Cleanup with a 3mL/mg AFFINIMIP SPE FumoZON cartridge 25g of ground samples were extracted with ml of Acetonitrile/Methanol/deionized Water (25/25/, 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 interferents 6mL 6/4 Water/ACN 2mL Methanol 2% Acetic Acid dissolved in water before HPLC analysis. HPLC Method with MS detection Column: Hypersil Gold C18 column mm 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. Intensity 9 8 7 6 4 3 2 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 cleanup and relative standard deviation calculated from results generated under reproducibility conditions Sample Zearalenon e Fumonisin B1 Fumonisin B1 Fumonisin B2 C µg/kg Mean µg/kg Recoveries % 38 39.2 13.2 248 22.2 83.1 4 41..2 63 684.6 18.7 3mL-mg sorbent PI-FS19-2 for 25 cartridges PI-FS19-3 for 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) 32

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 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 Recoveries % % RSD R Cleanup with a 3mL/mg AFFINIMIP SPE FumoZON cartridge 25g of ground samples were extracted with ml of Acetonitrile/Methanol/deionized Water (25/25/, 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 interferents 6mL 6/4 Water/ACN 2mL Methanol 2% Acetic Acid dissolved in water before HPLC analysis. Zearalenone 2 16.9 84.4 Fumonisin B1 2 168.6 84.3 Fumonisin B2 2 185.6 92.8 ION SUPPRESSION EVALUATION 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). HPLC Method with MS detection Column: Hypersil Gold C18 column mm 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. Analyte C µg/kg 3mL-mg sorbent PI-FS19-2 for 25 cartridges PI-FS19-3 for cartridges Ion suppression % Zearalenone 1 1% and 5% Zearalenone % and 5% Fumonisin B1 8% and 11% Fumonisin B1 12% and 14% 33

AFFINIMIP SPE ESTROGENS PH Estrogens are a group of compounds which play an important role in the estrous cycle. They are either natural (Estrone, Estriol, 17α- and 17β-Estradiol) or synthetic compounds (17α- EthinylEstradiol, Dienestrol, Diethylstilbestrol). Estrogens play a key role in developmental and reproductive functions. They also affect a diversity of biological processes involved in coronary artery disease, immunocompetence and cancer susceptibility. When they are present in wastewater, these endocrine disrupting chemicals (EDC) have adverse effects on endocrine systems of human beings and animals. 17β- Estradiol HO H H H OH In addition, because of their anabolic effects, estrogens have been used in animal fattening. Steroid hormones are used in animal fattening because of their capacity to increase weight gain and to reduce the feed conversion ratio which is the average feed intake in relation to the weight gain. For several years now, the use of anabolic steroids in animal fattening is prohibited in the European Community because of their possible toxic effects on public health (96/22/EC). Nevertheless, they are still offered on the black market for animal fattening purposes. AFFINIMIP SPE Estrogens are selective solid phase extraction cartridges that selectively clean and concentrate Estrogens prior to analysis by HPLC.AFFINIMIP SPE Estrogens are selective solid-phase extraction cartridges that selectively clean and concentrate the natural or synthetic estrogens family prior to further analysis from complex matrices such as Water, Plasma or Serum PI--2 for 25 cartridges, 3mL PI--3 for cartridges, 3mL PI--1.96W for 1 96-well plate

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 ppt 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 µl 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/mg AFFINIMIP SPE Estrogens cartridge 3mL Methanol 3mL Acetonitrile 3mL Water solution from sample preparation Washing of interferents 3mL Water 3mL Water/Acetonitrile (6/4) 3mL Methanol 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 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 PI-FS14-2 for 25 cartridges, 3mL PI-FS14-3 for cartridges, 3mL 35

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 HPLC dimethylaminopropyle 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 PI-FS14-2 for 25 cartridges, 3mL PI-FS14-3 for cartridges, 3mL 36

AFFINIMIP SPE Bisphenol A FS16 Bisphenol A (or BPA) is a molecule widely used in industry for the synthesis of polycarbonate plastics and epoxy resins. Polycarbonate plastics are used to make a variety of common products including baby and water bottles. Epoxy resins are used as coatings on the inside of almost all food and beverage cans. HO CH 3 CH 3 OH The migration of this endocrine disruptor compound from the packaging to food is the main source of consumers exposure to BPA. Its consumption is critical for babies. So, a European directive prohibits the use of BPA to manufacture infant feeding bottles (Directive 211/8/EU of 28 January 211). Progressively, countries become more and more restrictive on BPA use for food packaging. So, in October 211, French parliament voted a law for banning BPA from canned foods and plastic boxes applicable in 213 for infants and for all consumers on 1 st January 214. In the same way, Sweden bans BPA in food packaging for under-threes (212) and in Denmark since July 21, it has been illegal to sell infant feeding bottles and cups, and packaging for baby food, containing BPA. So, BPA is a topical issue with a worldwide regulation going to still lower concentrations of BPA allowed in food. Highly sensitive and reliable detection methods are required for routine analysis of BPA in food samples, particularly for baby food. In these application notes, we described protocols enabling the determination of very low concentration of BPA in liquid and powdered infant formula, and several other matrices. using AFFINIMIP SPE Bisphenol A cartridge. These methods show the determination of very low concentration of Bisphenol A with a fluorescence detector. Therefore, the use of AFFINIMIP SPE Bisphenol A enables to eliminate the tedious derivatization step required by gas chromatography. This method is also perfectly suitable for clean-up before GC-MS/MS or LC-MS/MS. 3mL-mg sorbent in a PP cartridge PI-FS16-2 for 25 cartridges PI-FS16-3 for cartridges 6mL-mg sorbent in a PP cartridge PI-FS16-2B for 25 cartridges PI-FS16-3B for cartridges 6mL-mg sorbent in a glass cartridge PI-FS16-2G for 25 cartridges PI-FS16-3G for cartridges 37

1 9 8 7 6 4 3 2 9 8 7 6 4 3 2 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 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, 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 mvolts Injection µl of Infant Formula before treatment Bisphenol A after treatment of 15mL of Infant Formula Cleanup with a 3mLor 6mL/mg AFFINIMIP SPE Bisphenol A cartridge 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water Up to 15mL of infant formula Washing of interferents 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 seconds 3mL Methanol dissolved in the mobile phase before HPLC analysis. mvolts 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 repeatability conditions (n=3, % RSD r ) HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 1mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 2 2.5 65 35 35 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: µl. C (µg/l) Mean (µg/l) Recoveries % % RSD r 1..9 88.4 1.5 2. 1.7 85.7 2.7 - 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-mg sorbent in a PP cartridge PI-FS16-2 for 25 cartridges PI-FS16-3 for cartridges 6mL-mg sorbent in a PP cartridge PI-FS16-2B for 25 cartridges PI-FS16-3B for cartridges 6mL-mg sorbent in a glass cartridge PI-FS16-2G for 25 cartridges PI-FS16-3G for cartridges 38

2-MD1.dat Fluo 2-md2.dat Fluo 4-md1.dat Fluo 4-md2.dat Fluo mnd.dat 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 14 Fluo 13 12 1 9 Bisphenol A 4.4g powdered infant milk was reconstituted in 3 ml of water and warmed up at ~ 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/mg AFFINIMIP SPE Bisphenol A cartridge 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water Up to 4mL of infant formula Washing of interferents 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 seconds 3mL Methanol dissolved in the mobile phase before HPLC analysis. mvolts 8 7 6 4 3 2-6,5 7, 7,5 8, 8,5 9, 9,5 1, 1,5 11, 11,5 12, 12,5 13, 13,5 14, 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/mg 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 Concentration 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 HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 1mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 2 2.5 65 35 35 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: µl. 3mL-mg sorbent in a PP cartridge PI-FS16-2 for 25 cartridges PI-FS16-3 for cartridges 6mL-mg sorbent in a PP cartridge PI-FS16-2B for 25 cartridges PI-FS16-3B for cartridges 6mL-mg sorbent in a glass cartridge PI-FS16-2G for 25 cartridges PI-FS16-3G for cartridges 39

AFFINIMIP SPE Bisphenol A PROTOCOL COMPARISON AFFINIMIP SPE Bisphenol A vs competitor POWDERED INFANT FORMULA ANALYSIS FS16 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 % MeOH Dilute supernatant with 7mL H2 Load on SPE cartridge OASIS HLB 3cc (7mL) Wash 2mL 4% Methanol ; Elute 1mL % 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-mg sorbent in a PP cartridge PI-FS16-2 for 25 cartridges PI-FS16-3 for cartridges 6mL-mg sorbent in a PP cartridge PI-FS16-2B for 25 cartridges PI-FS16-3B for cartridges 6mL-mg sorbent in a glass cartridge PI-FS16-2G for 25 cartridges PI-FS16-3G for cartridges 4

22 2 18 16 14 12 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, 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 mvolts Cleanup with a 3mL or 6mL/mg 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 interferents 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 seconds 3mL Methanol 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 reproducibility conditions (n=4). C (µg/l) Mean (µg/l) Recoveries % % RSD R 1. 1.5 15.1 5 - under reproducibility conditions (n=4). C (µg/l) Mean (µg/l) Recoveries % % RSD R 4 1. 1.4 14.3 1 EVALUATION OF BPA IN COMMERCIAL CANNED FOODS Bisphenol A 4 3 3 BPA : 2µg/L 2 mvolts 2 1 BPA :.5 µg/l HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 1mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 2 2.5 65 35 35 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: µl. - - BPA :.3 µg/l 3,5 4, 4,5 5, 5,5 6, 6,5 7, 7,5 8, 8,5 9, 9,5 1, 1,5 11, 11,5 12, Chromatograms after clean-up with AFFINIMIP SPE Bisphenol A of 1mL of canned salmon and tuna (liquid form). Blue: 1 st price canned salmon; Green: middle grade canned salmon: no BPA was detected; Red: premium canned salmon; Pink: canned tuna 3mL-mg sorbent in a PP cartridge PI-FS16-2 for 25 cartridges PI-FS16-3 for cartridges 6mL-mg sorbent in a glass cartridge PI-FS16-2G for 25 cartridges PI-FS16-3G for cartridges 41

MD1.dat Fluo md2.dat Fluo MD1.dat Fluo mnd.dat Fluo md2.dat Fluo mnd.dat 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 5 Fluo 3 1g of drained canned peas - carrots and 2 2mL of Water /ACN (/) are blended 2 during 2 min and centrifuged during 1min at 1 4rpm. The supernatant solution is collected, filtered (4-7µm) and diluted ½ with water to give the loading solution mvolts 4 4 3 ZOOM Bisphenol A Cleanup with a 3mL or 6mL/mg AFFINIMIP SPE Bisphenol A cartridge - 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 1 6 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water 2mL loading solution Washing of interferents 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 seconds 3mL Methanol dissolved in the mobile phase before HPLC analysis. mvolts 6 5 4 4 3 3 2 2 1 - Bisphenol A Recovery yield : 97-99% 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 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 1mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 2 2.5 65 35 35 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: µl. 3mL-mg sorbent in a PP cartridge PI-FS16-2 for 25 cartridges PI-FS16-3 for cartridges 6mL-mg sorbent in a PP cartridge PI-FS16-2B for 25 cartridges PI-FS16-3B for cartridges 6mL-mg sorbent in a glass cartridge PI-FS16-2G for 25 cartridges PI-FS16-3G for cartridges 42

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 Injection µl of beer before treatment Bisphenol A after treatment of 1mL of Beer The beer is degassed by sonication for 1 hour. Cleanup with a 3mL or 6mL/mg AFFINIMIP SPE Bisphenol A cartridge 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water 1mL of degassed beer Washing of interferents 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 seconds 3mL Methanol dissolved in the mobile phase before HPLC analysis. 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 repeatability conditions (n=3, % RSD r ) C (µg/l) Mean µg/l Recoveries % % RSD r 1. 1.1 16.9 1. 2. 1.9 93.4 1. - under reproducibility conditions ( % RSDR). HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 1mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 12.5 65 35 22 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: µl. C (µg/l) Mean µg/l Recoveries % % RSD R 1. 1. 99.3 8.9 2. 1.8 9.6 6. 3mL-mg sorbent in a PP cartridge PI-FS16-2 for 25 cartridges PI-FS16-3 for cartridges 6mL-mg sorbent in a glass cartridge PI-FS16-2G for 25 cartridges PI-FS16-3G for cartridges 43

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 1 Fluo MND.dat Fluo md1.dat Fluo md2.dat Fluo md3.dat Bisphenol A 9 8 7 Cleanup with a 3mL or 6mL/mg AFFINIMIP SPE Bisphenol A cartridge mvolts 6 4 3 2 3mL Methanol -2% Acetic Acid 3mL Acetonitrile 3mL Water Up to1mlof wine Washing of interferents 9mL Water 6mL Water/Acetonitrile (6/4) Drying 1 minute 3mL Methanol dissolved in the mobile phase before HPLC analysis. - 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, 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 concentration (µg/kg) Recoveries % HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 1mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 12.5 65 35 22 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: µl. Red wine 1 1.93 (n=2) 96.6 Red wine 2 2.13 (n=2) 16.5 Red wine 3 1.66 (n=2) 83. White wine 1.6 (n=3) 8. 3mL-mg sorbent in a PP cartridge PI-FS16-2 for 25 cartridges PI-FS16-3 for cartridges 6mL-mg sorbent in a PP cartridge PI-FS16-2B for 25 cartridges PI-FS16-3B for cartridges 6mL-mg sorbent in a glass cartridge PI-FS16-2G for 25 cartridges PI-FS16-3G for cartridges 44

AFFINIMIP SPE Bisphenol A DETERMINATION OF BISPHENOL A IN COLA DRINKS Comparison of the solution obtained before and after using AFFINIMIP SPE Bisphenol A Bisphenol A PROTOCOL OF CLEAUNP Cleanup with a 3mL or 6mL/mg 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 interferents 9mL Water 6mL Water/Acetonitrile (6/4) Drying 3 minute 3mL Methanol 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 5µg/kg after AFFINIMIP SPE Bisphenol A clean-up of 6mL of Cola drinks Mean concentration (µg/kg) Recoveries % RSDr % 1.93 (n=2) 96.6 1. HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 1mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 2 2.5 65 35 4 65 35 Flow rate: 1mL/min Fluorescence detection: excitation/emission wavelengths: 23 / 315nm Injection volume: µl. 3mL-mg sorbent in a PP cartridge PI-FS16-2 for 25 cartridges PI-FS16-3 for cartridges 6mL-mg sorbent in a PP cartridge PI-FS16-2B for 25 cartridges PI-FS16-3B for cartridges 6mL-mg sorbent in a glass cartridge PI-FS16-2G for 25 cartridges PI-FS16-3G for cartridges 45

AFFINIMIP SPE Bisphenol A DETERMINATION OF BISPHENOL A AND BADGE IN MILK 4 4 4 3 Bisphenol A BADGE 4 3 Bisphenol A Diglycidyl Ether (BADGE) 3 3 2 2 mvolts mvolts 2 2 1 1 Bisphenol A Cleanup with a 3mL or 6mL/mg AFFINIMIP SPE Bisphenol A cartridge - - 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 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). 3mL Methanol -2% Formic Acid 3mL Acetonitrile 3mL Water 9mL of Milk Washing of interferents 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. 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 HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 1mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN 65 35 2 65 35 12 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: µl. 3mL-mg sorbent in a PP cartridge PI-FS16-2 for 25 cartridges PI-FS16-3 for cartridges 6mL-mg sorbent in a PP cartridge PI-FS16-2B for 25 cartridges PI-FS16-3B for cartridges 6mL-mg sorbent in a glass cartridge PI-FS16-2G for 25 cartridges PI-FS16-3G for cartridges 46

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. Cleanup with a 6mL/mg 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. Intensity, cps Intensity, cps (a) (b) m/z 227.1 m/z 212.1 m/z 227.1 m/z 133.2 LC-MS/MS Chromatograms obtained after clean-up with AFFINIMIP SPE Bisphenol A (a) of children urine at.38ng/ml BPA, signal to noise (S/N) 13.9 (b) for the blank sample (neither urine nor BPA), S/N=1.9 Mean percentage recoveries of Bisphenol A spiked at different concentrations in 3mL of urine after AFFINIMIP SPE Bisphenol A clean-up: C (ng/ml) 1 1 Recoveries % 12.6 94.7 97.6 HPLC Method with LC-MS/MS HPLC Column: Kinetex 2.6µm PFP mm 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. 3mL-mg sorbent in a PP cartridge PI-FS16-2 for 25 cartridges PI-FS16-3 for cartridges 6mL-mg sorbent in a glass cartridge PI-FS16-2G for 25 cartridges PI-FS16-3G for cartridges 47

AFFINIMIP SPE Chloramphenicol FS11 Chloramphenicol: a major concern for human health and a challenge in food safety analysis Chloramphenicol is a broad-spectrum antibiotic widely used in the world in the past. Several health problems are related to its use. As a consequence, several countries (e.g. U.S.A, E.U, Canada ) have prohibited its use for food-producing animals. As no permitted limit has been established, E.U. has defined a Minimum Required Performance Limits (MRPLs) of.3µg/kg for product of animal origin (Commission decision 23/181/EC). However, due to its broad spectrum of activity and its availability, Chloramphenicol is still used in several countries to treat food-producing animals. Therefore, chloramphenicol analysis is still a current affair. In addition, food matrices are very complexes and induce ion-suppression phenomena which distort analysis results. For such a low MRPL threshold, a clean-up step is crucial in order to improve the sensitivity, the reliability and the specificity before analysis. It is therefore critical to develop a highly selective and sensitive analytical assay to control and monitor Chloramphenicol residues in difficult matrices such as food stuffs. AFFINISEP has developed AFFINIMIP SPE Chloramphenicol cartridge, a simple, fast, sensitive and selective tool for the extraction of Chloramphenicol from complex matrices. We demonstrate in these application notes that a reliable quantification of Chloramphenicol from honey and bovine urine at low concentrations using AFFINIMIP SPE Chloramphenicol and a single quadrupole mass detection is possible. In a very complex matrix such as honey, we obtained a high recovery yield (> 9%) with a low background, even with UV detection. The tests carried out on several kinds of honey demonstrated a good reproducibility, proving the efficiency of AFFINIMIP SPE Chloramphenicol cleanup. 1mL-mg sorbent PI-FS11-2A for 25 cartridges PI-FS11-3A for cartridges 48

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/mg AFFINIMIP SPE Chloramphenicol cartridge 2mL Acetonitrile 2mL Water 1mL of loading solution for 15µg/kg (or 1mL for.3µg/kg) Washing of interferents (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 interferents (W2).25mL Diethyl ether 2mL Methanol dissolved in the mobile phase before HPLC analysis..3µg/kg Chloramphenicol Chloramphenicol Intensity 9 9 8 8 7 7 6 6 5 4 4 3 3 2 2 1 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). Honey spiked at 15.7µg/kg Not spiked Chloramphenicol 2 3 4 5 6 7 8 9 1 Time (min) 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 repeatability conditions (n=3, % RSD r ) C (µg/kg) Mean (µg/kg) Recoveries % % RSDr 16. 15.4 96.1 3.3 SIM Chromatogram obtained after clean-up with AFFINIMIP SPE Chloramphenicol of 1g of Honey spiked with Chloramphenicol at.3µg/kg. HPLC Method with MS detection Column: Thermo Accucore C18 column mm 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. - under reproducibility conditions ( % RSD R ). C (µg/kg) Mean (µg/kg) Recoveries % % RSD R 15.7 16.9 18.1 6.5 (n=6) 18.2 16.6 91.4 11.4 (n=12) 1mL-mg sorbent PI-FS11-2A for 25 cartridges PI-FS11-3A for cartridges 49

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 Chloramphenicol 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/mg Chloramphenicol cartridge AFFINIMIP SPE 2mL Acetonitrile 2mL Water 1mL of loading solution Washing of interferents (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 interferents (W2).25mL Diethyl ether 2mL Methanol dissolved in the mobile phase before HPLC analysis. uau 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 1 9 8 7 6 4 3 2 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. NL: 1.32E5 Channel A UV UrineD1-E NL: 1.32E5 Channel A UV urined2-e NL: 1.32E5 Channel A UV UrineND-E C (µg/kg) Mean (µg/kg) Recovery % 17.6 16.7 9 HPLC Method with MS detection Column: Thermo Accucore C18 column mm 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-mg sorbent PI-FS11-2A for 25 cartridges PI-FS11-3A for cartridges

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/mg Chloramphenicol cartridge AFFINIMIP SPE 2mL Acetonitrile 2mL Water 1 or 2mL of loading solution Washing of interferents (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 interferents (W2).25mL Diethyl ether 2mL Methanol dissolved in the mobile phase before HPLC analysis. HPLC Method with MS detection Column: Thermo Accucore C18 column mm 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. Intensity 6 6 5 4 4 3 3 2 2 1mL-mg sorbent PI-FS11-2A for 25 cartridges PI-FS11-3A for cartridges Chloramphenicol 2 4 6 8 1 12 14 16 Time (min) 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) uau 9 8 7 6 4 3 2 - UV: VERY LOW BACKGROUND UV 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) 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 51

AFFINIMIP SPE Amphetamines DG12 Amphetamines are a class of illegal drugs that exhibit strong central nervous system stimulant effect. So, to determine if drivers are under influence of (Meth)amphetamine, several US and European states have set up a cut-off value in urine or blood [e.g. France and Virginia (respectively ng/ml and ng/ml of blood)]. For such low concentrations, a clean up step is crucial in order to improve the sensitivity, the reliability and the specificity before LC analysis. NH 2 N H O Amphetamine NH 2 O Methamphetamine H N O O 3,4-Methylenedioxyamphetamine MDA O 3,4-Methylenedioxymethamphetamine MDMA H N O 3,4-methylenedioxy-N-ethylamphetamine MDEA To do so, we have developed a AFFINIMIP SPE Amphetamines cartridge, a powerful technique for clean-up and pre-concentration applications of Amphetamines. These application notes describe the solid phase extraction of Amphetamines from human urine and human serum using AFFINIMIP SPE Amphetamines. 3mL-mg cartridge PI-DG12-2 for 25 cartridges PI-DG12-3 for cartridges 52

AFFINIMIP SPE Amphetamines DETERMINATION OF AMPHETAMINES IN HUMAN URINE Example of Regulations: France : prohibited cut-off limit of 1µg/mL in urine and ng/ml of blood Virginia (USA): ng/ml of blood 85 8 75 7 65 6 55 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 interferents (W1) 3mL Water 3mL Water/Acetonitrile (6/4) Drying 3 seconds 1.5mL Methanol 2% Formic acid dissolved in the mobile phase before HPLC analysis. HPLC Method with MS detection Column: Syncronis Aq column 1mm 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); 1 (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 Recoveries % 3mL-mg cartridge PI-DG12-2 for 25 cartridges PI-DG12-3 for cartridges % RSD R Amphetamine 17.5 87.5 8.9 (n=8) MDA 18.6 93.1 9.6 (n=8) Methamphetamine 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 53

AFFINIMIP SPE Amphetamines DETERMINATION OF AMPHETAMINES IN HUMAN SERUM Example of Regulations: France : prohibited cut-off limit of 1µg/mL in urine and ng/ml of blood Virginia (USA): ng/ml of blood 9 8 7 6 Amphetamine MDA 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. Cleanup with a 3mL AFFINIMIP SPE Amphetamines cartridge 1mL Acetonitrile 2mL Water 2.5mL of diluted serum Washing of interferents (W1) 3mL Water 3mL Water/Acetonitrile (6/4) Drying 3 seconds 1.5mL Methanol 2% Formic acid dissolved in the mobile phase before HPLC analysis. 4 3 2 1 Methamphetamine MDMA MDEA 2 4 6 8 1 12 14 16 18 2 22 24 Time (min) Mass Chromatogram (SIM) obtained after AFFINIMIP SPE Amphetamines clean-up of a human serum sample spiked at ng/ml with Amphetamine and its derivatives. Recovery of Amphetamines in human serum spiked at ng/ml after AFFINIMIP SPE Amphetamines clean-up and relative standard deviation calculated from results generated under reproducibility conditions (n=4). Sample Mean ng/ml Recoveries % % RSD R Amphetamine 87.9 87.9 5. MDA 94.4 94.4 3.7 Methamphetamine 9.7 9.7 2.2 MDMA 16.2 16.2 2.5 MDEA 111. 111. 4.9 HPLC Method with MS detection Column: Syncronis Aq column 1mm 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); 1 (Methamphetamine); 194 (MDMA); 28 (MDEA) Injection volume: 2µL. 3mL-mg cartridge PI-DG12-2 for 25 cartridges PI-DG12-3 for cartridges 54

AFFINIMIP SPE Tetracyclines FS112 Tetracyclines (TCs) like oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) are broad-spectrum antibiotics and are widely used as veterinary medicines and feed additives. These residues can cause toxic or allergic reactions in hypersensitive individuals and also transfer drug-resistant bacteria from food to humans. In response to these concerns and to prevent harmful effects of residual antibiotics in milk on the human health, various international health organizations have established the maximum residual limit (MRL) of TCs in all circulating milk in their countries. Worldwide maximum residue levels (MRL) for tetracycline antibiotics are ppb (µg/l). For such low concentrations, a clean up step is crucial in order to improve the sensitivity, the reliability and the specificity before LC analysis. Tetracycline Oxytetracycline Chlortetracycline To do so, we have developed a AFFINIMIP SPE Tetracyclines cartridge, a powerful technique for clean-up and pre-concentration applications of Tetracyclines. 1mL-1mg sorbent PI-FS112-2A for 25 cartridges 1mL PI-FS112-3A for cartridges 1mL kit of 12 reservoirs 15ml and adapters for use with 1, 3 & 6 ml columns PI-ACC-AR2 55

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 7µL 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 2g 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 2g, filter on Buchner. Add 7µL 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 interferents 1mL Water 2mL Water/Acetonitrile (6/4) Drying 3 minutes 2mL Methanol with 2% Formic acid dissolved in the mobile phase before HPLC analysis. HPLC Method with UV detection Column: Hypersil Gold C18 column 1mm x 2.1mm, 3µm Mobile phase: gradient profile Time ( min) % 1mM Oxalic Acid Water Flow rate:.2ml/min UV detection: 355nm Injection volume: µl. % 1mM Oxalic Acid ACN % MeOH 9 5 5 2 9 5 5 21 8 1 1 4 8 1 1 41 9 5 5 8 7 6 4 3 2 1-1 4-epiOTC TC 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48 UV Chromatograms (355nm) obtained after cleanup with AFFINIMIP SPE Tetracyclines of 1.5mL of Milk spiked with Tetracycline, Chlortetracycline and 4-epioxytetracycline (4-epiOTC) at µg/l (blue) or not spiked (red) or of 1.5mL of water spiked with Tetracycline, Chlortetracycline and 4- epioxytetracycline at µg/l (pink) Recovery of Tetracyclines after AFFINIMIP SPE Tetracyclines clean-up of Salmon or milk spiked at or µg/l and relative standard deviation calculated from results generated under repeatability conditions (n=3). Molecules Mean Milk Salmon (µg/l) 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 43.7 87.4 9.1 71 Doxycycline (DOX) 43.8 88. 2.9 89 1mL-1mg sorbent PI-FS112-2A for 25 cartridges 1mL PI-FS112-3A for cartridges 1mL kit of 12 reservoirs 15ml and adapters for use with 1, 3 & 6 ml columns PI-ACC-AR2 CTC 8 7 6 4 3 2 1-1 56

AFFINIMIP SPE Metanephrines DG11 Quantification of free Metanephrines in Plasma is considered to be the most accurate test for the clinical chemical diagnosis of Pheocromocytoma. The concentrations of these endogenous molecules are very low in serum and plasma (lower than 1nM). A clean up step is crucial in order to improve the sensitivity and the specificity before LC analysis. MeO HO OH N H Metanephrine (MN) MeO Me MeO HO HO NH 2 3-Methoxytyramine (3-MT) OH Normetanephrine (NMN) NH 2 Current method involves non specific sample preparation. To propose an accurate solution, we have developed AFFINIMIP SPE Metanephrines cartridges, a powerful technique for clean-up and pre-concentration applications of Metanephrines. This study describes the solid phase extraction of Metanephrines from plasma sample using AFFINIMIP SPE Metanephrines. In addition, a comparison with a Weak Cation Exchange SPE cartridge is shown. 1mL cartridge PI-DG11-2A for 25 cartridges PI-DG11-3A for cartridges 3mL cartridge PI-DG11-2 for 25 cartridges PI-DG11-3 for cartridges 57

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 interferents (W1) 1mL Water µl Water/Methanol (6/4) Drying 1 seconds Washing of interferents (W2) µl Methanol 1mL Methanol 5% Acetic acid 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. NMN WCX Recovery : 33% 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. Recoveries of MN and NMN at a contamination level of nm 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 1mm x 2.1mm Mobile phase: Water.1% Formic Acid flow rate:.2ml/min MS detection: m/z 322 (ESI + ) Injection volume: 2µL. 1mL cartridge PI-DG11-2A for 25 cartridges PI-DG11-3A for cartridges 3mL cartridge PI-DG11-2 for 25 cartridges PI-DG11-3 for cartridges 58

AFFINIMIP SPE Phenolics FS13 Class-selective extraction of Phenolic compounds from a variety samples: water, cosmectics, biological or food matrices. Phenolic compounds are important families of products found as natural substances in plants and life sciences or as synthetic products such as drugs. AFFINIMIP SPE Phenolics are class-selective solid phase extraction cartridges that selectively clean and concentrate a broad range of phenolic compounds prior to analysis. This treatment strongly reduces the amount of interferents and the matrix effects. Benefits Extraction of a broad family of phenolic compounds Reduction of matrix effects and of most interferents Several protocols proposed to purify your product 3mL-mg sorbent PI-FS13-2 for 25 cartridges PI-FS13-3 for cartridges 59

12 1 9 8 7 6 4 3 2 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 12 1 9 8 7 6 4 3 2 AFFINIMIP SPE Phenolics DETERMINATION OF PARABENS IN COSMETIC PRODUCTS Before After General structure of Parabens 1g of Lotion was mixed 1minute with 1mL of H2SO4 2M and ml 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/mg AFFINIMIP SPE Phenolics cartridge 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 8 6 4 Cream spiked at.8%, yield 91% (n=2) Cream spiked at.4%, yield 16 (n=2) Cream spiked at.2%, yield 16 (n=2) 18 16 14 12 8 6 4 3mL Acetonitrile 3mL Water Up to 5mL of loading solution Washing of interferents 3mL Water / Acetonitrile (75/25 v/v) 3mL Methanol The elution fraction was diluted by 2 with water prior to analysis. 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 Recovery yields and reproducibility after AFFINIMIP SPE Phenolics Clean-up. Recoveries % (n=6) RSD R % 11.1 8 2 HPLC-UV Method Column: Thermo Hypersil gold, 1mm x 2.1mm Mobile phase: 6/4 (v/v) Water/Methanol Flow rate:.2ml/min Detection: UV - 254nm Injection volume: 2µL. 3mL-mg sorbent PI-FS13-2 for 25 cartridges PI-FS13-3 for cartridges 6

3 3 2 2 1-1 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 8 6 4 2-2 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 3 3 2 2 1-2 DETERMINATION OF GUAIACOL AFFINIMIP SPE Phenolics General structure of Guaîacol Guaïacol Cleanup with a 3mL/mg AFFINIMIP SPE Phenolics cartridge 3mL Acetonitrile 3mL Water Up to 2mL of red or white wine Washing of interferents 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). Guaïacol Chromatograms obtained before (red) and after (blue) clean-up with AFFINIMIP SPE Phenolics of red wine spiked with Guaïacol (.1µM) Recovery yields and reproducibility evaluated with 3 cartridges and 3 different batches of AFFINIMIP SPE Phenolics by matrix (n=9) 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 White wine 2.2 93.5 2.6 HPLC-UV Method Column: Thermo Hypersil gold, 1mm x 4.6mm Mobile phase: 15/85 (v/v) Acetonitrile Water Flow rate: 1mL/min Detection: UV - 272nm Injection volume: µl. 3mL-mg sorbent PI-FS13-2 for 25 cartridges PI-FS13-3 for cartridges 61

AFFINIMIP SPE Phenolics DETERMINATION OF CARNOSIC ACID IN MEAT UV 6-23nm E ZON ethanol.dat 4 4 4 4 3 3 Carnosic acid 3 3 2 2 2 2 structure of Carnosic acid 1 1 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. Cleanup with a 3mL/mg AFFINIMIP SPE Phenolics cartridge 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 Chromatogram of a turkey containing ppm of Carnosic acid after clean-up with AFFINIMIP SPE Phenolics. Extraction of the turkey with Ethanol-.5% H3PO4 4 4 3 3 2 UV 6-23nm E ZON 75-25.dat Carnosic acid 4 4 3 3 2 2 2 3mL Acetonitrile 3mL Water Up to 8mL of loading solution Washing of interferents 3mL Water / Acetonitrile (6/4 v/v) 2mL Methanol -1% H 3 PO 4 1 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 Chromatogram of a turkey containing ppm 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 Clean-up. 1 Extraction solvent Recoveries % 74.5/25/.5 ACN/H 2 O/H 3 PO 4 >85% Ethanol-.5% H 3 PO 4 >8% HPLC-UV Method Column: Thermo Hypersil gold, 1mm 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. 3mL-mg sorbent PI-FS13-2 for 25 cartridges PI-FS13-3 for cartridges 62

AFFINIMIP SPE PRODUCT LIST FOR MYCOTOXINS ANALYSES Products Designation Definition Reference Nber of cartridges Zearalenone and Fumonisins AFFINIMIP SPE FumoZON 3mL Selective SPE cartridges for Zearalenone and Fumonisins PI-FS19-2 25 PI-FS19-3 Multimycotoxins AFFINIMIP SPE Multimyco1 3mL Selective SPE cartridges for analyses of Aflatoxins, Zearalenone, Ochratoxin A, T-2, HT-2, Fumonisins PI-FS114-3 PI-FS114-4 Patulin AFFINIMIP SPE Patulin AFFINIMIP SPE Patulin & Pectinase kit 3mL mg Selective SPE cartridges for Patulin 6mL 2mg Selective SPE cartridges for Patulin (for DRIED APPLE and higher enrichment with apple juice) Kit of 3mL selective SPE cartridges for Patulin + ml Pectinase enzyme solution Kit of 6mL - 2mg selective SPE cartridges for Patulin in dried apple + ml Pectinase enzyme solution PI-FS12-2 25 PI-FS12-3 PI-FS12-2B-2mg 25 PI-FS12-3B-2mg PI-FS12-2K 25 PI-FS12-3K PI-FS12-2KB-2mg 25 PI-FS12-3KB-2mg Ochratoxin A AFFINIMIP SPE Ochratoxin A 3mL Selective SPE cartridges for Ochratoxin A 6mL Selective SPE cartridges for Ochratoxin A PI-FS11-2 25 PI-FS11-3 PI-FS11-2B 25 PI-FS11-3B Deoxynivalenol AFFINIMIP SPE Deoxynivalenol 6mL -mg Selective SPE cartridges for Deoxynivalenol in FOOD 6mL -2mg Selective SPE cartridges for Deoxynivalenol in ANIMAL FEED PI-FS117-2B 25 PI-FS117-3B PI-FS11-2B-2mg 25 PI-FS11-3B-2mg Pectinase ml Pectinase enzyme solution PI-REA-1-mL Zearalenone AFFINIMIP SPE Zearalenone 3mL Selective SPE cartridges for ZON PI-FS-2 25 PI-FS-3 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 63