Customer Service About Canadian Life Science Au suject de Canadian Life Science Founded in 1997, Canadian Life Science (CLS) has steadily grown Fondée en 1997, Canadian Life Science (CLS) n’a cessé de croître to Canada’s leading supplier of LC/GC columns, chromatography pour devenir un chef de file canadien dans la vente de colonnes and dissolution accessories as well as 2d long-term sample HPLC / GC, d’accessoires de chromatographie et dissolution, storage tubes and racks for bio-banking and compound ainsi que dans la vente de tubes et systèmes d’entreposage long management. terme pour échantillons. For over 22 years, CLS has provided customers with unparalleled Depuis plus de 22 ans, CLS offre à ses clients un service et service, expertise and a wide selection of quality and cost- une expertise inégalés, en plus d’un large éventail de produits effective products that offer maximum flexibility to solve your économiques de qualité offrant une flexibilité maximale afin de analytical challenges. CLS is your foundation for what matters. résoudre vos problèmes analytique. CLS est votre pilier pour ce qui compte. Everyone is accessible to you Nous sommes très accessible We believe that our customers like our personal touch. Nous avons une approche personnelle et nous répondons à tous We take your calls personally, not a computer, not voicemail . les appels lorsque vous téléphonez au bureau chef. Nous n’avons We have dedicated inside customer service staff who care, and pas de boîte vocale. take care for specific areas. We have knowledgeable outside staff Notre personnel est dévoué au service à la clientèle et ce à based in most of the larger cities accross Canada. travers le Canada. Si vous désirez parler directement à notre We supply world class products Directrice de comptes au Québec, vous pouvez contacter Canadian Life Science distributes for top-quality manufacturers. Genevieve Lemieux au 514.428.8034. Our product offering has been chosen in a way that will give you Nous choisissons les meilleurs produits au monde the maximum flexibility solving your analytical challenges. En tant que distributeur nous avons l’avantage de choisir les Technical Support meilleurs fournisseurs et notre gamme complète de produit vous Our technical staff is happy to respond to customer enquiries. donne l’embarras du choix. We can also supply extensive application information from one Support Technique of the world’s largest data bases. Notre équipe technique se fait toujours un plaisir de vous aider à Plan the best delivery faire le meilleur choix pour vos applications. When you decide to purchase a product from Canadian Life Service et logistique Science our customer service department will find the fastest Lorsque vous placez une commande chez nous, nous nous and most economical way to send your order to you. For assurons de choisir le moyen le plus efficace et économique your convenience we have two warehouse locations, one in d’acheminer la marchandise. Edmonton, AB and one in Peterborough, ON. Nous avons deux entrepôts dont un en Alberta pour servir nos We’re glad to help clients de l’Ouest et un en Ontario pour servir nos clients de l’Est. We are never too busy to take your call. C’est un plaisir de vous servir Try us. You’ll notice the difference. Nous ne sommes jamais trop occupés pour vous répondre. Canadian Life Science, friendly people to do business with! Essayez-nous et vous verrez la différence. Il est agréable de faire affaire avec nous! I-22 CANADIAN Proud member of the Chrom4 buying group Fier membre du groupe dʼacheteur Chrom4 LIFE SCIENCE 1 888-226-2775 :: [email protected] :: www.lifescience.ca
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 ISO9001:2008 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 Multimyco10 8 Simultaneous determination of Aflatoxins, Fumonisins, Ochratoxin A, HT-2, T-2, Zearalenone in Wheat AFFINIMIP® SPE Patulin 9 10 Patulin in Baby food apple juice 11 Patulin in Apple juice 12 Patulin in Baby food apple puree 13 Patulin in Apple puree 14 Patulin in Apple – Fruit puree 15 Patulin in WHOLE apple 16 Patulin in Cider 17 Patulin in Alcohol Pommeau and Liquor 18 Patulin in Tomato Ketchup and Tomato Powder 19 Patulin in Blueberry juice 20 Patulin in Thick Juice and Concentrate Juice 21 Patulin in Apple puree Patulin in Dried apple 22 AFFINIMIP® SPE Ochratoxin A 23 Ochratoxin A in Cereals 24 Ochratoxin A in Paprika Ochratoxin A in Red and White wine 25 AFFINIMIP® SPE Zearalenone 26 Zearalenone in Maize and Rice 27 Zearalenone in Cereal-based Baby food Zearalenone in Edible corn oil 28 AFFINIMIP® SPE Deoxynivalenol 29 Deoxynivalenol in cereals for food (Water extraction) 30 Deoxynivalenol in babyfood cereals 31 Deoxynivalenol, 3-AcetylDON and 15-AcetylDON in cereals (Hydro-organic extraction) Deoxynivalenol in cereals for animal feeds 32 AFFINIMIP® SPE FumoZON 33 Fumonisins B1 + B2 and Zearalenone in Maize Flour 34 Fumonisins B1 + B2 and Zearalenone in Maize-based baby food 35 AFFINIMIP® SPE Estrogens 36 Determination of estrogens in plasma Protocol comparative - AFFINIMIP® SPE Estrogens vs usual protocol 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 40 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 48 AFFINIMIP® SPE Chloramphenicol 49 Chloramphenicol in Honey 50 Chloramphenicol in Bovine urine 51 Chloramphenicol in Shrimp 52 53 AFFINIMIP® SPE Amphetamines 54 Amphetamines in Human urine 55 Amphetamines in Human serum 56 57 AFFINIMIP® SPE Tetracyclines 58 Tetracyclines (Oxytetracycline, Chlortetracycline, Tetracycline), theirs epimers and Doxycycline in Milk and Salmon 59 60 AFFINIMIP® SPE Metanephrines 61 Methanephrines in Plasma – comparison with WCX cartridges 62 63 AFFINIMIP® SPE Phenolics 64 Parabens in cosmetic products Guaïacol Carnosic acid in meat AFFINIMIP® SPE PRODUCT LIST FOR MYCOTOXIN ANALYSES AFFINIMIP® SPE PRODUCT LIST (MISCELLANEOUS) 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® Compared to IAC, AFFINIMIP® SPE provides: High SPE Easier and faster protocol Selectivity 6µmol/g High Lower dilution 10- Easier automatisation Capacity Good 100µmol/g (Cf. Automated method for the selective SPE of Analyte recognition Poor Ochratoxin A from wheat Using Molecularly in water Poor Variable Imprinted Polymer; Gilson Application Notes Variable Handbook 2011; volume 1 Issue 4) Analyte recognition Expensive Good in Organics Stability Very High Good Reproducibility Cost Inexpensive PROTOCOL: Zearalenone (ZON) from maize PROTOCOL: Ochratoxin A (OTA) from wheat flour flour Step Vicam IAC AFFINIMIP®SPE Step Vicam IAC AFFINIMIP®SPE ZON OTA Extraction 25g sample in of target 100mL 90/10 25g sample in Extraction of 50g sample in 100mL 60/40 analyte Methanol/water 100mL 75/25 target analyte ACN/water Blender 3 minutes ACN/water Preparation Blender 1 minute + filtration loading + filtration Blender 3 solution minutes + Preparation 10mL extract + 10mL extract + Loading 4mL extract + filtration loading 40mL PBS 10mL HCl 0.1M 96mL water solution Washing 100mL Loading 10mL extract + pH=1 10mL Water Elution solution Loading 10mL Loading 4mL Loading 8mL Loading solution solution Protocol 20mL Water solution time Washing 10mL PBS 7mL 60/40 HCl 1.5mL Methanol 4mL 2/58/40 10mL Water 0.1M pH=1/ACN Acetic acid / 55min water / ACN Elution 1.5mL Methanol 2mL 98/2 Methanol/Acetic 2mL 98/2 Methanol/Acetic acid acid Protocol time 30min 20min 30min
AFFINIMIP® SPE CARTRIDGE VS IMMUNOAFFINITY COLUMN CHROMATOGRAM ASPECT Equivalent chromatograms AFFINIMIP® SPE IAC Figure 1. Chromatogram of Maize sample spiked with Zearalenone at 85 µg/kg obtained after cleanup by AFFINIMIP®SPE Zearalenone (red) or Vicam IAC (blue). Higher Recoveries RECOVERIES obtained with AFFINIMIP® SPE 52 Fluo Fluo 52 Recovery % 51 elution B06 elution immuno B02 50 49 51 48 100 98 97 50 10ng/g OTA ZON OTA 85% Recovery 85 79 90 49 80 48 70 47 47 60 46 46mVolts 50 45 AFFINIMIP® SPE 6ng/g OTA 45Area 40 mVolts 79% Recovery 30 Level found ng/g 44 IAC44 20 43 43 10 42 42 41 41 0 2 4 6 8 10 12 14 16 18 20 22 24 26 MIP IAC MIP IAC Minutes Figure 3. Recovery of Ochratoxin A or Zearalenone Figure 2. Chromatogram of wheat sample spiked obtained after cleanup by AFFINIMIP®SPE or Vicam with Ochratoxin A obtained after cleanup by IAC. AFFINIMIP®SPE Zearalenone (red, spiked at 10ng/g) or Vicam IAC (blue, spiked at 6ng/g). CAPACITY Capacity MIP > Capacity IAC 3,50E+08 5000 3,00E+08 AFFINIMIP® SPE 4500 2,50E+08 IAC 2,00E+08 4000 AFFINIMIP® SPE 1,50E+08 3500 1,00E+08 3000 IAC 5,00E+07 2500 0,00E+00 2000 4000 6000 8000 10000 2000 0 1500 Spiking level ng/g 1000 500 0 0 1000 2000 3000 4000 5000 6000 Spiking level ng/g Figure 4. Comparison of capacity between Figure 5. Comparison of capacity between AFFINIMIP®SPE Zearalenone (red) and Vicam AFFINIMIP®SPE OTA (red) and Vicam IAC (blue). 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-FS102) AFFINIMIP® SPE Ochratoxin A (PI-FS101) AFFINIMIP® SPE Zearalenone (PI-FS100) 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-FS109) for the analyses of Fumonisins Zearalenone AFFINIMIP® SPE Multimyco10 (PI-FS114) for the analyses of Fumonisins Aflatoxins Ochratoxin A T-2 and HT-2 Zearalenone 7
SIMULTANEOUS DETERMINATIONAFOFIFNMIMUILPT®IMSPYECMOTuOltXimINyScoIN1F0S114 WHEAT AFFINIMIP® SPE Multimyco10 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. PROTOCOL OF CLEANUP WHEAT Sample preparation 25g of ground wheat were extracted with MRM parameters for mycotoxins analyses 100mL of Acetonitrile/Water (50/50, v/v/v) for 2 min using a blender. The extract was filtered Compound name RT MRM transition through a folded filter paper and 4mL of the filtrate were diluted with 16mL of water. Then, Aflatoxin B1 1.4 312.8 > 285.0 this solution was filtered through a filter paper. Positive ion mode 2 Fumonisin B1 1.7 722.4 > 141.1 722.4 > 334.5 Positive ion mode 1 722.4 > 352.3 Cleanup with a 3mL/60mg AFFINIMIP® SPE HT-2 1.7 441.9 > 104.9 Multimyco10 cartridge 441.9 > 214.7 Positive ion mode 6 441.9 > 263.2 Equilibration •2mL MeOH -2% Formic acid T-2 1.8 484.0 > 105.0 •3mL Acetonitrile Positive ion mode 9 484.0 > 185.1 •2mL water 484.0 > 305.1 Loading Zearalenone 2.0 316.8 > 131.1 •3mL of loading solution 316.8 > 174.9 Negative ion mode 5 316.8 > 273.1 Washing of interferents (W1) •3mL Water Ochratoxin A 2.1 403.8 > 221.1 •3mL Water/Acetonitrile (85/15 v/v) 403.8 > 239.2 Positive ion mode 3 403.8 > 356.1 Drying by applying vacuum 3 minutes Elution (E) Recovery yield •3mL Methanol/ACN/Formic acid Recovery of multimycotoxins extracted from wheat (48.5/48.5/3, v/v/v) The elution fraction was then evaporated and and analyzed after AFFINIMIP® SPE Multimyco10 dissolved in water containing 0.1% acetic acid before HPLC analysis. cleanup Compound C° Mean R% name µg/k µg/kg g Aflatoxin B1 2 1.6 85 Fumonisin B1 1000 937 94 UFLC Method HT-2 100 119 119 Column: Phenomenex Kinetix XB-C18 T-2 50 56.5 113 Detection: LC-MS/MS with ESI source - MRM mode Zearalenone 50 54 108 Injection volume: 20µL. Ochratoxin A 3 2.6 88 Catalog number: PI-FS114-03 for 50 cartridges, 3mL PI-FS114-04 for 100 cartridges, 3mL 8
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN BABY FOOD APPLE JUICE Regulations for apple juice: RESULTS Europe (EC 1881/2006) : 50µg/Kg USA (FDA CPG Sec.510.150) : 50µg/Kg 40 40 Regulations for apple juice for infants and young children: 35 Patulin 35 Europe (EC 1881/2006) : 10µg/Kg 30 30 PROTOCOL OF CLEANUP Sample preparation 25 25 Loading solution: 2.5mL apple juice and 2.5mL of water-2% acetic acid are mixed. 20 20 Cleanup with a 3mL/100mg AFFINIMIP® SPE 15 15 Patulin cartridge 10 10 Equilibration •2mL Acetonitrile mAU •1mL water mAU Loading 55 •4mL of loading solution 00 Washing of interferents (W1) •1mL NaHCO3 -5 -5 •2mL Water -10 -10 Drying by applying vacuum 10 seconds Washing of interferents (W2) -15 -15 •1mL Diethyl Ether -20 -20 Elution (E) -25 -25 •2mL Ethyl Acetate The elution fraction was then evaporated and 9 10 11 12 13 14 15 16 17 18 19 20 dissolved in water containing 0.1% acetic acid Minutes before HPLC analysis. Chromatograms obtained after AFFINIMIP® SPE Patulin Clean-up of an apple juice spiked at 10µk/kg with Patulin (Green and blue) or not spiked (Red) Recovery of Patulin (n=9) at a contamination level of 10µg/kg in apple Juice after AFFINIMIP® SPE Patulin Clean-up. Recoveries % (n=9) % RSDR 97.9 11 HPLC Method Catalog number: Column: Atlantis T3 column, 150mm x 2.1mm 3mL-100mg sorbent for apple juice and Mobile phase: Deionized water/ACN (95/5, v/v) puree Flow rate: 0.2mL/min PI-FS102-02 for 25 cartridges Detection: UV - 276nm PI-FS102-03 for 50 cartridges Injection volume: 100µL. 6mL-200mg sorbent for a apple-based products, fruit juice and concentrate PI-FS102-02B -200mg for 25 cartridges PI-FS102-03B -200mg for 50 cartridges 9
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN APPLE JUICE Regulations for apple juice: RESULTS Europe (EC 1881/2006) : 50µg/Kg USA (FDA CPG Sec.510.150) : 50µg/Kg UV 6000-276nm UV6000-276nm Regulations for apple juice for infants and Brut carrefour.dat C1 C05 young children: Europe (EC 1881/2006) : 10µg/Kg 2000 2000 PROTOCOL OF CLEANUP 1800 1800 Sample preparation Loading solution: 2.5mL apple juice and 2.5mL 1600 1600 of water-2% acetic acid are mixed. Before clean- Clean-up with a 3mL/100mg AFFINIMIP® SPE Patulin cartridge 1400 1400 Equilibration up •2mL Acetonitrile •1mL water 1200 1200 Loading mAU mAU •4mL of loading solution 1000 1000 Washing of interferents (W1) •1mL NaHCO3 in Water Patulin800 800 •2mL Water 600 600 Drying by applying vacuum 10 seconds Washing of interferents (W2) 400 400 •1mL Diethyl Ether 200 200 Elution (E) 00 •2mL Ethyl Acetate The elution fraction was then evaporated and 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 dissolved in water containing 0.1% acetic acid Minutes before HPLC analysis. Chromatograms of apple juice containing 25µg/kg HPLC Method of Patulin before (Red) and after (Blue) AFFINIMIP® Column: Atlantis T3 column, 150mm x 2.1mm SPE Patulin Clean-up Mobile phase: Deionized water/ACN (95/5, v/v) Flow rate: 0.2mL/min 70 70 Detection: UV - 276nm Injection volume: 100µL. 60 60 50 50 HMF 40 40 30 30 mAU mAU 20 20 10 10 00 -10 -10 -20 -20 9 10 11 12 13 14 15 16 17 18 19 Minutes Chromatograms obtained after AFFINIMIP® SPE Patulin Clean-up of an apple juice spiked at 40µg/kg (tested twice, red) or at 10µg/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 Recoveries % RSDR Patulin (ng/mL) % 10 97.9 11 (n=9) 40 90.6 11 (n=41) Catalog number: 3mL-100mg sorbent for apple juice and puree PI-FS102-02 for 25 cartridges PI-FS102-03 for 50 cartridges 6mL-200mg sorbent for a apple-based products, fruit juice and concentrate PI-FS102-02B -200mg for 25 cartridges PI--FS102-03B -200mg for 50 cartridges 10
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN APPLE PUREE Regulations for apple puree: RESULTS Europe (EC 1881/2006) : 25µg/Kg Regulations for apple juice for infants and UV 6000-276nm UV 6000-276nm UV 6000-276nm young children: C07 C10 C15 Europe (EC 1881/2006) : 10µg/Kg 140 140 120 120 Patulin 100 100 80 80 mAU mAU PROTOCOL OF CLEANUP 60 60 Sample preparation 10g of apple puree, 150µL of a pectinase 40 40 enzyme solution and 10mL water are mixed. Leave solution at room temperature overnight 20 20 or for 2h at 40°C. Centrifuge at 4500g for 5min and then filter the solution with a 0.2µm filter. 00 This solution is used as the loading solution. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Cleanup with a 3mL/100mg AFFINIMIP® SPE Minutes Patulin cartridge Chromatograms of apple puree containing 40µg/kg Equilibration or 80µg/kg of Patulin before (Red) and after (Blue) •2mL Acetonitrile AFFINIMIP® SPE Patulin Clean-up •1mL Water UV 6000-276nm UV 6000-276nm UV 6000-276nm Loading C4-E-bis C21 C06 C11 •5mL of loading solution 120 120 Washing of interferents (W1) 100 •4mL Water -1%Acetic acid Patulin •1mL NaHCO3 1% solution •3mL Water 100 Drying by applying vacuum 10 seconds 80 80 Washing of interferents (W2) mAU 60 60 mAU •500µL Diethyl Ether Elution (E) 40 40 •2mL Ethyl Acetate 20 20 The elution fraction was then evaporated and dissolved in water containing 0.1% acetic acid 00 before HPLC analysis. 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Minutes Chromatograms of apple puree containing 0µg/kg (blue) or 20µg/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 20µg/kg in apple puree after AFFINIMIP® SPE Patulin Clean-up. HPLC Method Concentration of Recoveries % % RSDr Column: Atlantis T3 column, 150mm x 2.1mm Patulin (µg/kg) (n=3) 4.5 Mobile phase: gradient 20 84 Time (min) % water % ACN Catalog number: 0 98 2 20 98 2 3mL-100mg sorbent 21 50 50 PI-FS102-02 for 25 cartridges 25 50 50 PI-FS102-03 for 50 cartridges 26 98 2 PI-FS102-02K for a kit of 25 cartridges + 50mL Pectinase Flow rate: 0.2mL/min PI-FS102-03K for a kit of 50 cartridges + 50mL Detection: UV - 276nm Pectinase Injection volume: 100µL. PI-REA-001-50mL for 50mL Pectinase solution 11
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN BABY FOOD APPLE PUREE Regulations for apple puree: RESULTS Europe (EC 1881/2006) : 25µg/Kg Regulations for apple puree for infants and Patulin young children: Europe (EC 1881/2006) : 10µg/Kg PROTOCOL OF CLEANUP Chromatograms obtained after AFFINIMIP® SPE Sample preparation Patulin Clean-up of different apple puree. 10g of apple puree, 150µL of a pectinase enzyme solution and 10mL water are mixed. In the lower part, clean-up of an apple puree from Leave solution at room temperature overnight a well-known brand spiked at 25µg/kg (orange), or for 2h at 40°C. Centrifuge at 4500g for 5min 10µk/kg with Patulin (pink, tested twice) or not and then filter the solution with a 0.2µm filter. spiked (red). This solution is used as the loading solution. In the top part, clean-up of an apple puree second well known brand spiked at 25µg/kg (green), Cleanup with a 3mL/100mg AFFINIMIP® SPE 10µk/kg with Patulin (blue, tested twice) or not Patulin cartridge spiked (turquoise). Equilibration Recovery and repeatability of Patulin (n=4) at a •2mL Acetonitrile contamination level of 10µg/kg in apple puree after •1mL Water AFFINIMIP® SPE Patulin Clean-up. Loading Recoveries % (n=4) % RSDR •5mL of loading solution 81.2 2.1 Washing of interferents (W1) •4mL Water -1%Acetic acid •1mL NaHCO3 1% solution •3mL Water Drying by applying vacuum 10 seconds Washing of interferents (W2) •500µL Diethyl Ether Elution (E) •2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing 0.1% acetic acid before HPLC analysis. HPLC Method Catalog number: Column: Atlantis T3 column, 150mm x 2.1mm Mobile phase: gradient 3mL-100mg sorbent PI-FS102-02 for 25 cartridges Time (min) % water % ACN PI-FS102-03 for 50 cartridges 0 98 2 PI-FS102-02K for a kit of 25 cartridges + 50mL 20 98 2 Pectinase 21 50 50 PI-FS102-03K for a kit of 50 cartridges + 50mL 25 50 50 Pectinase 26 98 2 PI-REA-001-50mL for 50mL Pectinase solution Flow rate: 0.2mL/min Detection: UV - 276nm Injection volume: 100µL. 12
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN APPLE – FRUIT PUREE Regulations for apple puree: RESULTS Europe (EC 1881/2006) : 25µg/Kg Regulations for apple puree for infants and UV 6000-276nm UV 6000-276nm UV 6000-276nm young children: C4-E-bis C21 C06 C11 Europe (EC 1881/2006) : 10µg/Kg 120 120 100 100 80 80 Patulin 60 60 mAU mAU mAU mAU 40 40 PROTOCOL OF CLEANUP 20 20 Sample preparation 10g of apple puree, 150µL of a pectinase 00 enzyme solution and 10mL water are mixed. Leave solution at room temperature overnight 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 or for 2h at 40°C. Centrifuge at 4500g for 5min Minutes and then filter the solution with a 0.2µm filter. This solution is used as the loading solution. Chromatograms of apple puree containing 0µg/kg (blue) or 20µg/kg (tested twice, green and red) of Cleanup with a 3mL/100mg AFFINIMIP® SPE Patulin after AFFINIMIP® SPE Patulin Clean-up. Patulin cartridge 250 250 Equilibration 225 •2mL Acetonitrile Apple-pear puree with Patulin (25µg/kg) •1mL Water 225 Loading •5mL of loading solution 200 Apple puree with Patulin (40µg/kg) 200 Washing of interferents (W1) 175 Apple-strawberry puree with Patulin (40µg/kg) 175 •4mL Water -1%Acetic acid •1mL NaHCO3 1% solution 150 150 •3mL Water 125 125 Drying by applying vacuum 10 seconds Washing of interferents (W2) 100 100 75 •500µL Diethyl Ether Patulin Elution (E) 75 •2mL Ethyl Acetate The elution fraction was then evaporated and 50 50 dissolved in water containing 0.1% acetic acid before HPLC analysis. 25 25 00 -25 -25 -50 -50 -75 -75 26 27 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Minutes SPE Chromatograms obtained after AFFINIMIP® 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 Recoveries % RSDR Patulin (µg/kg) % 10 (n=9) 77.4 8.1 25 (n=8) 90.9 11.4 40 (n=6) 86.0 11.9 HPLC Method Catalog number: Column: Atlantis T3 column, 150mm x 2.1mm Mobile phase: gradient 3mL-100mg sorbent PI-FS102-02 for 25 cartridges Time (min) % water % ACN PI-FS102-03 for 50 cartridges 0 98 2 PI-FS102-02K for a kit of 25 cartridges + 50mL 20 98 2 Pectinase 21 50 50 PI-FS102-03K for a kit of 50 cartridges + 50mL 25 50 50 Pectinase 26 98 2 PI-REA-001-50mL for 50mL Pectinase solution Flow rate: 0.2mL/min Detection: UV - 276nm Injection volume: 100µL. 13
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN WHOLE APPLE Regulations for solid apple products: RESULTS Europe (EC 1881/2006) : 25µg/Kg PROTOCOL OF CLEANUP mAU 90 90 Sample preparation 80 Preparation with microwave 80 70 Whole apple is cut into pieces and put in a 60 microwave for 90s before crushing the pieces. 70 Patulin 50 15g sample and 7.5mL water are mixed with 40 150µL pectinase solution and put overnight at 60 30 room temperature or for 2h at 40°C before a 20 filtration with filter 4-7µm to obtain the loading 50 BLENDER 10 solution. 40 PREPARATION 0 Preparation with a blender -10 Whole apple is cut into pieces, put in a blender 30 -20 with Water (2:1 Apple: Water) and mix for -30 1min. 15g sample and 300µL pectinase solution 20 are put overnight at room temperature or for 2h at 40°C before a filtration with filter 4-7µm 10 MICROWAVE to obtain the loading solution. 0 PREPARATION Cleanup with a 3mL/100mg AFFINIMIP® SPE Patulin cartridge -10 Equilibration -20 •2mL Acetonitrile •1mL Water -30 Loading 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 •3mL of loading solution Minutes Washing of interferents (W1) Chromatograms obtained after AFFINIMIP® SPE •3mL Water-2% Acetic Acid Patulin Clean-up of whole apple spiked at 40µg/kg with Patulin (dark colors) or not spiked (light colors). Drying by applying vacuum 10 seconds Washing of interferents (W2) Recovery yields obtained after AFFINIMIP® SPE Patulin Clean-up of spiked whole apple with •250µL Diethyl Ether 40µg/kg of Patulin. Whole apples are prepared Drying by applying vacuum 10 seconds according to 2 different methods Elution (E) Whole apple prepared Whole apple prepared •1mL Ethyl Acetate The elution fraction was then evaporated and with blender with microwave dissolved in water containing 0.1% acetic acid before HPLC analysis. 96 96 95 88 HPLC Method Catalog number: Column: Atlantis T3 column, 150mm x 2.1mm Mobile phase: gradient profile 3mL-100mg sorbent PI-FS102-02 for 25 cartridges Time (min) % water % ACN PI-FS102-03 for 50 cartridges 0 98 2 PI-FS102-02K for a kit of 25 cartridges + 50mL 20 98 2 Pectinase 21 50 50 PI-FS102-03K for a kit of 50 cartridges + 50mL 25 50 50 Pectinase 26 98 2 PI-REA-001-50mL for 50mL Pectinase solution Flow rate: 0.2mL/min Detection: UV - 276nm Injection volume: 100µL. 14
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN CIDER Regulations for cider: RESULTS Europe (EC 1881/2006) : 50µg/Kg Patulin PROTOCOL OF CLEANUP Chromatograms obtained after AFFINIMIP® SPE Sample preparation Patulin Clean-up of a cider spiked at 40µg/kg (tested The cider is degassed by sonicating sample for twice, pink) or at 10µg/kg (tested twice, blue) with 1 hour. Then the degas cider is diluted by 2 Patulin or not spiked (red). with water containing 2% of acetic acid. This solution is mixed and used as the loading Recovery of Patulin at a contamination level of solution. 10µg/kg and 40µg/kg in cider after AFFINIMIP® SPE Patulin Clean-up and relative standard deviation Cleanup with a 3mL/100mg AFFINIMIP® SPE calculated from results generated under Patulin cartridge reproducibility conditions. Equilibration Concentration of Recoveries % % RSDR •2mL Acetonitrile Patulin (ng/mL) - •1mL Water 87.5 (n=2) 7.5 10 80.5 (n=5) Loading •4mL of loading solution 40 Washing of interferents (W1) •1mL NaHCO3 1% in Water •2mL Water Drying by applying vacuum 10 seconds Washing of interferents (W2) •500µL Diethyl Ether Elution (E) •2mL Ethyl Acetate The elution fraction was then evaporated and dissolved in water containing 0.1% acetic acid before HPLC analysis. HPLC Method Catalog number: Column: Atlantis T3 column, 150mm x 2.1mm Mobile phase: Deionized water/ACN (95/5, v/v) 3mL-100mg sorbent Flow rate: 0.2mL/min PI-FS102-02 for 25 cartridges Detection: UV - 276nm PI-FS102-03 for 50 cartridges Injection volume: 100µL. PI-FS102-02K for a kit of 25 cartridges + 50mL Pectinase PI-FS102-03K for a kit of 50 cartridges + 50mL Pectinase PI-REA-001-50mL for 50mL Pectinase solution 15
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN ALCOHOL POMMEAU AND LIQUOR Regulations for apple based beverage : RESULTS Europe (EC 1881/2006) : 50µg/Kg 300 300 Manzella liquor contains 20% alcohol and 2.1% of concentrated apple juice. 275 275 Alcohol Pommeau is a mixture of Calvados and Apple Juice. It contains 17% Alcohol. 250 250 PROTOCOL OF CLEANUP 225 Patulin 225 Sample preparation To 1mL of Manzella Liquor or Alcohol 200 200 Pommeau, add 2mL Water to obtain the loading solution. mAU 175 175 m Cleanup with a 3mL/100mg AFFINIMIP® SPE 150 150 Patulin cartridge 125 125 Equilibration •2mL Acetonitrile 100 100 •1mL Water 75 75 Loading •3mL of loading solution 50 50 Washing of interferents (W1) 25 25 •3mL Water (containing 2% Acetic Acid for AA W1 protocol) 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Minutes Drying by applying vacuum 10 seconds Washing of interferents (W2) Chromatograms obtained after AFFINIMIP® SPE Patulin Clean-up of Manzella liquor spiked at •250µL Diethyl Ether 40µg/L with Patulin (dark blue for Water in W1 and Drying by applying vacuum 10 seconds red for Water –AA in W1) or not spiked (light blue Elution (E) and pink). Washing with Acetic acid is more efficient. •2mL Ethyl Acetate The elution fraction was then evaporated and CUV106000-276UCnV1m26000-276UCnV1m16000-276UCnV1m36000-276CUnV1m56000-276CUnV1m46000-276nm 160 dissolved in water containing 0.1% acetic acid 160 before HPLC analysis. 140 140 mAU 120 120 100 Patulin 100 80 80 60 60 40 40 20 20 00 8 9 10 11 12 13 14 15 16 17 18Minu1t9es 20 21 22 23 24 25 26 27 28 29 Chromatograms obtained after AFFINIMIP® SPE Patulin Clean-up of Pommeau spiked at 40µg/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 HPLC Method Pommeau Water for W1 Water-AA for W1 Column: Atlantis T3 column, 150mm x 2.1mm Manzella 101 101 90 93 Mobile phase: gradient profile 102 106 87 90 Time (min) % water % ACN Catalog number: 0 98 2 20 98 2 3mL-100mg sorbent 21 50 50 PI-FS102-02 for 25 cartridges 25 50 50 PI-FS102-03 for 50 cartridges 26 98 2 PI-FS102-02K for a kit of 25 cartridges + 50mL Pectinase ) Flow rate: 0.2mL/min PI-FS102-03K for a kit of 50 cartridges + 50mL Detection: UV - 276nm Pectinase Injection volume: 100µL. PI-REA-001-50mL for 50mL Pectinase solution 16
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN TOMATO KETCHUP AND TOMATO POWDER UV 6000-276nm UV 6000-276nm B14 B15 110 110 100 PROTOCOL OF CLEANUP 100 RESULTS 90 Sample preparation Preparation OF TOMATO KETCHUP 90 10g tomato ketchup and 10mL water are mixed with 150µL pectinase solution and left 80 80 overnight at RT before a filtration with filter 70 0.2µm to obtain the loading solution. TOMATO KETCHUP Preparation OF TOMATO POWDER 70 10g tomato ketchup and 20mL water are mixed. 10g of the mixture, 10mL water and UV 6000-276nm UV 6000-276nm Patulin 60 150µL pectinase solution are left overnight at B14 B15 60 50 RT before a centrifugation at 4500rpm during 5 40 min. Then the mixture is filtered with filter 1100 mAU 1100 mAU 30 0.2µm to obtain the loading solution. 1000 1000 mAU 20 50 900 10 Cleanup with a 3mL/100mg AFFINIMIP® SPE 900 800 0 Patulin cartridge 40 700 600 Equilibration mAU 30 500 •2mL Acetonitrile 800 •1mL Water 700 Recove20 ry yield Loading 80%600 10 •5mL of loading solution from tomato ketchup or 2mL from tomato powder 500 0 Washing of interferents (W1) Patulin400 6 7 8 9 10 11 12 13 14 15 16 17 18 19 42000 21 22 23 24 25 •4mL Water-1% Acetic Acid Minutes •4mL Water 300 300 Drying by applying vacuum 10 seconds 200 200 Washing of interferents (W2) 100 100 •500µL Diethyl Ether 00 Elution (E) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 •2mL Ethyl Acetate Minutes The elution fraction was then evaporated and dissolved in water containing 0.1% acetic acid Chromatograms obtained after AFFINIMIP® SPE before HPLC analysis. Patulin Clean-up of TOMATO KETCHUP spiked at 40µg/kg with Patulin (red) or not spiked (light blue). TOMATO POWDER110 UV 6000-276nm UV 6000-276nm 110 B 16 B 17 100 90 100 80 UV 6000-276nm UV 6000-276nm 90 B16 B17 1100 80 1100 1000 70 1000 70 900 60 900 Recovery60 800 50 mAU 700 40 mAU 600 30 20 mAU mAU 50 yield800 40 70%700 30 600 20 500 10 500 10 400 0 0 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 400 26 27 28 29 30 9 Minutes Patulin300 300 200 200 100 100 00 -100 -100 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes Chromatograms obtained after AFFINIMIP® SPE Patulin Clean-up of TOMATO POWDER spiked at 36µg/kg with Patulin (red) or not spiked (light blue). HPLC Method Catalog number: Column: Atlantis T3 column, 150mm x 2.1mm Mobile phase: gradient profile 3mL-100mg sorbent PI-FS102-02 for 25 cartridges Time (min) % water % ACN PI-FS102-03 for 50 cartridges 0 98 2 PI-FS102-02K for a kit of 25 cartridges + 50mL 20 98 2 Pectinase 21 50 50 PI-FS102-03K for a kit of 50 cartridges + 50mL 25 50 50 Pectinase 26 98 2 PI-REA-001-50mL for 50mL Pectinase solution Flow rate: 0.2mL/min Detection: UV - 276nm Injection volume: 100µL. 17
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN BLUEBERRY JUICE PROTOCOL OF CLEANUP RESULTS Sample preparation 5mL Blueberry juice is diluted with 5mL water 70 Recovery yields: 90 and 96% 70 containing 2% of acetic acid to obtain the loading solution. 60 60 Cleanup with a 3mL/100mg AFFINIMIP® SPE 50 50 Patulin cartridge 40 40 Equilibration •2mL Acetonitrile m Patulin 30 •1mL Water A U Loading 30 •4mL of loading solution 20 20 Washing of interferents (W1) •1mL NaHCO3 1% in Water 10 10 •2mL Water 00 Drying by applying vacuum 10 seconds -10 -10 Washing of interferents (W2) -20 -20 •500µL Diethyl Ether 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Elution (E) Minutes •2mL Ethyl Acetate Chromatograms obtained after AFFINIMIP® SPE The elution fraction was then evaporated and Patulin Clean-up of Blueberry juice spiked at 40µg/L dissolved in water containing 0.1% acetic acid with Patulin (red) or not spiked (light blue). before HPLC analysis. HPLC Method Column: Atlantis T3 column, 150mm x 2.1mm Mobile phase: gradient profile Time (min) % water % ACN Catalog number: 0 98 2 20 98 2 3mL-100mg sorbent 21 50 50 PI-FS102-02 for 25 cartridges 25 50 50 PI-FS102-03 for 50 cartridges 26 98 2 PI-FS102-02K for a kit of 25 cartridges + 50mL Pectinase ) Flow rate: 0.2mL/min PI-FS102-03K for a kit of 50 cartridges + 50mL Detection: UV - 276nm Pectinase Injection volume: 100µL. PI-REA-001-50mL for 50mL Pectinase solution 18
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN CONCENTRATE JUICE AND THICK JUICE PROTOCOL OF CLEANUP mAU RESULTS Preparation of fruit juice concentrate samples 2.5g of fruit juice concentrate are mixed with THICK JUICE 10mL water and 100µL Pectinase. (REA-001- 50mL). Leave the solution at room temperature 450 overnight or for 2h at 40°C. Centrifuge at 4000g for 10min and collect the supernatant. 400 Dilute the supernatant by 2 with Acetic Acid 2% in water. This solution is used as the loading 350 Patulin solution. 300 Preparation of thick fruit juice samples 15mL of thick fruit juice are mixed with 120µL 250 Pectinase (REA-001-50mL). Leave the solution at room temperature overnight or for 2h at 200 40°C. Centrifuge at 4000g for 10min and collect the supernatant. Dilute the supernatant by 2 150 with acetic acid 2% in water. This solution is used as the loading solution. 100 Cleanup with a 6mL/200mg AFFINIMIP® SPE 50 Patulin cartridge 0 Equilibration 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 •4mL Acetonitrile Minutes •4mL Water Chromatograms obtained after AFFINIMIP® SPE Loading Patulin clean-up of apple mango juice spiked at •4 to 6mL of loading solution 20µg/kg (blue) with Patulin or not spiked (red). In green, Patulin solution at 50ng/mL. prepared by Washing of interferents (W1) dilution of a 100µg/mL Patulin standard solution •2mL NaHCO3 1% in Water (REA-PAT-1mL) in mobile phase. •4mL Water CONCENTRATE JUICE Drying by applying vacuum 30 seconds Washing of interferents (W2) 160 160 •1mL Diethyl Ether 140 140 Elution (E) 120 120 •2mL Ethyl Acetate The elution fraction was then evaporated and 100 100 dissolved in water containing 0.1% acetic acid before HPLC analysis. 80 80 mAU mAU 60 60 40 40 20 20 00 HPLC Method -20 -20 Column: Atlantis T3 column, 150mm x 2.1mm 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Mobile phase: gradient profile Minutes Chromatograms obtained after AFFINIMIP® SPE Patulin Clean-up of grapefruit juice concentrate spiked at 10µg/kg (blue) with Patulin or not spiked (red). Time (min) % water % ACN 0 98 2 Catalog number: 20 98 2 6mL-200mg sorbent PI-FS102-02B-200mg for 25 cartridges 21 50 50 PI-FS102-03B-200mg for 50 cartridges PI-FS102-02KB-200mg for a kit of 25 cartridges 25 50 50 + 50mL Pectinase PI-FS102-03KB-200mg for a kit of 50 cartridges 26 98 2 + 50mL Pectinase PI-REA-001-50mL for 50mL Pectinase solution ) Flow rate: 0.2mL/min Detection: UV - 276nm Injection volume: 100µL. 19
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN APPLE PUREE A format tailored for the larger liquid volume required for apple puree protocol Regulations for apple puree: RESULTS Europe (EC 1881/2006) : 25µg/Kg Regulations for apple juice for infants and 800 800 young children: Europe (EC 1881/2006) : 10µg/Kg 700 700 PROTOCOL OF CLEANUP 600 600 Sample preparation 10g of apple puree, 150µL of a pectinase 500 500 enzyme solution and 10mL water are mixed. Leave solution at room temperature overnight 400 400 or for 2h at 40°C. Centrifuge at 4500g for 5min and then filter the solution with a 0.2µm filter. Patulin This solution is used as the loading solution. 300 300 Cleanup with a 6mL/200mg AFFINIMIP® SPE Patulin cartridge 200 200 100 100 4 5 6 7 8 9 10 11 12 13 14 15 Minutes Chromatograms of apple puree spiked with 20µg/kg of Patulin (Red) and not spiked (blue) after AFFINIMIP® SPE Patulin Clean-up mAU mAU Equilibration Recovery and repeatability of Patulin (n=6) at a •2mL Acetonitrile contamination level of 10µg/kg in apple puree after •1mL Water AFFINIMIP® SPE Patulin Clean-up. Loading Concentration of Recoveries % % RSDr •5mL of loading solution Patulin (µg/kg) Washing of interferents (W1) 10 (n=6) 90 9 •4mL Water -1%Acetic acid •1mL NaHCO3 1% solution 20 (n=3) 92 11 •3mL Water Catalog number: Drying by applying vacuum 10 seconds Washing of interferents (W2) 6mL - 200mg sorbent PI-FS102-02B-200mg for 25 cartridges •500µL Diethyl Ether PI-FS102-03B-200mg for 50 cartridges Elution (E) PI-FS102-02KB-200mg for a kit of 25 cartridges + 50mL Pectinase •2mL Ethyl Acetate PI-FS102-03KB-200mg for a kit of 50 cartridges The elution fraction was then evaporated and + 50mL Pectinase dissolved in water containing 0.1% acetic acid PI-REA-001-50mL for 50mL Pectinase solution before HPLC analysis. HPLC Method Column: Atlantis T3 column, 150mm x 2.1mm Mobile phase: gradient Time (min) % water % ACN 0 98 2 20 98 2 21 50 50 25 50 50 26 98 2 Flow rate: 0.2mL/min Detection: UV - 276nm Injection volume: 100µL. 20
AFFINIMIP® SPE Patulin DETERMINATION OF PATULIN IN DRIED APPLE Regulations for solid apple products: RESULTS Europe (EC 1881/2006) : 25µg/Kg UV6000-276nm UV6000-276nm UV6000-276nm PROTOCOL OF CLEANUP Sample preparation B16 B17 B13 80 3g of dried apple dices, 30mL of water and 80 150µL of pectinase are mixed and left at room temperature overnight. Then, they are 70 70 centrifuged at 4500rpm during 5min and filtered with 0.2µm filter to obtain the loading 60 60 solution. 50 Patulin 50 Cleanup with a 6mL/200mg AFFINIMIP® SPE Patulin cartridge 40 Recovery >90% 40 Equilibration 30 30 •4mL Acetonitrile •2mL Water 20 20 Loading mAU •10mL of loading solution mAU Washing of interferents (W1) 10 10 •5mL Water-2% Acetic Acid •5mL Water 00 Drying by applying vacuum 30 seconds -10 -10 Washing of interferents (W2) -20 -20 •500µL Diethyl Ether Elution (E) -30 -30 •2mL Ethyl Acetate -40 -40 The elution fraction was then evaporated and dissolved in water containing 0.1% acetic acid -50 -50 before HPLC analysis. 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Minutes Chromatograms obtained after AFFINIMIP® SPE Patulin Clean-up of dried apple dices spiked at 20µg/kg (red) or at 10µg/kg (blue) with Patulin or not spiked (green). HPLC Method Catalog number: Column: Atlantis T3 column, 150mm x 2.1mm 6mL - 200mg sorbent for apple-based Mobile phase: gradient profile products and fruit juice, concentrate PI-FS102-02B-200mg for 25 cartridges Time (min) % water % ACN PI-FS102-03B-200mg for 50 cartridges 0 98 2 PI-FS102-02KB-200mg for a kit of 25 cartridges 20 98 2 + 50mL Pectinase 21 50 50 PI-FS102-03KB-200mg for a kit of 50 cartridges 25 50 50 + 50mL Pectinase 26 98 2 PI-REA-001-50mL for 50mL Pectinase solution Flow rate: 0.2mL/min Detection: UV - 276nm Injection volume: 100µL. 21
AFFINIMIP® SPE Ochratoxin A DETERMINATION OF OCHRATOXIN A IN CEREALS Regulations for umprocessed cereals: Intensity RESULTS Europe (EC 1881/2006) : 5µg/Kg Codex Alimentarius Standard: 5µg/Kg for 6000 raw wheat 4000 PROTOCOL OF CLEANUP Sample preparation Ochratoxin A 50g of finely ground wheat are mixed during 1 minute in a blender with 100mL of extraction 2000 solvent (60/40 Acetonitrile/deionized Water). The extract is filtered through a filter paper. 0 Then, 5mL of the extract is diluted with 5mL of 0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 HCl solution pH=1, 0.1M. After a filtration through a filter paper, this solution is used as Chromatogram obtained after Cleanup of wheat the loading solution. (spiked at 5µg / kg (pink) or not contaminated (orange)) with AFFINIMIP® SPE Ochratoxin A Cleanup with a 3mL/100mg AFFINIMIP® SPE Ochratoxin A cartridge Recoveries of Ochratoxin A after AFFINIMIP® SPE Ochratoxin A Clean-up in wheat (n=6) Equilibration •3mL Acetonitrile C° (µg/kg) Recoveries % % RSD •3mL Water 5 96.3 7.7 Loading •4mL of loading solution (eq. 1g wheat) Washing of interferents •6mL 60/40 HCl solution pH 1, 0.1M/ACN Elution (E) •2mL Methanol – 2% Acetic acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. HPLC Method with Fluorescence detection Catalog number: Column: Hypersil Gold C18 column 150mm x 2.1mm PI-FS101-02 for 25 cartridges Mobile phase: water/acetic acid/MeOH (39/1/60, PI-FS101-03 for 50 cartridges v/v) Flow rate: 0.2mL/min Fluorescence detection: excitation/emission wavelengths: 333 / 460nm Injection volume: 20µL. 22
AFFINIMIP® SPE Ochratoxin A DETERMINATION OF OCHRATOXIN A IN PAPRIKA Regulations for paprika: RESULTS Europe (EC 594/2012) : 30µg/Kg until 31.12.14 then 15µg/Kg 20000 PROTOCOL OF CLEANUP Intensity Ochratoxin A Sample preparation 10g of paprika are shaken during 30 minutes 0 with 100mL of NaHCO3 1% in water. The 0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 extract is centrifuged for 30 minutes at 4000 rpm at room temperature then filtered through Chromatogram obtained after Cleanup of paprika a filter paper. (spiked at 30µg / kg (pink) or not contaminated 25mL of the extract is diluted with 25mL of HCl (orange)) with AFFINIMIP® SPE Ochratoxin A solution pH=1, 0.1M. After a filtration through a filter paper, this solution is used as the Recoveries of Ochratoxin A after AFFINIMIP® SPE loading solution. Ochratoxin A Clean-up in paprika (n=4). Cleanup with a 3mL/100mg AFFINIMIP® SPE C° (µg/kg) Recoveries % % RSD Ochratoxin A cartridge 30 93.3 3.4 Equilibration •3mL Acetonitrile •3mL Water Loading •4mL of loading solution (eq. 1g sample) Washing of interferents •6mL 60/40 HCl solution pH 1, 0.1M/ACN Elution (E) •2mL Methanol – 2% Acetic acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. HPLC Method with Fluorescence detection Catalog number: Column: Hypersil Gold C18 column 150mm x 2.1mm PI-FS101-02 for 25 cartridges Mobile phase: water/acetic acid/MeOH (39/1/60, PI-FS101-03 for 50 cartridges v/v) Flow rate: 0.2mL/min Fluorescence detection: excitation/emission wavelengths: 333 / 460nm Injection volume: 20µL. 23
AFFINIMIP® SPE Ochratoxin A DETERMINATION OF OCHRATOXIN A IN RED AND WHITE WINE Regulations for wine: RESULTS Europe (EC 1881/2006) : 2µg/L 10000 5000Intensity PROTOCOL OF CLEANUP Ochratoxin A Sample preparation Ochratoxin A 10mL of wine is diluted with 10mL of HCl solution pH=1, 0.1M. This solution is used as 0 the loading solution. 0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 Cleanup with a 3mL/100mg AFFINIMIP® SPE Chromatograms obtained after Cleanup of white Ochratoxin A cartridge wine spiked at 2µg/kg (loading with 5mL (blue); loading with 10mL (pink)) and after a loading of 5mL Equilibration of not contaminated white wine (orange) with •3mL Acetonitrile AFFINIMIP® SPE Ochratoxin A •3mL Water 6000Intensity 60000 Loading 4000 Intensity40000 •2 to 10mL of loading solution (eq. 1 to 20000 5mL sample) 0 0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 Washing of interferents •6mL 60/40 HCl solution pH 1, 0.1M/ACN 2000 Ochratoxin A Elution (E) 0 •2mL Methanol – 2% Acetic acid 0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 The elution fraction was then evaporated and dissolved in water before HPLC analysis. Chromatograms obtained after Cleanup of red wine spiked at 2µg / kg (loading with 2mL (orange); HPLC Method with Fluorescence detection loading with 5mL (blue); loading with 10mL (pink)) Column: Hypersil Gold C18 column 150mm x 2.1mm and after a loading of 2mL of not contaminated red Mobile phase: water/acetic acid/MeOH (39/1/60, wine (grey) with AFFINIMIP® SPE Ochratoxin A v/v) Flow rate: 0.2mL/min Recoveries of Ochratoxin A after AFFINIMIP® SPE Fluorescence detection: excitation/emission Ochratoxin A Clean-up in wine (white and red). wavelengths: 333 / 460nm Injection volume: 20µL. Matrix C° Recoveries % (µg/kg % RSD ) White wine 2 91.3 6.2 (n=10) Red wine (n=4) 2 78.8 2.8 Catalog number: PI-FS101-02 for 25 cartridges PI-FS101-03 for 50 cartridges 24
AFFINIMIP® SPE Zearalenone DETERMINATION OF ZEARALENONE IN MAIZE AND RICE Regulations for unprocessed cereal except RESULTS maize: Europe (EC 1126/2007) : 100µg/Kg Regulations for maize: Europe (EC 1126/2007) : 350µg/Kg PROTOCOL OF CLEANUP ZON Sample preparation Chromatogram obtained after Cleanup of Maize Cleanup with a 3mL/100mg AFFINIMIP® SPE (contamined at 41 µg / kg) with AFFINIMIP® SPE Zearalenone cartridge Zearalenone 25g of ground cereal-based samples were extracted with 100 mL of ZON acetonitrile/deionized water (75/25, v/v) for 3 min. The extract was filtered through a folded Chromatogram obtained after Cleanup of Rice filter paper and 10 mL of the filtrate were (contamined at 41 µg / kg) with AFFINIMIP® SPE diluted with 10 mL of deionized water. Then, Zearalenone . this solution was filtered through a filter paper. This solution was used as the loading solution. Recoveries of Zearalenone at a contamination level of 41µg / kg after AFFINIMIP® SPE Zearalenone . Equilibration Clean-up in Maize (n=9) •3mL Acetonitrile •3mL Water Loading •12mL of loading solution (eq. 1.5g sample) Washing of interferents (W1) •3mL 58/2/40 Water/Acetic Acid/ACN Elution (E) •2mL Methanol – 2% Acetic Acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. Recoveries % % RSD 86 8 HPLC Method with Fluorescence detection Catalog number: Column: Hypersil Gold C18 column 150mm x 4.6mm 3mL-100mg sorbent Mobile phase: water/MeOH (40/60, v/v) PI-FS100-02 for 25 cartridges Flow rate: 1mL/min PI-FS100-03 for 50 cartridges Fluorescence detection: excitation/emission wavelengths: 275 / 450nm Injection volume: 100µL. 25
AFFINIMIP® SPE Zearalenone DETERMINATION OF ZEARALENONE IN CEREAL-BASED BABY FOOD Regulations for processed cereal based RESULTS food for baby food: Europe (EC 1126/2007) : 20µg/Kg PROTOCOL OF CLEANUP ZON Sample preparation Chromatogram obtained after Cleanup of Cleanup with a 3mL/100mg AFFINIMIP® SPE Cereal-based babyfood (contamined at 41µg / Zearalenone cartridge kg) AFFINIMIP® SPE Zearalenone (after dilution 25g of ground cereal-based samples were by 2 of the elution fraction with water). extracted with 100 mL of acetonitrile/deionized water (75/25, v/v) for 3 ZON min. The extract was filtered through a folded filter paper and 10 mL of the filtrate were Chromatograms obtained after Cleanup of Cereal- diluted with 10 mL of deionized water. Then, based babyfood (contamined at 10µg/kg (blue) or this solution was filtered through a filter paper. 0µg/kg (red)) with AFFINIMIP® SPE Zearalenone This solution was used as the loading solution. (after evaporation of the elution fraction and dissolution in 1mL of the mobile phase). Equilibration •3mL Acetonitrile •3mL Water Loading •12mL of loading solution (eq. 1.5g sample) Washing of interferents (W1) •3mL 58/2/40 Water/Acetic Acid/ACN Elution (E) •2mL Methanol – 2% Acetic Acid The elution fraction was then evaporated and dissolved in water before HPLC analysis. 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 80 3 HPLC Method with Fluorescence detection Catalog number: Column: Hypersil Gold C18 column 150mm x 4.6mm 3mL-100mg sorbent Mobile phase: water/MeOH (40/60, v/v) PI-FS100-02 for 25 cartridges Flow rate: 1mL/min PI-FS100-03 for 50 cartridges Fluorescence detection: excitation/emission wavelengths: 275 / 450nm Injection volume: 100µL. 26
AFFINIMIP® SPE Zearalenone DETERMINATION OF ZEARALENONE IN EDIBLE CORN OIL Regulations for processed cereal based RESULTS food for baby food: Europe (EC 1126/2007) : 20µg/Kg 4 5 PROTOCOL OF CLEANUP Sample preparation 4 0 Cleanup with a 3mL/100mg AFFINIMIP® SPE Zearalenone cartridge 3 Zearalenone Corn oil is diluted 1/3 in Diethyl Ether to obtain 5 the loading solution. 3 Equilibration 0 •3mL Diethyl Ether mVolt 2 Loading s 5 •3mL of loading solution (eq. 1mL of corn oil) 2 0 Washing of interferents (W1) •6mL Diethyl ether 1 5 Drying 30 seconds Washing of interferents (W2) 1 0 •6mL 58/2/40 Water/Acetic Acid/ACN Elution (E) 5 •4mL Methanol – 2% Acetic Acid 0 The elution fraction was then evaporated and dissolved in water before HPLC analysis. 0 2 4 6 81 1 1 1 1 2 2 2 2 2 3 0 2 4 Minutes 6 8 0 2 4 6 8 0 Chromatograms of Corn Oil spiked with Zearalenone at 400µg/L (blue) or not spiked (orange) obtained after cleanup by AFFINIMIP®SPE Zearalenone. Zearalenone Chromatograms obtained after cleanup by AFFINIMIP®SPE Zearalenone of Corn Oil spiked with Zearalenone at 200µg/L (red), 400µg/L (green), 600 µg/L (blue) or not spiked (purple). Recoveries of Zearalenone in Corn Oil at various contamination levels after AFFINIMIP®SPE Zearalenone cleanup. HPLC Method with Fluorescence detection C° (µg/L) Mean C° (µg/L) Recoveries % Column: Hypersil Gold C18 column 150mm x 4.6mm 200 230 115 Mobile phase: water/MeOH (40/60, v/v) 400 440 110 Flow rate: 1mL/min 600 678 113 Fluorescence detection: excitation/emission wavelengths: 275 / 450nm Catalog number: Injection volume: 100µL. 3mL-100mg sorbent PI-FS100-02 for 25 cartridges PI-FS100-03 for 50 cartridges 27
AFFINIMIP® SPE Deoxynivalenol DETERMINATION OF DEOXYNIVALENOL IN CEREALS FOR FOOD (Water extraction) Regulations for unprocessed corn or durum RESULTS wheat for food: Europe (EC 1126/2007) : 1750µg/Kg Deoxynivalenol (DON) PROTOCOL OF CLEANUP UV chromatograms obtained after WATER Sample preparation with EXTRACTION WITH WATER extraction of DON from corn and clean-up with 20g of cereals were ground in a blender for 1 minute. Then, 80 ml of deionized water were AFFINIMIP®SPE Deoxynivalenol : added. This mixture was then ground for 2 • black, red and green spiked with DON at additional minutes. After grinding the mixture was placed in a beaker and left stirred under 800µg/kg magnetic agitation for 30 minutes. • dark yellow not spiked Then the whole mixture was transferred in a • blue, a standard solution of DON at 200ng/mL is centrifuge vial and centrifuged at 2500 rpm for 15 minutes. After centrifugation the prepared by dilution of a 100µg/mL supernatant was filtered through filter paper. This solution was then diluted 5 times using Deoxynivalenol standard solution (reference : deionized water. REA-DON-1mL) in mobile phase Cleanup with a 6mL/100mg AFFINIMIP® SPE Deoxynivalenol cartridge Recovery of Deoxynivalenol after AFFINIMIP®SPE . Deoxynivalenol clean-up and relative standard deviation (repeatability conditions). Equilibration •2mL Acetonitrile Matrix Detec Mean R% %RSDr •2mL Water tion µg/kg Loading Corn UV 623.4 78.0 1.4 •6mL of loading solution (800µg/kg) (n=6) Washing of interferents (W1) Corn MS 642.7 80.3 3.4 •3mL NaHCO3 1% in water (800µg/kg) (n=6) Drying 30 seconds Wheat (n=3) MS 540.0 90.0 9.8 Washing of interferents (W2) (600µg/kg) (n=3) •1mL Diethylether Recovery of Deoxynivalenol after AFFINIMIP®SPE Elution (E) Deoxynivalenol clean-up and relative standard deviation (reproducibility conditions). •4mL Ethyl Acetate The elution fraction was then evaporated and Matrix Detect Mean R% %RSDR dissolved in water -0,1% HCOOH before HPLC ion µg/kg analysis. Corn (n=6) UV 658.7 82.3 7.5 HPLC Method with MS or UV detection Column: Hypersil Gold C18 column 50mm x 2,1mm Corn (n=6) MS 659.5 82.4 7.8 Mobile phase: water with 0,1% formic acid/ACN (95/5, v/v) Catalog number: Flow rate: 0,2mL/min 6mL-100mg sorbent for (baby)food MS detection: m/z 265 (ESI-) PI-FS117-02B for 25 cartridges UV detection: 220nm PI-FS117-03B for 50 cartridges Injection volume: 20µL. 6mL-200mg sorbent for feed PI-FS117-02B-200mg for 25 cartridges PI-FS117-03B-200mg for 50 cartridges 28
AFFINIMIP® SPE Deoxynivalenol DETERMINATION OF DEOXYNIVALENOL IN BABYFOOD CEREALS Regulations for cereal based food for baby RESULTS food: Europe (EC 1126/2007) : 200µg/Kg Deoxynivalenol (DON) PROTOCOL OF CLEANUP MS chromatograms obtained after water extraction Sample preparation 150 ml of deionized water were added to 20g of Deoxynivalenol from cereals - based babyfoods of cereals - based babyfood. This mixture was and clean-up with AFFINIMIP® SPE Deoxynivalenol: then placed in a beaker and left stirring under • black, red and green spiked with Deoxynivalenol magnetic agitation for 30 minutes. Then, the whole mixture was centrifuged at at 150µg/kg 2500 g for 15 minutes. After centrifugation, the • dark yellow not spiked supernatant was filtered through filter paper. • blue, a standard solution of Deoxynivalenol at Cleanup with a 6mL/100mg AFFINIMIP® SPE 200ng/mL is prepared by dilution of a 100µg/mL Deoxynivalenol cartridge . Deoxynivalenol standard solution (reference : Equilibration REA-DON-1mL) in mobile phase •2mL Acetonitrile •2mL Water Recovery of Deoxynivalenol after AFFINIMIP®SPE Deoxynivalenol clean-up and relative standard Loading deviation calculated from results generated under •6mL of loading solution repeatability conditions. Washing of interferents (W1) Matrix C° Mean R% %RSDr •3mL NaHCO3 1% in water µg/kg µg/kg Drying 30 seconds Babyfood 150 136.5 91 0.4 Washing of interferents (W2) (n=3) •1mL Diethylether Recovery of Deoxynivalenol after AFFINIMIP®SPE Elution (E) Deoxynivalenol clean-up and relative standard deviation calculated from results generated under •4mL Ethyl Acetate reproducibility conditions. The elution fraction was then evaporated and dissolved in water -0,1% HCOOH before HPLC Matrix C° Mean R% %RSDR analysis. µg/kg µg/kg 91 0.4 Babyfood HPLC Method with MS detection (n=3) 150 136.5 Column: Hypersil Gold C18 column 50mm x 2,1mm Mobile phase: water with 0,1% formic acid/ACN Catalog number: (95/5, v/v) 6mL-100mg sorbent for (baby)food Flow rate: 0,2mL/min PI-FS117-02B for 25 cartridges MS detection: m/z 265 (ESI-) PI-FS117-03B for 50 cartridges Injection volume: 20µL. 6mL-200mg sorbent for feed PI-FS117-02B-200mg for 25 cartridges PI-FS117-03B-200mg for 50 cartridges 29
AFFINIMIP® SPE Deoxynivalenol DETERMINATION OF DEOXYNIVALENOL, 3-AcetylDON AND 15-AcetylDON IN CEREALS (Hydro-organic extraction) Regulations for unprocessed corn or durum RESULTS wheat for food: Europe (EC 1126/2007) : 1750µg/Kg 3-AcetylDeoxynivalenol PROTOCOL OF CLEANUP 200 (3-AcDON) Sample preparation WITH HYDROORGANIC EXTRACTION 180 20g of cereals were ground in a blender for 1 minute. Then, a solution of deionized water: 160 acetonitrile (50:50) was added. This mixture was then ground for 2 additional minutes. After 140 grinding, the mixture was placed in a beaker and left stirred under magnetic agitation for 30 120 minutes. Then the mixture was centrifuged at 2500 g for 100 15 minutes. After centrifugation, the supernatant was filtered through filter paper. 80 This solution was then diluted 10 times using deionized water. 60 Cleanup with a 6mL/100mg AFFINIMIP® SPE 40 Deoxynivalenol cartridge . 20 Equilibration 0 5 10 15 20 25 30 •2mL Acetonitrile 0 Time (min) •2mL Water MS chromatograms obtained after hydro-organic Loading •6mL of loading solution extraction of 3-acetylDON from corn and clean-up Washing of interferents (W1) with AFFINIMIP®SPE Deoxynivalenol : •3mL NaHCO3 1% in water -black, red and green: spiked with Deoxynivalenol Drying 30 seconds Washing of interferents (W2) at 800µg/kg •1mL Diethylether -dark yellow: not spiked Elution (E) -blue: a standard solution of 3-AcetylDON at •4mL Ethyl Acetate The elution fraction was then evaporated and 200ng/mL is prepared by dilution of a 100µg/mL 3- dissolved in water-0.1% formic acid before HPLC analysis. AcetylDeoxynivalenol standard solution (reference HPLC Method with MS detection : REA-3AcDON-1mL) in mobile phase Column: Hypersil Gold C18 column 50mm x 2,1mm Mobile phase for DON analyses: water with 0,1% Recovery obtained for DON, 3-acetylDON and 15- formic acid/ACN (95/5, v/v) acetylDON after AFFINIMIP®SPE Deoxynivalenol Mobile phase for 3-AcDON and 15-AcDON analyses: clean-up of Corn and relative standard deviation - water with 0.1% formic acid/ACN (90/10, v/v) repeatability conditions (n=3). Flow rate: 0.2mL/min MS detection: m/z 265 (ESI-) Compound C° µg/kg Mean R% %RSDr Injection volume: 20µL. µg/kg DON 800 653.7 81.7 0.3 3-AcetylDON 800 601.0 75.1 2.3 15- 800 641.8 80.2 3.4 AcetylDON Catalog number: 6mL-100mg sorbent for (baby)food PI-FS117-02B for 25 cartridges PI-FS117-03B for 50 cartridges 6mL-200mg sorbent for feed PI-FS117-02B-200mg for 25 cartridges PI-FS117-03B-200mg for 50 cartridges 30
AFFINIMIP® SPE Deoxynivalenol DETERMINATION OF DEOXYNIVALENOL IN CEREALS FOR ANIMAL FEED Regulations for DON in animal feed: RESULTS Europe (EC 576/2006) : 8mg/Kg for cereals and cereals products Deoxynivalenol (DON) 12mg/Kg for maize by-products PROTOCOL OF CLEANUP UV chromatograms obtained after WATER Sample preparation with EXTRACTION WITH WATER extraction of DON from wheat (animal feed) and 20g of animal feed were ground in a blender for 1 minute. Then, 80 ml of deionized water clean-up with AFFINIMIP®SPE Deoxynivalenol : were added. This mixture was then ground for • black, red and green spiked with DON at 6mg/kg 2 additional minutes. After grinding the • dark yellow not spiked mixture was placed in a beaker and left stirred • blue, a standard solution of DON at 1µg/mL is under magnetic agitation for 30 minutes. Then, the whole mixture was centrifuged at prepared by dilution of a 100µg/mL 2500 g for 15 minutes. After centrifugation the supernatant was filtered through filter paper. Deoxynivalenol standard solution (reference : This solution was then diluted 5 times using deionized water. REA-DON-1mL) in mobile phase Cleanup with a 6mL/200mg AFFINIMIP® SPE abc Deoxynivalenol cartridge . Analysis of Whiskas: a. Extraction solution with water Equilibration b. Loading solution •2mL Acetonitrile c. Elution solution •2mL Water Recovery of Deoxynivalenol after AFFINIMIP®SPE Loading Deoxynivalenol clean-up and relative standard •2mL of loading solution deviation - repeatability conditions (n=3). Washing of interferents (W1) Feed C° Mean R% %RSDr •3mL NaHCO3 1% in water Matrices mg/kg mg/kg Drying 30 seconds Washing of interferents (W2) Wheat 6 5.7 94 0.1 •1mL Diethylether Whiskas 0.8 0.73 91 2.4 Elution (E) Catalog number: •4mL Ethyl Acetate 6mL-100mg sorbent for (baby)food The elution fraction was then evaporated and PI-FS117-02B for 25 cartridges dissolved in water -0,1% HCOOH before HPLC PI-FS117-03B for 50 cartridges analysis. 6mL-200mg sorbent for feed PI-FS117-02B-200mg for 25 cartridges HPLC Method with UV detection PI-FS117-03B-200mg for 50 cartridges Column: Hypersil Gold C18 column 50mm x 2,1mm Mobile phase: water with 0,1% formic acid/ACN (95/5, v/v) Flow rate: 0,2mL/min UV detection: 220nm Injection volume: 20µL. 31
AFFINIMIP® SPE FumoZON DETERMINATION OF FUMONISINS B1 / B2 AND ZEARALENONE IN MAIZE FLOUR Regulations for cereal flour: RT: 0.08 - 20.00Intensity RESULTS Zearalenone 220000 Europe (EC 1126/2007) : 75µg/Kg 210000 Fumonisin B2 Fumonisins 200000 Europe (EC 1126/2007) : 1000µg/Kg for 190000 3.52 maize flour 180000 USA: FDA advisory 2000µg/Kg 170000 2 46 8 10 12 14 16 18 20 160000 6.19 Time (min) PROTOCOL OF CLEANUP 150000 Sample preparation 140000Intensity Fumonisin B1 130000 Cleanup with a 3mL/100mg AFFINIMIP® SPE 120000Intensity Zearalenone FumoZON cartridge 110000 25g of ground samples were extracted with 100000 26.95 100 mL of Acetonitrile/Methanol/deionized 90000 Water (25/25/50, v/v/v) for 3 min using a 80000 5 10 15 20 25 30 blender. The extract was filtered through a 70000 Time (min) folded filter paper and 10 mL of the filtrate 60000 were diluted with 10 mL of deionized water. 50000 Then, this solution was filtered through a filter 40000 paper. 30000 This solution was used as the loading solution. 20000 10000 Equilibration 0 •2mL Acetonitrile •2mL Water RT: 0.08 - 30.00 400000 Loading •6mL of loading solution 350000 Washing of interferents 300000 •6mL 60/40 Water/ACN 250000 Elution (E) •2mL Methanol – 2% Acetic Acid 200000 The elution fraction was then evaporated and 150000 dissolved in water before HPLC analysis. 100000 HPLC Method with MS detection Column: Hypersil Gold C18 column 50mm x 2.1mm 50000 Mobile phase ZON AND FB1: Water-Formic Acid 0.1%/ACN (73/27) 0 Mobile phase FB2: Water-Formic Acid 0.1%/ACN 12000 (65/35) 11000 Flow rate: 0.2mL/min 10000 MS detection: m/z 722 for Fumonisin B1 (ESI+) m/z 706 for Fumonisin B2 (ESI+) 9000 m/z 317 for Zearalenone (ESI-) 8000 Injection volume: 20µL. 7000 6000 5000 4000 3000 2000 1000 0 Chromatograms obtained after AFFINIMIP® SPE FumoZON Clean-up of a maize flour spiked at 38µg/kg with Zearalenone, 2408µk/kg with Fumonisin B1 and 630µg/kg with Fumonisin B2. Recovery of Zearalenone, Fumonisins B1 and B2 in maize flour after AFFINIMIP® SPE FumoZON clean- up and relative standard deviation calculated from results generated under reproducibility conditions Sample C° Mean Recoveries % µg/kg µg/kg % RSDR Zearalenon e 38 39.2 103.2 8.5 (n=8) Fumonisin 2408 2002.2 83.1 10.3 B1 (n=8) Fumonisin 400 401.0 100.2 - B1 (n=2) Fumonisin 630 684.6 108.7 11.5 B2 (n=3) Catalog number: 3mL-100mg sorbent PI-FS109-02 for 25 cartridges PI-FS109-03 for 50 cartridges 32
AFFINIMIP® SPE FumoZON DETERMINATION OF FUMONISINS B1 / B2 AND ZEARALENONE IN MAIZE-BASED BABY FOOD Regulations for maize-based baby food: RESULTS Zearalenone Europe (EC 1126/2007) : 20µg/Kg Recovery of Zearalenone, Fumonisins B1 and B2 in Fumonisins maize-based baby food after AFFINIMIP® SPE Europe (EC 1126/2007) : 200µg/Kg FumoZON clean-up and relative standard deviation calculated from results generated under PROTOCOL OF CLEANUP reproducibility conditions. Sample preparation Sample C° Mean Recoveries % RSDR Cleanup with a 3mL/100mg AFFINIMIP® SPE µg/kg µg/kg % FumoZON cartridge 25g of ground samples were extracted with Zearalenone 20 16.9 84.4 1.6 100 mL of Acetonitrile/Methanol/deionized (n=4) Water (25/25/50, v/v/v) for 3 min using a blender. The extract was filtered through a Fumonisin B1 200 168.6 84.3 1.4 folded filter paper and 10 mL of the filtrate (n=3) were diluted with 10 mL of deionized water. Then, this solution was filtered through a filter Fumonisin B2 200 185.6 92.8 1.9 paper. (n=3) This solution was used as the loading solution. ION SUPPRESSION EVALUATION Equilibration •2mL Acetonitrile Ion suppression phenomenon can induce an •2mL Water erroneous quantification. To evaluate the ion- suppression, blank maize-based baby food Loading samples were cleaned up with AFFINIMIP® SPE •6mL of loading solution FumoZON . The SPE extracts were spiked with a mixture of Fumonisin B1 and Zearalenone at Washing of interferents 2 different concentrations. The standard •6mL 60/40 Water/ACN calibration curves were compared to the matrix SPE extracts. The use of AFFINIMIP® SPE Elution (E) FumoZON strongly reduces ion-suppression •2mL Methanol – 2% Acetic Acid phenomena with a maximum of 15% observed for Fumonisins. The elution fraction was then evaporated and dissolved in water before HPLC analysis. Ion suppression percentage obtained in Maize- based baby food (tested twice). HPLC Method with MS detection Column: Hypersil Gold C18 column 50mm x 2.1mm Analyte C° Ion Mobile phase ZON AND FB1: Water-Formic Acid µg/kg suppression 0.1%/ACN (73/27) Mobile phase FB2: Water-Formic Acid 0.1%/ACN % (65/35) Flow rate: 0.2mL/min Zearalenone 10 1% and 5% MS detection: m/z 722 for Fumonisin B1 (ESI+) m/z 706 for Fumonisin B2 (ESI+) Zearalenone 50 0% and 5% m/z 317 for Zearalenone (ESI-) Injection volume: 20µL. Fumonisin B1 100 8% and 11% Fumonisin B1 500 12% and 14% Catalog number: 3mL-100mg sorbent PI-FS109-02 for 25 cartridges PI-FS109-03 for 50 cartridges 33
PH100 AFFINIMIP® SPE ESTROGENS Estrogens are a group of compounds which play an important 17β- Estradiol OH role in the estrous cycle. They are either natural (Estrone, Estriol, 17α- and 17β-Estradiol) or synthetic compounds (17α- HO H EthinylEstradiol, Dienestrol, Diethylstilbestrol). HH 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. 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 Catalog number: PI-100-02 for 25 cartridges, 3mL PI-100-03 for 50 cartridges, 3mL PI-100-1.96W for 1 96-well plate
AFFINIMIP® SPE Estrogens DETERMINATION OF ESTROGENS IN PLASMA Regulations for Estrogens: RESULTS Europe (EC directive) : 40pg/mL of plasma or serum of bovine animals Blank plasma 10ppt 40ppt 100ppt 17β-Estradiol-d3 419>285 17 α/β-Estradiol 416>129 17 α/β-Estradiol 416>285 PROTOCOL OF CLEANUP MRM chromatograms from GC-MS/MS analysis of Sample preparation fortified calves’ plasma samples at 0, 10, 40 and 100 2mL serum samples spiked with 40pg 17β- pg.mL-1 with 17α-estradiol, 17β-estradiol and estrone. Estradiol-d3. Then 2mL of Acetate buffer Chromatograms obtained after a clean-up with (0.8M, pH 6.8) and 100µL β-glucuronidase AFFINIMIP® SPE Estrogens (Courtesy of Emmanuelle were added. Hydrolysis performed overnight at Bichon - LABERCA) 37°C and samples centrifuged at 4000 rpm for 10min. Upper layer was used as loading Data extracted from Quantification of estrogens at solution. ppt levels in bovine plasma by Molecularly Cleanup with a 3mL/100mg AFFINIMIP® SPE Imprinted Solid Phase Extraction and GC-MS/MS Estrogens cartridge analysis, Emmanuelle Bichon et al. (LABERCA) Poster session, HTSP-2 and HTC 2012 Equilibration •3mL Methanol Catalog number: •3mL Acetonitrile PI-FS104-02 for 25 cartridges, 3mL •3mL Water PI-FS104-03 for 50 cartridges, 3mL Loading solution from sample preparation Washing of interferents •3mL Water •3mL Water/Acetonitrile (60/40) Elution (E) 3mL Methanol The elution fraction was then evaporated and estrogens were derivatised 40min at 60°C with BSTFA before GC-MS/MS analysis. GC-MS/MS Analysis Column: RTX-1614 Resteck 15m x 0.25mm x 0.10µm Gradient temperature: 80 to 320°C (15°C/min) 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 AFFINIMIP® SPE Estrogens 3cc Copolymeric SPE 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 2012 Catalog number: PI-FS104-02 for 25 cartridges, 3mL PI-FS104-03 for 50 cartridges, 3mL 36
FS106 AFFINIMIP® SPE Bisphenol A Bisphenol A (or BPA) is a molecule widely used in industry HO CH3 OH for the synthesis of polycarbonate plastics and epoxy CH3 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. 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 2011/8/EU of 28 January 2011). Progressively, countries become more and more restrictive on BPA use for food packaging. So, in October 2011, French parliament voted a law for banning BPA from canned foods and plastic boxes applicable in 2013 for infants and for all consumers on 1st January 2014. In the same way, Sweden bans BPA in food packaging for under-threes (2012) and in Denmark since July 2010, 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. Catalog number: 3mL-100mg sorbent in a PP cartridge PI-FS106-02 for 25 cartridges PI-FS106-03 for 50 cartridges 6mL-100mg sorbent in a PP cartridge PI-FS106-02B for 25 cartridges PI-FS106-03B for 50 cartridges 6mL-100mg sorbent in a glass cartridge PI-FS106-02G for 25 cartridges PI-FS106-03G for 50 cartridges 37
AFFINIMIP® SPE Bisphenol A DETERMINATION OF BISPHENOL A IN LIQUID INFANT FORMULA Regulations for Bisphenol A: RESULTS Europe (directive 2011/8/EU) : forbiden in infant feeding bottles mVolts mVolts 1100 PROTOCOL OF CLEANUP 1000 Sample preparation 900 Injection 50µL of Infant Cleanup with a 3mLor 6mL/100mg AFFINIMIP® SPE Bisphenol A cartridge 800 Equilibration Formula before treatment •3mL Methanol -2% Acetic Acid •3mL Acetonitrile 700 •3mL Water 600 Loading Up to 15mL of infant formula Bisphenol A after treatment of Washing of interferents 500 •9mL Water •6mL Water/Acetonitrile (60/40) 400 15mL of Infant Formula Drying 30 seconds 300 Elution (E) 200 3mL Methanol The elution fraction was then evaporated and 100 dissolved in the mobile phase before HPLC analysis. 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Minutes Chromatograms of Infant Formula containing 1µg/L of Bisphenol A before clean-up (Red) and after clean-up (Blue) with AFFINIMIP® SPE Bisphenol A. 900 800 700 Bisphenol A 600 500 400 300 200 100 0 9,2 9,4 9,6 9,8 10,0 10,2 10,4 10,6 10,8 11,0 11,2 11,4 11,6 11,8 12,0 12,2 12,4 12,6 12,8 13,0 9,0 Minutes 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, % RSDr) % RSDr C° (µg/L) Mean (µg/L) Recoveries % 1.5 1.0 0.9 88.4 2.0 1.7 85.7 2.7 - under reproducibility conditions ( % RSDR). HPLC Method with Fluorescence detection C° (µg/L) Mean (µg/L) Recoveries % % RSDR Column: Hypersil Gold C18 column 150mm x 4.6mm 7.4 Mobile phase: gradient profile 1.0 0.8 84.4 2.0 1.7 85.8 5.3 Time (min) % water % ACN Catalog number: 0 65 35 2 65 35 3mL-100mg sorbent in a PP cartridge 12 50 50 PI-FS106-02 for 25 cartridges 20 50 50 PI-FS106-03 for 50 cartridges 65 35 6mL-100mg sorbent in a PP cartridge 20.5 65 35 PI-FS106-02B for 25 cartridges 35 PI-FS106-03B for 50 cartridges 6mL-100mg sorbent in a glass cartridge Flow rate: 1mL/min excitation/emission PI-FS106-02G for 25 cartridges Fluorescence detection: PI-FS106-03G for 50 cartridges wavelengths: 230 / 315nm Injection volume: 50µL. 38
AFFINIMIP® SPE Bisphenol A DETERMINATION OF BISPHENOL A IN POWDERED INFANT FORMULA Regulations for Bisphenol A: RESULTS Europe (directive 2011/8/EU) : forbiden in infant feeding bottles 1400 Fluo Fluo Fluo Fluo Fluo 1300 2-MD1.dat 2-md2.dat 4-md1.dat 4-md2.dat mnd.dat 1200 1100 Bisphenol A 1000 PROTOCOL OF CLEANUP mVolts Sample preparation 900 4.4g powdered infant milk was reconstituted in 800 30 mL of water and warmed up at ~ 50°C 700 during 20 seconds using microwaves. Then 20 600 mL of acetonitrile were added to 20 mL of 500 warm milk and centrifuged at 4000 rpm during 400 10 minutes. The supernatant was collected and 300 filtered on filter paper (4-7µm). This extract 200 was diluted 1:1 with water to form the loading 100 solution. Cleanup with a 3mL or 6mL/100mg 0 AFFINIMIP® SPE Bisphenol A cartridge -100 Equilibration 6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0 10,5 11,0 11,5 12,0 12,5 13,0 13,5 14,0 •3mL Methanol -2% Acetic Acid Minutes •3mL Acetonitrile •3mL Water Chromatograms obtained after clean-up with AFFINIMIP® SPE Bisphenol A of equivalent at 10mL Loading of Infant Formula spiked with Bisphenol A at Up to 40mL of infant formula 4.3µg/L (tested twice, red) or at 2.1µg/L (tested twice, blue) or not spiked (pink). Washing of interferents •9mL Water Recovery of Bisphenol A spiked at different •6mL Water/Acetonitrile (60/40) concentrations after 3mL/100mg AFFINIMIP® SPE Bisphenol A clean-up of 40mL of loading solution Drying 30 seconds (equivalent to 10mL of reconstituted Infant milk) Elution (E) and relative standard deviation calculated from results generated under repeatability conditions 3mL Methanol The elution fraction was then evaporated and Concentration of Mean Recoveries RSDr % dissolved in the mobile phase before HPLC BPA in concentration % analysis. 8.7 reconstituted (µg/L) 108 3.7 milk (µg/L) 95 2.1 2.3 (n=5) 4.3 4.0 (n=4) HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 150mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN Catalog number: 0 65 35 2 65 35 3mL-100mg sorbent in a PP cartridge 12 50 50 PI-FS106-02 for 25 cartridges 20 50 50 PI-FS106-03 for 50 cartridges 65 35 6mL-100mg sorbent in a PP cartridge 20.5 65 35 PI-FS106-02B for 25 cartridges 35 PI-FS106-03B for 50 cartridges 6mL-100mg sorbent in a glass cartridge Flow rate: 1mL/min excitation/emission PI-FS106-02G for 25 cartridges Fluorescence detection: PI-FS106-03G for 50 cartridges wavelengths: 230 / 315nm Injection volume: 50µL. 39
PROTOCOL COMPARISON – AFFINIMIP® SPE Bisphenol FAS106 AFFINIMIP® SPE Bisphenol A vs competitor POWDERED INFANT FORMULA ANALYSIS AFFINISEP method WATERS method* Precipitation (15g sample + 15mL ACN) Precipitation (10g sample + 10mL ACN) Centrifuge and collect supernatant Centrifuge and collect supernatant Dilute 20mL supernatant with 20mL H20 Quecher 1 Load on AFFINIMIP® SPE Bisphenol A Add contents from DisQue tube 1. 3cc (40mL) Shake. Centrifuge and collect 10mL Wash 10mL H20 and 6mL 40% ACN ; Elute 3mL 100% MeOH Quecher 2 Add contents from DisQue tube 2. Shake. Centrifuge and collect supernatant Dilute supernatant with 70mL H20 Load on SPE cartridge OASIS HLB 3cc (70mL) Wash 2mL 40% Methanol ; Elute 1mL 100% MeOH HPLC analysis : HPLC analysis : LC-MS/MS LC Fluorescence detector or GC-MS/MS or LC-MS/MS •Extract from Waters application note, published 2012 : Rapid analysis of Bisphenol A Performance. Save your time. Catalog number: 3mL-100mg sorbent in a PP cartridge PI-FS106-02 for 25 cartridges PI-FS106-03 for 50 cartridges 6mL-100mg sorbent in a PP cartridge PI-FS106-02B for 25 cartridges PI-FS106-03B for 50 cartridges 6mL-100mg sorbent in a glass cartridge PI-FS106-02G for 25 cartridges PI-FS106-03G for 50 cartridges 40
AFFINIMIP® SPE Bisphenol A DETERMINATION OF BISPHENOL A IN CANNED FOOD (Liquid form) Regulations for Bisphenol A: RESULTS Europe (directive 2011/8/EU) : Specific migration limit in food from packaging of 220 0.6mg/kg 200 PROTOCOL OF CLEANUP Sample preparation 180 Bisphenol A Cleanup with a 3mL or 6mL/100mg 160 AFFINIMIP® SPE Bisphenol A cartridge 140 Equilibration •3mL Methanol -2% Acetic Acid 120 •3mL Acetonitrile •3mL Water 100 Loading mVolts 80 10mL liquid from canned food after filter paper filtration 60 Washing of interferents 40 •9mL Water •6mL Water/Acetonitrile (60/40) 20 Drying 30 seconds 0 Elution (E) -20 3mL Methanol The elution fraction was then evaporated and -40 dissolved in the mobile phase before HPLC analysis. -60 -80 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0 10,5 11,0 Minutes Chromatograms after clean-up with AFFINIMIP® SPE Bisphenol A of 10mL 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 10mL 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 % % RSDR 5 1.0 1.05 105.1 - under reproducibility conditions (n=4). C° (µg/L) Mean (µg/L) Recoveries % % RSDR 10 1.0 1.04 104.3 EVALUATION OF BPA IN COMMERCIAL CANNED FOODS 450 Bisphenol A 400 350 BPA : 2µg/L 300 250 mVolts 200 150 100 BPA : 0.5 µg/L 50 0 BPA : 0.3 µg/L -50 HPLC Method with Fluorescence detection -100 Column: Hypersil Gold C18 column 150mm x 4.6mm Mobile phase: gradient profile 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0 10,5 11,0 11,5 12,0 Chromatograms after clean-upMinutes with AFFINIMIP® SPE Bisphenol A of 10mL of canned salmon and tuna Time (min) % water % ACN (liquid form). 0 65 35 2 65 35 Blue: 1st price canned salmon; Green: middle grade 12 50 50 20 50 50 canned salmon: no BPA was detected; Red: 65 35 20.5 65 35 premium canned salmon; Pink: canned tuna 35 Catalog number: Flow rate: 1mL/min excitation/emission 3mL-100mg sorbent in a PP cartridge Fluorescence detection: PI-FS106-02 for 25 cartridges wavelengths: 230 / 315nm PI-FS106-03 for 50 cartridges Injection volume: 50µL. 6mL-100mg sorbent in a glass cartridge PI-FS106-02G for 25 cartridges PI-FS106-03G for 50 cartridges 41
AFFINIMIP® SPE Bisphenol A DETERMINATION OF BISPHENOL A IN CANNED FOOD (Vegetable) Regulations for Bisphenol A: RESULTS ZOOM Europe (directive 2011/8/EU) : Specific Bisphenol A migration limit in food from packaging of 0.6mg/kg 550 F luo F luo F luo MD 1. dat m d2. dat m nd. dat 500 PROTOCOL OF CLEANUP 450 400 Sample preparation 350 150g of drained canned peas - carrots and300 200mL of Water /ACN (50/50) are blende2d50mVolts during 2 min and centrifuged during 10min at200 150 4000rpm. The supernatant solution is collected 100 , filtered (4-7µm) and diluted ½ with water to50 give the loading solution 0 -50 Cleanup with a 3mL or 6mL/100mg0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 AFFINIMIP® SPE Bisphenol A cartridge Minut es Equilibration Fluo Fluo Fluo •3mL Methanol -2% Acetic Acid MD1.dat md2.dat mnd.dat •3mL Acetonitrile •3mL Water 650 Loading 600 20mL loading solution 550 Recovery yield : 97-99% Washing of interferents •9mL Water 500 •6mL Water/Acetonitrile (60/40) 450 Drying 30 seconds Elution (E) 400 3mL Methanol Bisphenol A The elution fraction was then evaporated and dissolved in the mobile phase before HPLC 350 analysis. mVolts 300 250 200 150 100 50 0 -50 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Minutes Chromatograms after clean-up with AFFINIMIP® SPE Bisphenol A of 20mL 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 150mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN Catalog number: 0 65 35 2 65 35 3mL-100mg sorbent in a PP cartridge 12 50 50 PI-FS106-02 for 25 cartridges 20 50 50 PI-FS106-03 for 50 cartridges 65 35 6mL-100mg sorbent in a PP cartridge 20.5 65 35 PI-FS106-02B for 25 cartridges 35 PI-FS106-03B for 50 cartridges 6mL-100mg sorbent in a glass cartridge Flow rate: 1mL/min excitation/emission PI-FS106-02G for 25 cartridges Fluorescence detection: PI-FS106-03G for 50 cartridges wavelengths: 230 / 315nm Injection volume: 50µL. 42
AFFINIMIP® SPE Bisphenol A DETERMINATION OF BISPHENOL A IN BEER Regulations for Bisphenol A: RESULTS Europe (directive 2011/8/EU) : Specific migration limit in food from packaging of Injection 50µL of beer 0.6mg/kg before treatment Bisphenol A after PROTOCOL OF CLEANUP treatment of 10mL of Beer Sample preparation The beer is degassed by sonication for 1 hour. Chromatograms of beer containing 1µg/L of Bisphenol A before (Red) and after (Blue) Cleanup with a 3mL or 6mL/100mg AFFINIMIP® SPE Bisphenol A Clean-up. AFFINIMIP® SPE Bisphenol A cartridge Bisphenol A Equilibration •3mL Methanol -2% Acetic Acid Chromatograms obtained after AFFINIMIP® SPE •3mL Acetonitrile Bisphenol A Clean-up of 10mL of beer spiked at •3mL Water 2µg/L (tested 3 times, orange) or at 1µg/L (tested 3 times, blue) with Bisphenol A or not spiked (red) Loading 10mL of degassed beer Recovery of Bisphenol A in spiked beer after AFFINIMIP® SPE Bisphenol A clean-up and relative standard deviation Washing of interferents calculated from results generated: •9mL Water •6mL Water/Acetonitrile (60/40) - under repeatability conditions (n=3, % RSDr) Drying 30 seconds Elution (E) 3mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. C° (µg/L) Mean µg/L Recoveries % % RSDr 1.0 1.1 106.9 1.0 2.0 1.9 93.4 1.0 - under reproducibility conditions ( % RSDR). HPLC Method with Fluorescence detection C° (µg/L) Mean µg/L Recoveries % % RSDR Column: Hypersil Gold C18 column 150mm x 4.6mm 1.0 1.0 99.3 8.9 Mobile phase: gradient profile 2.0 1.8 90.6 6.0 Time (min) % water % ACN Catalog number: 0 65 35 3mL-100mg sorbent in a PP cartridge 2 65 35 PI-FS106-02 for 25 cartridges 12 50 50 PI-FS106-03 for 50 cartridges 65 35 6mL-100mg sorbent in a glass cartridge 12.5 65 35 PI-FS106-02G for 25 cartridges 22 PI-FS106-03G for 50 cartridges Flow rate: 1mL/min excitation/emission Fluorescence detection: wavelengths: 230 / 315nm Injection volume: 50µL. 43
AFFINIMIP® SPE Bisphenol A DETERMINATION OF BISPHENOL A IN RED/WHITE WINES Regulations for Bisphenol A: RESULTS Europe (directive 2011/8/EU) : Specific migration limit in food from packaging of Fluo Fluo Fluo Fluo 0.6mg/kg MND.dat md1.dat md2.dat md3.dat PROTOCOL OF CLEANUP 1100 Bisphenol A Cleanup with a 3mL or 6mL/100mg 1000 AFFINIMIP® SPE Bisphenol A cartridge 900 Equilibration •3mL Methanol -2% Acetic Acid 800 •3mL Acetonitrile •3mL Water 700 Loading 600 Up to 10mL of wine mVolts 500 Washing of interferents •9mL Water 400 •6mL Water/Acetonitrile (60/40) 300 Drying 1 minute Elution (E) 200 3mL Methanol 100 The elution fraction was then evaporated and dissolved in the mobile phase before HPLC 0 analysis. -100 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0 10,5 11,0 11,5 12,0 12,5 13,0 Minutes Chromatograms obtained after clean-up with AFFINIMIP® SPE Bisphenol A of 10mL 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 10mL of white wine. HPLC Method with Fluorescence detection Matrice Mean Recoveries Column: Hypersil Gold C18 column 150mm x 4.6mm Spiked at 2µg/kg concentration % Mobile phase: gradient profile Red wine 1 (µg/kg) 96.6 Red wine 2 1.93 (n=2) 106.5 Red wine 3 83.0 White wine 2.13 (n=2) 80.0 1.66 (n=2) 1.60 (n=3) Time (min) % water % ACN Catalog number: 0 65 35 2 65 35 3mL-100mg sorbent in a PP cartridge 12 50 50 PI-FS106-02 for 25 cartridges 65 35 PI-FS106-03 for 50 cartridges 12.5 65 35 6mL-100mg sorbent in a PP cartridge 22 PI-FS106-02B for 25 cartridges PI-FS106-03B for 50 cartridges Flow rate: 1mL/min excitation/emission 6mL-100mg sorbent in a glass cartridge Fluorescence detection: PI-FS106-02G for 25 cartridges wavelengths: 230 / 315nm PI-FS106-03G for 50 cartridges Injection volume: 50µL. 44
AFFINIMIP® SPE Bisphenol A DETERMINATION OF BISPHENOL A IN COLA DRINKS Comparison of the solution obtained before RESULTS and after using AFFINIMIP® SPE Bisphenol A Bisphenol A PROTOCOL OF CLEAUNP Chromatograms obtained after clean-up with AFFINIMIP® SPE Bisphenol A of 10mL of white wine spiked with Cleanup with a 3mL or 6mL/100mg Bisphenol A at 2µg/kg (tested three times, blue) or not AFFINIMIP® SPE Bisphenol A cartridge spiked (red). The white wine naturally contained 2µg/kg of BPA Equilibration Recovery of Bisphenol A spiked at 5µg/kg after •3mL Methanol -2% Acetic Acid AFFINIMIP® SPE Bisphenol A clean-up of 6mL of Cola •3mL Acetonitrile drinks •3mL Water Mean Recoveries RSDr % Loading concentration % 1.0 6mL of Cola drinks after 30min degassing with ultrasounds (µg/kg) 96.6 Washing of interferents 1.93 (n=2) •9mL Water •6mL Water/Acetonitrile (60/40) Drying 3 minute Elution (E) 3mL Methanol The elution fraction was then evaporated and dissolved in the mobile phase before HPLC analysis. HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 150mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN Catalog number: 0 65 35 2 65 35 3mL-100mg sorbent in a PP cartridge 12 50 50 PI-FS106-02 for 25 cartridges 20 50 50 PI-FS106-03 for 50 cartridges 65 35 6mL-100mg sorbent in a PP cartridge 20.5 65 35 PI-FS106-02B for 25 cartridges 40 PI-FS106-03B for 50 cartridges 6mL-100mg sorbent in a glass cartridge Flow rate: 1mL/min excitation/emission PI-FS106-02G for 25 cartridges Fluorescence detection: PI-FS106-03G for 50 cartridges wavelengths: 230 / 315nm Injection volume: 50µL. 45
AFFINIMIP® SPE Bisphenol A DETERMINATION OF BISPHENOL A AND BADGE IN MILK RESULTS 500 500 450 450 400 400 350 350 Bisphenol A BADGE Bisphenol A Diglycidyl Ether (BADGE) 300 300 250 250 mVolts mVolts 200 200 150 150 100 100 50 50 Bisphenol A 00 PROTOCOL OF CLEANUP -50 -50 Cleanup with a 3mL or 6mL/100mg 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 AFFINIMIP® SPE Bisphenol A cartridge Minutes Fluorescence chromatograms obtained after clean-up with AFFINIMIP® SPE Bisphenol A of 9mL of milk spiked with 10µg/kg Bisphenol A and 10µg/kg BADGE (tested twice, blue) or not spiked (red). Equilibration Recovery of Bisphenol A and BADGE spiked at •3mL Methanol -2% Formic Acid 10ng/mL after AFFINIMIP® SPE Bisphenol A clean-up of •3mL Acetonitrile 9mL of milk. •3mL Water Matrice Mean Recoveries Loading Spiked at concentration % 9mL of Milk 10ng/mL (µg/kg) 108.5 Washing of interferents BPA 75 •9mL Water 10.85 •6mL Water/Acetonitrile (60/40) BADGE 7.5 Drying 3 minute Elution (E) 3mL Methanol (E1) 3mL Acetonitrile (E2) The elution fractions E1 and E2 were gathered, evaporated and dissolved in the mobile phase before HPLC analysis. HPLC Method with Fluorescence detection Column: Hypersil Gold C18 column 150mm x 4.6mm Mobile phase: gradient profile Time (min) % water % ACN Catalog number: 0 65 35 2 65 35 3mL-100mg sorbent in a PP cartridge PI-FS106-02 for 25 cartridges 12 50 50 PI-FS106-03 for 50 cartridges 20 20 80 6mL-100mg sorbent in a PP cartridge 25 20 80 PI-FS106-02B for 25 cartridges 30 65 35 PI-FS106-03B for 50 cartridges 40 65 35 6mL-100mg sorbent in a glass cartridge PI-FS106-02G for 25 cartridges Flow rate: 1mL/min excitation/emission PI-FS106-03G for 50 cartridges Fluorescence detection: wavelengths: 230 / 315nm Injection volume: 50µL. 46
AFFINIMIP® SPE Bisphenol A DETERMINATION OF TOTAL BISPHENOL A IN HUMAN URINE PROTOCOL OF CLEANUP RESULTS Sample preparation 3mL urine sample, 1mL of sodium acetate m/z 227.1 → m/z 212.1 buffer 0.1M at pH 5.0 and 20µL of β- glucuronidase/sulfatase Helix pomatia enzyme (a) m/z 227.1 → m/z 133.2 solution at 1.0mg/mL in the same buffer were mixed thoroughly by vortex. The enzymatic Intensity, cps reaction was carried out for 2h at 37°C to obtain the loading solution. (b) Cleanup with a 6mL/100mg AFFINIMIP® SPE Intensity, cps Bisphenol A glass cartridge LC-MS/MS Chromatograms obtained after clean-up with Equilibration AFFINIMIP® SPE Bisphenol A •3mL Methanol -2% Acetic Acid •3mL Acetonitrile (a) of children urine at 0.38ng/mL BPA, signal to noise •3mL Water (S/N) 13.9 Loading solution (b) for the blank sample (neither urine nor BPA), S/N=1.9 Up to 12mL of loading solution (Equivalent to around 9mL of urine) Mean percentage recoveries of Bisphenol A spiked at different concentrations in 3mL of urine after Washing of interferences AFFINIMIP® SPE Bisphenol A clean-up: •4mL Water •4mL Water/Acetonitrile (60/40) Elution (E) 3mL Methanol The elution fraction was then concentrated and diluted to 1mL before HPLC analysis. C° (ng/mL) 1 10 100 Recoveries % 102.6 94.7 97.6 HPLC Method with LC-MS/MS By courtesy of Nadia Diano, Dept. of Experimental HPLC Column: Kinetex 2.6µm PFP 100mm x 4.6mm Medicine, Second University of Naples (Italy) Mobile phase: gradient profile 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, 2013. Time (min) % water % Methanol Catalog number: 0 70 30 3mL-100mg sorbent in a PP cartridge 1 70 30 PI-FS106-02 for 25 cartridges 2 5 95 PI-FS106-03 for 50 cartridges 5 5 95 6mL-100mg sorbent in a glass cartridge 6 70 30 PI-FS106-02G for 25 cartridges 9 70 30 PI-FS106-03G for 50 cartridges Flow rate: 0.5mL/min Injection volume: 20µL. Detector: ESI-MS/MS 47
FS110 AFFINIMIP® SPE Chloramphenicol 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 0.3µg/kg for product of animal origin (Commission decision 2003/181/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 (> 90%) 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. Catalog number: 1mL-50mg sorbent PI-FS110-02A for 25 cartridges PI-FS110-03A for 50 cartridges 48
AFFINIMIP® SPE Chloramphenicol DETERMINATION OF CHLORAMPHENICOL IN HONEY Regulations for Chloramphenicol in RESULTS residues in food of animal origin: Europe 2003/181/EC prohibited with a SIM Chromatograms obtained after clean-up with minimum required performance limits of AFFINIMIP® SPE Chloramphenicol of 1g of Honey 0.3µg/Kg spiked with Chloramphenicol at 15.7µg/kg (red) or not spiked (blue). PROTOCOL OF CLEANUP Sample preparation Intensity Honey spiked at 15.7µg/kg 10g of honey and 10mL Water were mixed under magnetic stirring during 10 minutes and 10000 used as the loading solution. 9500 Cleanup with a 1mL/50mg AFFINIMIP® SPE Chloramphenicol cartridge 9000 Not spiked Equilibration 8500 •2mL Acetonitrile 8000 •2mL Water 7500 7000 Loading 6500 1mL of loading solution for 15µg/kg (or 10mL for 0.3µg/Kg) 6000 Chloramphenicol Washing of interferents (W1) 5500 •1mL Water 5000 •1mL (Water - 0.5% AA)/ACN (95/5) 4500 •2mL of Ammonia (1%) in Water 4000 •2mL (Water-1% Ammonia)/ACN (80/20) 3500 3000 Drying 1 min 2500 Washing of interferents (W2) 2000 0.25mL Diethyl ether 1500 Elution (E) 1000 2mL Methanol The elution fraction was then evaporated and 2 3 4 5 6 7 8 9 10 dissolved in the mobile phase before HPLC Time (min) analysis. SIM Chromatograms obtained after clean-up with 0.3µg/Kg Chloramphenicol AFFINIMIP® SPE Chloramphenicol of 1g of Honey spiked with Chloramphenicol at 15.7µg/kg (red) or Chloramphenicol not spiked (blue). SIM Chromatogram obtained after clean-up with Recovery of Chloramphenicol spiked at 16µg/kg AFFINIMIP® SPE Chloramphenicol of 10g of after AFFINIMIP® SPE Chloramphenicol clean-up of Honey spiked with Chloramphenicol at 0.3µg/kg. 1g of Honey and relative standard deviation calculated from results generated:. HPLC Method with MS detection Column: Thermo Accucore C18 column 50mm x - under repeatability conditions (n=3, % RSDr) 2.1mm Mobile phase: Ammonium acetate (10mM) in C° (µg/kg) Mean (µg/kg) Recoveries % % RSDr water /Methanol (75/25) flow rate: 0.2mL/min 16.0 15.4 96.1 3.3 MS detection: m/z 322 (ESI-) Injection volume: 20µL. - under reproducibility conditions ( % RSDR). C° Mean (µg/kg) Recoveries % % RSDR (µg/kg) 15.7 16.9 108.1 6.5 (n=6) 18.2 16.6 91.4 11.4 (n=12) Catalog number: 1mL-50mg sorbent PI-FS110-02A for 25 cartridges PI-FS110-03A for 50 cartridges 49
AFFINIMIP® SPE Chloramphenicol DETERMINATION OF CHLORAMPHENICOL IN BOVINE URINE Regulations for Chloramphenicol in RESULTS residues in food of animal origin: Europe (2003/181/EC) : prohibited with a Chloramphenicol Minimum Required Performance Limits of 0.3µg/Kg SIM Chromatograms obtained after clean-up with USA FDA: prohibited AFFINIMIP® SPE Chloramphenicol of 1 mL of Urine spiked with Chloramphenicol at 17.6µg/kg (red and PROTOCOL OF CLEANUP blue) or not spiked (green). Sample preparation 10 mL of urine were adjusted at pH 7 with RT: 0.00 - 30.00 NL: Ammonia 1%. This solution was mixed and 1.32E5 used as the loading solution. 14000 Channel A Cleanup with a 1mL/50mg AFFINIMIP® SPE 13000 UV: VERY LOW BACKGROUND UV Chloramphenicol cartridge UrineD1-E 12000 Equilibration NL: •2mL Acetonitrile 11000 1.32E5 •2mL Water 10000 Channel A Loading UV 1mL of loading solution urined2-e Washing of interferents (W1) NL: •1mL (Water - 0.5% Acetic 1.32E5 Acid)/Acetonitrile (95/5) •2mL of Ammonia (1%) in Water Channel A •2mL (Water-1% Ammonia)/Acetonitrile UV (80/20)) UrineND-E Drying 1 min 9000 Washing of interferents (W2) 8000 0.25mL Diethyl ether Elution (E) uAU 7000 2mL Methanol 6000 The elution fraction was then evaporated and dissolved in the mobile phase before HPLC 5000 analysis. 4000 3000 2000 1000 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Time (min) UV Chromatograms of Urine spiked with Chloramphenicol at 17.6 µg/kg (red and black) or not spiked (green) after clean-up with AFFINIMIP® SPE Chloramphenicol Recovery of Chloramphenicol spiked at 17.6µg/kg after AFFINIMIP® SPE Chloramphenicol clean-up of 1 mL of Urine. C° (µg/kg) Mean (µg/kg) Recovery % 17.6 16.7 90 HPLC Method with MS detection Catalog number: Column: Thermo Accucore C18 column 50mm x 1mL-50mg sorbent 2.1mm PI-FS110-02A for 25 cartridges Mobile phase: Ammonium acetate (10mM) in PI-FS110-03A for 50 cartridges water /Methanol (75/25) flow rate: 0.2mL/min MS detection: m/z 321 (ESI-) Injection volume: 20µL. 50
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