Innovative vs Traditional

And in the red corner, introducing the challenger NASDAQ April 4 2011 243.47 $ 1.38 $ 0.58 % 0.07 $ 0.02 $ 39.4 % Technology DVD Digital Streamline Instant Play Internet Access Unlimited Selection No Late Fees Instant Play Technology DVD Vinyl DVD reader Retail Stores Limited Selection Late Fees Innovative vs Traditional

Commodities Class A Class B Class C Low Complexity Intermediate Complexity High Complexity

Traditional in 2000 Chemistries/Consumables Liquid Chromatography Mass Spectrometry Alliance 2695 Ultima MS/MS Class A: low complexity Class B: intermediate complexity Class C: high complexity 2.5-3 hrs per sample 3-4 hrs per sample up to 8 hours per sample could be 2 days in some instance

After several hours of hard work. collect samples measure, weight and manipulate extract, clean, filter Analyze 1 ml aqueous extract evaporate, reconstitute.

Traditional Chemistries/Consumables HPLC Mass Spectrometry Alliance 2695 Ultima MS/MS UPLC ASMS 2010 Acquity XEVO TQS

How high end MS are utilized today A B C D A - Homogenization B - Liquid-liquid extraction / salting out C - Dispersive SPE / clean up D - Filtration particulate E - 100-1000 X dilution

Innovative Chemistries/Consumables Liquid Chromatography Mass Spectrometry Alliance 2695 Ultima MS/MS PittCon 2011 ASMS 2010 + + Cap n Flip Acquity 2D UPLC XEVO TQS

Multi-Dimension Chromatography Common Perception LC x LC GC X GC Benefits Drawbacks Wide selection of combinations Increase separation/resolution High level of automation Increase throughput Cost reduction time & process Highly complex instruments Highly trained resources needed

Multi-Dimension Chromatography Current reality. 1D chromatography 2D chromatography

1D vs 2D chromatography At column dilution BSM Pump Eluting Stream BSM Pump Eluting Stream Injector Loop Injector Loop waste 50uL mixer Inj Inj V1 D1 2.1 mm ID A1 D1 2.1 mm ID MS BSM Pump Loading Stream B1 D2 2.1 mm ID MS up to 100 µl injection volume aqueous extracts only up to 20 000 µl injection volume aqueous and organic extracts

1D vs 2D chromatography 100% acn extract 100 250 ul 2D with AT-column dilution 6.31 MRM of 6 Channels ES+ 327 > 269.9 1.88e6 100 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 MRM of 6 Channels ES+ 4.32 327 > 269.9 3.31e5 250 ul 2D 0 100 0 100 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 MRM of 6 Channels ES+ 327 > 269.9 20 ul 1D 1.89e5 % % % 0 % 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 MRM of 6 Channels ES+ 0.86 327 > 269.9 7.23e5 250 ul 1D 0.72 0.33 0.92 2.73 0.32 0 0.40 2.14 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Time

1D vs 2D Extraction Protocol Class A Traditional Innovation 0 min Condition 5 ml ACN/Dichloromethane (1:1) 6 min Condition 5 ml Methanol Condition 5 ml Water 0 min Condition 52 ml ACN/Dichloromethane (1:1) 6 min Condition 52 ml Methanol Condition 52 ml Water Load Sample 200 1000 ml ml @ 10 10 ml/min Load Sample 20 ml 16 min 106 min 108min 118 min Wash 5 ml Water Air Dry 10 min 18 min 0.3 hrs Wash2 ml water Elute 1mL ACN Inject 1000 µl into LC/LC/MS/MS (2D) 120 min 150 min Elute 35 ml ACN/Dichloromethane (1:1) Evaporate to 0.1 ml ~30min (N, 35 2 C, ~30min ) Reconstitute 1 ml ACN/Water (1:1) (1:9) 20:1 enrichment from sample protocol 100:1 enrichment from injection volume 2000:1 final enrichment 100% of final extract use for analysis 2.5hrs Inject 10 µl into LC/MS/MS 1000:1 enrichment from sample protocol 1% of final extract use for analysis

Cap n Flip concept Liquid Liquid Extraction Filtration Centrifugation Solid Phase Extraction Dispersive SPE Homogenization

Cap n Flip Concept Novel dual cap format Current SPE Barrel Format Cap n flip concept Reservoir Filter/sorbent + Incorporated in a dual vial cap

Cap n Flip Concept Filtration & Separation Formats A Liquid-Liquid separation Liquid-solid separation Filtration (coarse / intermediate / fine ) Double end cap Filter/membrane B Double end cap Dispersive or Captive - PSA - GCB - Oasis HLB - Silica - Silica-C18, C8, C4, phenyl, cyano - Mixed-Mode (MCX, MAX, WAX, WCX) Sorbent Filter/membrane

Cap n Flip Concept Vacuum & Centrifuge compatible Vacuum Manifold Benchtop Centrifuge

Cap n Flip Features: Collection, Shipping, Receiving Collection Shipping Receiving Septum Cap for direct injection analysis or Proceed with Extraction Protocol Solid Cap

Class B Matrix Traditional Extraction Protocol Published Extraction Protocol Step 1: Pipette 5 ml milk into 50 ml centrifuge tube Step 2: Add 15 ml acetonitrile Step 3: Shake and centrifuge 3200 RPM for 15 min Step 4: Transfer supernatant into 50 ml centrifuge tube Barrels or Syringe Step 5: Add 20 ml hexane Step 6: Shake 15 min Step 7: Centrifuge 32100 RPB for 15 min Step 8: Remove hexane layer Step 9: Evaporate ACN/milk supernatant to 3 ml Step 10: Add 15 ml 0.1 M phosphate buffer ph 8.0 Step 11: Condition SPE (6 cc) with 10 ml Methanol Step 12: Condition SPE (6 cc) with 10 ml Water Step 13: Condition SPE (6 cc) with 10 ml 2% NaCl Step 14: Condition SPE (6 cc) with 2 ml 0.1 M PO 4 buffer Step 15: Load reconstituted sample (step 10) Step 16: Wash 5 ml water Step 17: Wash 5 ml 40 % Methanol in water Step 18: Dry SPE for 5 min Step 19: Elute with 5 ml 95 % Methanol in water Step 20: Evaporate close to dryness (45 min) Step 21: Reconstitute in 1 ml initial mobile phase condition Step 22: Filter extract with 0.45 µm PVDF Legend: Red = Quechers and/or PPT Blue = LLE Green = SPE Black = Filtration OFF-line SPE Extraction Manifold

Quechers + Dispersive SPE 10 g Homogenate 10 ml ACN ACN salting-out A B C D Transfer DISQUE TUBE 1 G Ready for next step F PSA/C18 second clean E Double layer DISQUE Tube 2

LLE, Transfer & SPE A B C Transfer supernatant separate aqueous/organic phase Evaporate supernatant D E F Add 15 ml phosphate buffer SPE 6 cc cartridge Elute, Evaporate & reconstitute

Class B matrix Traditional Vs Innovative Published Extraction Protocol Step 1: Pipette 5 ml milk into 50 ml centrifuge tube Step 2: Add 15 ml acetonitrile Step 3: Shake and centrifuge 3200 RPM for 15 min Step 4: Transfer supernatant into 50 ml centrifuge tube Step 5: Add 20 ml hexane Step 6: Shake 15 min Step 7: Centrifuge 3200 RPM for 15 min Step 8: Remove hexane layer Step 9: Evaporate ACN/milk supernatant to 3 ml Step 10: Add 15 ml 0.1 M phosphate buffer ph 8.0 Step 11: Condition SPE (6 cc) with 10 ml Methanol Step 12: Condition SPE (6 cc) with 10 ml Water Step 13: Condition SPE (6 cc) with 10 ml 2% NaCl Step 14: Condition SPE (6 cc) with 2 ml 0.1 M PO 4 buffer Step 15: Load reconstituted sample Step 16: Wash 5 ml water Step 17: Wash 5 ml 40 % Methanol in water Step 18: Dry SPE for 5 min Step 19: Elute with 5 ml 95 % Methanol in water Step 20: Evaporate close to dryness (45 min) Step 21: Reconstitute in 1 ml initial mobile phase condition Step 22: Filter extract with 0.45 µm PVDF Cap n Flip Extraction Protocol Step 1: Pipette 1 ml milk into 20 ml vial Step 2: Add 2 ml acetonitrile Step 3: Shake and centrifuge 3200 RPM for 5 min Step 4: Filter supernatant into 20 ml vial **** Step 5: Add 2 ml hexane Step 6: Shake 5 min Step 7: Centrifuge 3200 RPM for 5 min Step 8: Filter hexane layer **** Step 9: Dilute ACN supernatant 18 ml phosphate buffer ph 8.0 Step 10: Condition SPE (3 cc) with 2 ml Methanol Step 11: Condition SPE (3 cc) with 2 ml Water Step 12: Condition SPE (3 cc) with 2 ml 2% NaCl Step 13: Condition SPE (3 cc) with 2 ml 0.1 M PO 4 buffer Step 14: Load reconstituted sample **** Step 15: Wash 2 ml water Step 16: Wash 2 ml 40 % Methanol in water Step 17: Elute with 1 ml 95 % Methanol in water Step 18: Filter extract with 0.45 µm PVDF **** Total Time: 35 min (0.58 hrs) Total Time: 190 min (3.2 hrs)

Cap n Flip Extraction Protocol Step 1 & 2 A B Aliquot 1 ml of sample Add 2 ml acetonitrile C D Cap Shake or vortex

Cap n Flip Extraction Protocol Step 3 A Centrifuge 15 min @ 3 500 rpm B C D

Cap n Flip Extraction Protocol Step 4 pellet supernatant flip pellet Centrifuge 5 min @ 4 000 rpm supernatant

Class C matrix Traditional Extraction Protocol Comminute: > 1kg sample with vertical cutter Homogenize: 200 g with probe blender Sample representation Transfer: 15 g homogenate in 50 ml Tube Liquid-solid extraction Add: 15 ml ACN + 1% Hac 1.5 g Anh. NaAcetate 6 g Anh. MgSO 4 Shake: 1 min Centrifuge: 1500 rcf 5 min Transfer: 10 ml ACN supernatant 15 ml Tube Dispersive SPE Add: 900 mg Anh. MgSO4 150 mg PSA Centrifuge: 1500 rcf 1 min Transfer: 4 ml ACN supernatant 15 ml Tube Transfer: 0.1 ml ACN supernatant 2 ml vial Add: 1 ml Toluene Dilute: 0.9 ml aqueous initial mobile phase Evaporate, dry & reconstitute: 1 ml toluene LC/MS/MS analysis GC/MS analysis

Class C matrix Innovative Option A: Representative Sampling Comminute: > 1kg sample with vertical cutter Homogenize: 200 g with probe blender Option B: Spatial Sampling Comminute: > 1kg sample with vertical cutter Transfer: 1.5 g in 10 ml Tube x10 Transfer: 1.5 g homogenate in 10 ml Tube 1/10 th Homogenize: 2 ml ACN ( up to 10 ml) Add: 2 ml ACN + 1% Hac 0.15 g Anh. NaAcetate 0.6 g Anh. MgSO 4 etc 1/10 th Add: 1 pill or as many as needed etc Solid sample Completed in < 30 sec Single process Stainless Steel Ball bearings Multi process Orbital Shaker Powder Format Solid format

Cap n Flip Concept Feedback from Major Food Company Food Products : Bottle Water (matrix A), Fruit Juice (Matrix B) and Fruit snacks (Matrix C) Cost Benefits with Shipping Currently shipping upwards 4 liters of water in glass bottles to support global monitoring program Significant cost reduction in shipping and sample bottles Reduction in sample volume Reduction in logistics by tracking smaller sample kits instead of larger kits (3 x2 x2 ) Time Benefits on Sample Preparation Reduction of consumables for Quechers and filtration to support Class B and C sample types Reduction in time for extraction based on elimination of steps in traditional methodology Reduction of additional preparation and test using 2D technology for the analysis of different class of compounds (i.e. acid/base/neutral analytes)

Final thought. Traditional Class A Innovation 2-3 hrs per sample 15 min per sample Class B 3-4 hrs per sample 30 min per sample Class C 8 hrs per sample 60 min per sample