Zoe Grosser, Vinson Leung, Jim Fenster, Brian LaBrecque Horizon Technology, Inc., Salem, NH USA

Zoe Grosser, Vinson Leung, Jim Fenster, Brian LaBrecque Horizon Technology, Inc., Salem, NH USA

To develop an automated SPE method for the extraction of 20 organochlorine pesticides using an established, regulated manual-spe method for wine testing Demonstrate comparability of automation and manual SPE methodologies through good recoveries Show improvement or optimization to existing extraction methodology Establish an efficient workflow for transitioning from manual to automated SPE 2

Manual SPE Vacuum Manifold SmartPrep Extractor Automated SPE System 3

Condition Cleans sorbent bed and activates sorbent chemistry Load Introduces sample to sorbent to begin binding at consistent flow rate Wash Selectively removes interferences Elute Collects compounds in smallest volume for further analysis 4

ALCOHOL AND TOBACCO TAX AND TRADE BUREAU U.S. Department of the Treasury TTB routinely conducts pesticide analysis of domestic and imported wines Unauthorized pesticides or quantities outlined in FD&C Act, 21 U.S.C. 346a Authorizes EPA to set maximum residue levels for pesticides in/on foods Adulterated wines are determined by consultation with EPA and FDA based on determined pesticides and levels 5

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Matrix BAREFOOT Moscato (ALC. 9%) Livingston Cellars Chardonnay (ALC. 12%) Cartridges Waters Oasis HLB 6 cc, 200 mg Supelco Aminopropyl LC-NH2 3 cc, 500 mg Solvents 50:50 Ethyl Acetate:Hexane 80:20 Ethyl Acetate:Hexane Methanol Dionized Water Pesticide Spike Restek #32415 Other Magnesium Sulfate 7

Aldrin (309-00-2) α-bhc (319-84-6) β-bhc (319-85-7) δ-bhc (319-86-8) γ-bhc (Lindane) (58-89-9) cis-chlordane (5103-71-9) trans-chlordane (5103-74-2) 4,4 -DDD (72-54-8) 4,4 -DDE (72-55-9) 4,4 -DDT (50-29-3) Dieldrin (60-57-1) Endosulfan I (959-98-8) Endosulfan II (33213-65-9) Endosulfan sulfate (1031-07-8) Endrin (72-20-8) Endrin aldehyde (7421-93-4) Endrin ketone (53494-70-5) Heptachlor (76-44-8) Heptachlor epoxide (isomer B) (1024-57-3) Methoxychlor (72-43-5) 8

Modified TTB Method Procedure 1. Mix 20 ml wine with 20 ml DI Water (total volume 40 ml) 2. Spike sample with 50 µl of pesticide mix (50 µg/ml) 3. Condition Oasis HLB cartridge with 10 ml of: i. 50:50 Ethyl Acetate:Hexane ii. Methanol iii. DI Water 4. Load sample at 5 ml/min and vent remaining sample of 1.1 ml to cartridge 5. Air dry for 10 mins 6. Elute cartridge with 5 ml of i. 80:20 Ethyl Acetate:Hexane ii. 50:50 Ethyl Acetate:Hexane iii. 50:50 Ethyl Aceate:Hexane 7. Load aminopropyl cartridge with 1/3 column volume of magnesium sulfate (drying agent) 8. Condition aminopropyl cartridge with 5 ml 50:50 Ethyl Acetate:Hexane 9. Load and collect extract 10. Evaporate with XcelVap to 1.0 ml TTB Method Procedure 1. Mix 20 ml wine with 20 ml DI Water (total volume 40 ml) 2. Spike sample with 50 µl of pesticide mix (50 µg/ml) 3. Condition Oasis HLB cartridge with 10 ml of: i. 50:50 Ethyl Acetate:Hexane ii. iii. Methanol DI Water 4. Load sample by gravity (initial vacuum pull) 5. Air dry for 15 mins 6. Elute cartridge with 5 ml of i. 80:20 Ethyl Acetate:Hexane ii. iii. 50:50 Ethyl Acetate:Hexane 20:80 Ethyl Aceate:Hexane 7. Load aminopropyl cartridge with 1/3 column volume of magnesium sulfate (drying agent) 8. Condition aminopropyl cartridge with 5 ml 50:50 Ethyl Acetate:Hexane 9. Load and collect extract 10. Evaporate with Nitrogen to 0.1 ml 11. Add 1 ml 0.1% corn oil/etoac 9

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Sample line rinse Sample bottle rinse 11

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OVEN Initial temp: 70 'C Initial time: 0.50 min Ramps: # Rate ( /min) Final temp Final time 1 16.00 190 C 0.00 2 8.00 290 C 0.00 3 25.00 325 C 3.00 FRONT INLET (SPLIT/SPLITLESS) Mode: Pulsed Splitless Initial temp: 245 C Pressure: 8.71 psi Pulse pressure: 25.0 psi Pulse time: 1.00 min Gas type: Helium COLUMN 1 Capillary Column Model Number: Phenomenex ZB-SemiVolatiles Nominal length: 30.0 m Nominal diameter: 250.00 µm Nominal film thickness: 0.25 µm Mode: constant flow Initial flow: 1.0 ml/min MSD Transfer Line Heater held at 325 C GC Injector Injection Volume 1.00 microliters 14

Manual Manifold SmartPrep Compounds Water Wine (Moscato) Water Wine (Moscato) a-hch 89 90 82 101 102 99 93 91 95 85 76 92 b-hch 101 100 95 109 112 110 90 91 95 88 81 92 g-hch 87 89 79 97 99 97 91 90 91 85 77 91 d-hch 94 93 82 104 114 105 95 92 96 90 84 95 Heptachlor 83 87 78 57 42 50 90 84 89 42 42 49 Aldrin 79 80 76 50 34 44 88 81 88 36 36 40 Heptachlor epoxide (B) 84 87 79 84 77 78 85 82 93 67 56 77 t-chlordane 79 84 75 55 40 50 86 79 91 39 38 46 c-chlordane 74 79 72 55 42 44 86 78 90 40 39 46 Endosulfan I 84 85 81 74 69 75 89 82 90 60 51 71 4,4`-DDE 74 79 72 47 32 45 92 80 95 33 35 39 Dieldrin 82 89 79 76 70 73 86 81 95 56 51 68 Endrin 84 92 79 59 64 60 101 94 96 67 61 82 Endosulfan II 87 87 79 98 96 94 88 83 93 80 66 89 4,4`-DDD 81 87 74 49 32 39 77 69 79 36 35 40 Endrin Aldehyde 90 93 77 17 30 22 84 88 99 43 42 74 Endosulfan Sulfate 86 89 73 98 98 97 87 83 87 89 73 95 4,4`-DDT 72 77 68 57 45 55 95 86 96 44 42 51 Endrin Ketone 86 93 77 115 114 115 83 84 88 85 73 91 Methoxychlor 83 90 77 71 59 69 76 71 81 57 51 67 15

Manual Manifold SmartPrep Manual Manifold SmartPrep Compounds Water Water Wine (Moscato) Wine (Moscato) a-hch b-hch g-hch d-hch Heptachlor Aldrin Heptachlor epoxide (B) t-chlordane c-chlordane Endosulfan I 4,4`-DDE Dieldrin Endrin Endosulfan II 4,4`-DDD Endrin Aldehyde Endosulfan Sulfate 4,4`-DDT Endrin Ketone Methoxychlor Statistics 87 93 101 85 99 92 111 87 85 90 98 84 90 94 108 90 83 88 50 44 78 85 42 37 83 87 80 67 80 85 48 41 75 84 47 42 83 87 72 61 75 89 42 36 84 87 73 58 85 97 61 70 84 88 96 78 80 75 40 37 87 90 23 53 83 86 98 86 72 92 52 46 85 85 114 83 83 76 66 58 MEAN STDEV %CV MEAN STDEV %CV MEAN STDEV %CV MEAN STDEV %CV 83 6 7 88 5 6 71 28 39 62 20 31 16

SmartPrep Compounds Wine (Moscato) Wine (Chardonnay) a-hch b-hch g-hch d-hch Heptachlor Aldrin Heptachlor epoxide (B) t-chlordane c-chlordane Endosulfan I 4,4`-DDE Dieldrin Endrin Endosulfan II 4,4`-DDD Endrin Aldehyde Endosulfan Sulfate 4,4`-DDT Endrin Ketone Methoxychlor 85 94 87 98 84 91 90 101 44 55 37 48 67 80 41 55 42 55 61 72 36 50 58 75 70 64 78 95 37 49 53 18 86 90 46 60 83 104 58 68 17

Compound: Endrin Aldehyde Wine Matrix: Moscato Mean % Recovery: 53% Compound: Endrin Aldehyde Wine Matrix: Chardonnay Mean % Recovery: 18% 18

Manual Manifold vs. SmartPrep Demonstrated comparability - automation and manual SPE Horizon Technology SmartPrep Extractor produced comparable recoveries to manual Manual Manifold SmartPrep Manual Manifold SmartPrep Water Wine (Moscato) MEAN STDEV %CV MEAN STDEV %CV MEAN STDEV %CV MEAN STDEV %CV 83.0 5.7 6.9 87.5 5.3 6.1 71.1 27.9 39.3 62.1 19.5 31.4 Established efficient workflow from manual to automated SPE SmartPrep software enabled consistent programming to TTB method Automated SPE and bottle rinsing process XcelVap performed consistent sample eluent dry-down Results show there is a likely wine matrix effect Affecting recoveries of certain compounds vs. water recoveries Room for improvement with future SPE optimization Moscato vs. Chardonnay Higher sugar content may hinder recoveries (approx. 10% difference) 19

Index M Index A Index B Index C Index D Index E Index F Index G Index I Index K Index H Index J Index L 20

Achieve higher recoveries by resolving matrix effect with optimization Automate the optimization process using the SmartPrep software Test elution flow rates and elution volumes to determine optimal values Use optimal values to test elution solvents for 1 st and 3 rd fractions Elution Flow Rates 10 ml/min 7 ml/min 5 ml/min Elution Volumes 5 ml 7 ml 1 st Elution Solvent 80:20 EtOAc:Hexane To 75:25 EtOAc:Hexane 85:15 EtOAc:Hexane 3 rd Elution Solvent 50:50 EtOAC:Hexane To 35:65 EtOAc:Hexane 25:75 EtOAc:Hexane 21