Fundamentals of Solid-Phase Extraction (SPE): Principles and Practical Tips for Developing Sample Preparation Methods

Thank you for joining us! The Webinar will begin shortly. Fundamentals of Solid-Phase Extraction (SPE): Principles and Practical Tips for Developing Sample Preparation Methods 2013 Waters Corporation 1

Introduction Joe Arsenault This webinar will be presented by Joseph Arsenault. With more than 37 years of chromatography experience at Waters, Joseph Arsenault was a part of the team that introduced Solid Phase Extraction (SPE) technology for analytical laboratories in 1978. He has been involved in all aspects of SPE products from manufacturing, quality control, quality assurance, having spent numerous years as a Product Manager for SPE products. Over the last 14 years, he has devoted himself to the training and education of chromatographers around the world. In addition to his presentations, he has also authored and co-authored 3 handbooks/primers for chromatographers: Beginners Guide to Solid Phase Extractions (SPE) Beginners Guide to Liquid Chromatography Beginners Guide to Ultra Performance Liquid Chromatography (UPLC) o All Available via www.waters.com 2013 Waters Corporation 2

Friendly Reminders Please use text chat functionality to submit questions during the Webinar. Upon conclusion, follow up information will be available: http://www.waters.com/spepart1 Recorded version of today s presentation Copies of today s slides Product discount offers Product specific information Reference material 2013 Waters Corporation 3

Agenda The Importance of Solid Phase Extraction How SPE Works Device Design and Tips for Processing Samples Chromatographic Sorbent Choices Typical SPE Strategies Was My SPE Method Successful? Appendix 2013 Waters Corporation 4

The New SPE Textbook Part #: 715003405 212 pages, paperback Size: 8.25 x 11 >150 full color figures and diagrams Chapter Titles Benefits of SPE in Sample Preparation SPE is LC Key Terms and Calculations In the Lab Method Development Troubleshooting Appendix: Glossary of SPE and LC Terms Appendix: Oasis Sorbent Technology for SPE Appendix: Applications Appendix: Additional Reference Materials 2013 Waters Corporation 5

Why do Sample Preparation? Issue: Plugging Cause: solid particulates Impact: may limit instrument/system up-time 2013 Waters Corporation 6

Sample Prep Tools For solid particulates: Filters Centrifuge For sample matrix components: Precipitation Liquid-Liquid Extraction {LLE} Solid-Phase Extraction {SPE} 2013 Waters Corporation 7

Why do Sample Prep? Issue: Complicated analytical results / too much variability and potential resolution issues 0.08 0.06 AU 0.04 0.02 0.00 Cause: analytes contained in a complex sample matrix Impact: 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Minutes Quantitation errors: slight changes might result in loss of resolution of critical pairs May reduce instrument/system up-time 2013 Waters Corporation 8

Improve Analytical Results: Example #1 0.08 Improvement: Remove unnecessary peaks 0.06 AU 0.04 0.02 0.00 0.08 0.06 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Only need to analyze these peaks Minutes AU 0.04 0.02 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Minutes 2013 Waters Corporation 9

Improve Analytical Results: Example #2 Improvement: Remove Baseline Interferences PDA (0.0 01 AUFS) 1 2 Sample Sample Prep 1 2 0 5 10 Minutes 2013 Waters Corporation 10

Improve Analytical Results: Example #3 Improvement: Remove Baseline Interferences 0.004 AU 1 2 3 Spiked Sample 0.0 00 2.0 00 4.0 00 Peak 1 has baseline contamination from sample matrix Minute es 6.0 00 8.0 00 Peaks 2 and 3 are clean 10.00 Blank Sample Matrix With SPE Clean-up 1 2 0.004 AU 3 Spiked Sample 0 2 4 6 min. Peak 1 is clean 8 10 Blank Sample Matrix 2013 Waters Corporation 11

Why do Sample Prep? Issue: not enough sensitivity Cause: Analyte concentration in original sample matrix TOO LOW to measure by instrument Impact: Quantitation errors 2013 Waters Corporation 12

Improve Analytical Results: Example #4 Concentration of Analyte in Original Sample TOO LOW (difficult to quantitate) Time 0 1 2 3 4 5 2013 Waters Corporation 13

Improve Analytical Results: Example #4 Utilize SPE Chromatographic Bed to Trace Concentrate the Original Sample for that Analyte - Obtain Good Response Concentration of Analyte in Original Sample TOO LOW (difficult to quantitate ) Time 0 1 2 3 4 5 2013 Waters Corporation 14

Why do Sample Prep? Issue: Not enough sensitivity Cause: For MS detection: minimize ion suppression/enhancement Impact: Quantitation Errors 2013 Waters Corporation 15

What is Ion Suppression? 100 % 50/50 Water/ACN + human plasma supernatant Analytes in human plasma with only Protein Precipitation 591.7 Scan ES+ Standards in aqueous solution give acceptable response 260.2-97 % 291.2-96 % 354.4-86 % 411.4-93 % 472.6-93 % 485.6-95 % 591.6-89 % 609.5-93 % 0 100 354.4 518.5 472.6 485.5 609.6 354.4 50/50 Water/ACN Scan ES+ 609.6 260.2 472.6 485.6 % 291.3 411.5 591.6 0 m/z 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 2013 Waters Corporation 16

Complex Sample Matrix: Ion Suppression 100 % 50/50 Water/ACN + human plasma supernatant Analytes in human plasma with only Protein Precipitation 591.7 Scan ES+ % Loss 260.2-97 % 291.2-96 % 354.4-86 % 411.4-93 % 472.6-93 % 485.6-95 % 591.6-89 % 609.5-93 % 0 100 354.4 518.5 472.6 485.5 609.6 354.4 50/50 Water/ACN Scan ES+ 609.6 % 260.2 291.3 411.5 472.6 485.6 591.6 Analyte standards in aqueous solution 0 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 m/z 2013 Waters Corporation 17

Improve Analytical Results: Example #5 100 % MRM for Terfenadine Significant ion suppression observed for analytes that co-elute with residual matrix components using just PPT. Protein Precipitation (PPT) 80% ion suppression MRM 472.2 > 436.4 1.27e6 1.89 % 0 100 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 SPE with Oasis MCX 1.91 Minimal ion suppression MRM 472.2 > 436.4 1.27e6 0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Note: These samples are dried and reconstituted. Gradient time = 1.5 min 2013 Waters Corporation 18

Do all Sample Preparation Techniques Give the Same Result? MRM for Terfenadine 100 A (PPT) 80% ion suppression MRM 472.2 > 436.4 1.27e6 % 1.89 0 100 % 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 B (SPE) (Oasis MCX) 1.91 Minimal ion suppression MRM 472.2 > 436.4 1.27e6 0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 No loss in signal observed for analytes when the interferences, which cause the suppression, are removed by SPE. Note: These samples are dried and reconstituted. Gradient time = 1.5 min 2013 Waters Corporation 19

Goals of Sample Preparation: Summary To remove interferences for Better chromatography More confident analytical results Longer column lifetime Less instrument downtime To enrich sample for Higher detection sensitivity To make sample more compatible for separation and detection Matching solvent strength Eliminating ion-suppression in LC/MS analysis The Ultimate Goal is to make your analytical lab more productive - Lower Limits of Detection - Run more samples with less time - Minimize costs in manpower and equipment maintenance 2013 Waters Corporation 20

How a Chromatographic Column Works -- BANDS x Yellow is the earliest eluting analyte band, it likes the mobile phase, Mix Yellow, Red and Blue Dyes together to create what appears, Food has very low k (little retention) Dyes to our eyes Blue is well retained, it likes the particles, has high k (high retention - captured ) as a Black Sample 2013 Waters Corporation 22

How a Chromatographic Column Works -- BANDS Yellow is the earliest eluting analyte band - it likes the mobile phase, has very low k (little retention) Blue is well retained - it likes the particles, has high k (high retention - captured ) 2013 Waters Corporation 23

Band Migration on an SPE Cartridge SPE follows the same principles as LC Sample Blue Red Yellow 2013 Waters Corporation 24

SPE Configurations from Waters Syringe Barrel Cartridge Standard 96-Well Plate µelution 96-Well Plate On-line SPE Devices 2013 Waters Corporation 26

Sample Processing: Gravity 2013 Waters Corporation 27

Sample Processing: Glass Vacuum Manifold Syringe Adapter Stop Cock flow control valvesto prevent drying out effect Male-male Adapter Vacuum Manifold 2013 Waters Corporation 28

Sample Processing: Large Liquid Volumes Vacuum Manifold 2013 Waters Corporation 29

Sample Processing: 96 96--well Plate Collection Plate Set-up for typical 96-well plates Under vacuum, a properly seated well-plate should move down and the tips will extend into the wells of the collection plate. 2013 Waters Corporation 30

Sample Processing: Positive Pressure 2013 Waters Corporation 31

Tip: Cartridge Stacking 2 of the SAME for More Capacity 2 Different Sorbents for 2 Dimensional Selectivity Multiple cartridges can be stacked for a mixed-mode approach 2013 Waters Corporation 32

Sample Processing: Positive Pressure Manifold 2013 Waters Corporation 33

SPE Terminology Sorbent -- Chromatographic packing material (Stationary Phase) Syringe Barrel Style Filter/Frit Retention Mechanisms: 1. Reversed Phase (RP) 2. Normal Phase (NP) 3. Ion-Exchange (IEX) Sorbent Filter/Frit 2013 Waters Corporation 35

Sorbents for SEP-PAKPAK Products Silica and Alumina Based 2013 Waters Corporation 36

Sorbents for SEP-PAKPAK Products Reversed-Phase 2013 Waters Corporation 37

Sorbents for SEP-PAKPAK Products Normal Phase 2013 Waters Corporation 38

Sorbents for SEP-PAKPAK Products Ion-Exchange (Silica Based) 2013 Waters Corporation 39

Sorbents for SEP-PAKPAK Products Specialty Phases 2013 Waters Corporation 40

Polymeric SPE Sorbent Chemistry: Oasis HLB Hydrophilic-Lipophilic Balanced Copolymer N O Hydrophilic monomer Lipophilic monomer Retention of Polars Reversed-phase Retention Water wettable Polar retention Stable across ph 1-14 No silanol interactions High recoveries for acids, bases and neutrals 2013 Waters Corporation 41

Polymeric SPE Sorbent Chemistries: Oasis Mixed-mode Ion Exchangers 2013 Waters Corporation 42

Polymeric SPE Sorbent Chemistries: Oasis Mixed-mode Ion Exchangers Strong Cation RP Weak Cation RP Reversed Phase RP Strong Anion RP Weak Anion RP 2013 Waters Corporation 43

Agenda The Importance of Solid Phase Extraction How SPE Works Device Design and Tips for Processing Samples Chromatographic Sorbent Choices Typical SPE Protocol and Strategies Was My SPE Method Successful? Appendix 2013 Waters Corporation 44

SPE Strategies Typical SPE Protocol and Strategies 1) Pass Through 2) Clean-up (retention of analytes of interest) 3) Fractionation 4) Trace Concentration 2013 Waters Corporation 45

SPE Protocol (Steps) Conditioning and Equilibration Steps 2013 Waters Corporation 46

SPE Protocol Conditioning and Equilibration Steps 2013 Waters Corporation 47

Loading of Sample Successful Loading Proper Wetting Good Capture 2013 Waters Corporation 48

SPE Protocol Wash Step Wash Solvent 2013 Waters Corporation 49

SPE Protocol Elute Step 2013 Waters Corporation 50

SPE Protocol Dry Down and Reconstitution These steps are performed for some applications where the mobile phase conditions, for the actual analytical separation, will require a change in the sample solvent. Typically, the strong elution solvent from the SPE Protocol is carefully evaporated away and the analytes are reconstituted with the mobile phase used in the instrument. 2013 Waters Corporation 51

Tip: Contact Time Use slower flow rate to increase contact time 2013 Waters Corporation 52

Tip: Solvent Volume Solvent volume is directly proportional to bed mass Sorbent per Well Maximum Mass Capacity Typical Sample Volume (Pre- Dilution) Typical Elution Volume 2 mg 0.03 to 0.05 mg 5 to 200 µl 50 µl 5 mg 0.15 to 1 mg 10 to 200 µl 150 µl 10 mg 0.35 to 2 mg 50 to 400 µl 250 µl 30 mg 1 to 5 mg 100 µl to 1 ml > 400 µl 60 mg 2 to 10 mg 200 µl to 2 ml > 800 µl 2013 Waters Corporation 53

SPE Strategies Typical SPE Protocol and Strategies 1) Pass Through 2) Clean-up (retention of analytes of interest) 3) Fractionation 4) Trace Concentration 2013 Waters Corporation 54

SPE Strategy #1: Pass Through Purification Pass Through Approach: Load: want k = high for interferences (max retention) want k = 0 for analytes -- no retention Advantages: clean-up is easy, fast, with little or no method development Disadvantages: no concentration of analytes; no solvent switch 2013 Waters Corporation 55

Pass Through SPE: Load and Collect Analyte is a Purple Compound k=0 Same Sample Solvent k=0 for analyte 2013 Waters Corporation 56

Ostro Plate Technology Pass Through Strategy Specifically Designed for Phospholipid Removal which, when present, results in Significant Ion-Suppression 2013 Waters Corporation 57

Ostro Plate Technology Pass Through Strategy Methodology Place Ostro plate onto collection plate Precipitate Proteins Held up by Filters Phospholipids Retained by SPE Sorbent Bed, Analytes Pass Through Pipette 50-200µL of plasma into wells Forcefully add 2% formic acid in acetonitrile, 3:1 solvent:plasma (methanol not recommended) Mix thoroughly by aspirating 3x with pipette Filter samples using vacuum manifold or positive pressure manifold Analyze samples It is possible to work with lower sample volumes (such as 25µL). When doing so you will need a higher organic solvent to sample ratio, such as 10:1 or 20:1. The well volume is 1.9 ml, however in order to mix by aspiration, the maximum volume is 1.4 ml. This translates to a maximum sample size of 350µL. 2013 Waters Corporation 58

Ostro Plate Technology Pass Through Strategy OSTRO Plate - Phospholipid Removal - No Method Development - Superior Performance Competitors 2013 Waters Corporation 59

Competitive Techniques: Phospholipids Remaining MRM of m/z 184-184 100 % LLE with 5% NH 4 OH in MTBE 1.90 2.29 2.21 2.10 184.4 > 184.4 (Lipid 184) 2.00e8 2.60 2.78 2.72 2.88 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 100 LLE with MTBE 2.27 2.56 2.62 2.682.80 184.4 > 184.4 (Lipid 184) 2.00e8 % 1.90 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 100 % Ostro 184.4 > 184.4 (Lipid 184) 2.00e8 0 1.90 1.77 1.96 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 100 % PPT 1.381.42 1.51 1.32 1.63 1.75 1.96 2.21 184.4 > 184.4 (Lipid 184) 2.00e8 2.84 0 Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 2013 Waters Corporation 60

SPE Strategy #2: Capture Retain Analytes of Interest Capture Approach: Load: want k = high for analyte (max retention capture) Wash: want k = 0 for interferences -- no retention want k = high for analyte so we don t lose to waste Elution: want k = 0 for analyte (no retention - strong solvent) to elute and collect ^Çî~åí~ÖÉëW=ÅäÉ~åJìéI=ÇÉë~äíáåÖI=ÅçåÅÉåíê~íáçåI=ëçäîÉåí=ëïáíÅÜáåÖ= Z=ÅäÉ~å=Éñíê~Åíë aáë~çî~åí~öéëw=êéèìáêéë=ãéíüçç=çéîéäçéãéåí 2013 Waters Corporation 61

Capture SPE Method (RP): Conditioning 2013 Waters Corporation 62

Capture SPE Method (RP): Equilibration 2013 Waters Corporation 63

Capture SPE Method (RP): Load Proper Wetting Conditioning and Equilibration Good Capture 2013 Waters Corporation 64

Capture SPE Method (RP): Wash Wash Solvent 2013 Waters Corporation 65

Capture SPE Method (RP): Elution 2013 Waters Corporation 66

Capture SPE Method (RP): Dry down and reconstitute These steps are performed for some applications where the mobile phase conditions, for the actual analytical separation, will require a change in the sample solvent. Typically, the strong elution solvent from the SPE Protocol is carefully evaporated away and the analytes are reconstituted with the mobile phase used in the instrument. 2013 Waters Corporation 67

Tip: Solvent Strength Optimizing Peak Shape Poor Peak Shape Sample Use the Solvent correct TOO solvent Strong strength to optimize peak shape Sample: 80% Organic Mobile Phase: 40% Result: Poor Peak Shapes 2013 Waters Corporation 68

Tip: Solvent Strength Same Column, Same Mobile Phase Weaker Sample Solvent Sample: 0% Organic Mobile Phase: 40% 2013 Waters Corporation 69

SPE Protocol: Reversed-Phase Condition/Equilibrate cartridge strongest solvent first (DCM, MTBE, ethyl acetate) intermediate solvent next (methanol) weak solvent last (water) Load Sample dissolve in or exchange to weak solvent (water or water/methanol) Wash Cartridge use strongest possible solvent without eluting analyte (methanol/water) Elute Cartridge with strong solvent* methanol, IPA,MTBE, DCM *Oasis HLB elute solvent should have at least 5 % methanol or IPA as polar modifier Oasis HLB SPE Protocol Prepare Sample Condition/Equilibrate 1 ml methanol, 1 ml water Load Sample Wash 1 ml 5% methanol/water Elute 2 ml methanol Evaporate, Reconstitute Conditions for 3 cc 60 mg cartridges 2013 Waters Corporation 70

SPE Protocol: Reversed-Phase vs. Mixed-Mode Mode IEX Oasis HLB SPE Protocol Oasis MCX SPE Protocol Prepare Sample Condition/Equilibrate methanol, water Load Sample Wash 5% methanol/water Elute methanol Evaporate, Reconstitute Prepare Sample Condition/Equilibrate Load Sample Wash: 2% Formic acid Wash 2: 100% MeOH Elute: 5% NH 4 OH in MeOH Reversed-Phase only Ion-Exchange AND Reversed-Phase 2013 Waters Corporation 71

Oasis 2x4 Method: Acids and Bases For Bases: pka 2-10 Use Oasis MCX For Strong Acids pka <1.0 Use Oasis WAX For Strong Bases pka >10 Use Oasis WCX For Acids pka 2-8 Use Oasis MAX Protocol 1 Prepare Sample Condition/Equilibrate Load Sample Wash: 2% Formic acid Elute 1: 100% MeOH Elute 2: 5% NH 4 OH in MeOH Neutrals Protocol 2 Prepare Sample Condition/Equilibrate Load Sample Wash: 5% NH 4 OH Elute 1: 100% MeOH Elute 2: 2% Formic Acid in MeOH Bases Strong Acids Strong Bases Acids 2013 Waters Corporation 72

Oasis 2x4 Method: Neutrals For Bases: pka 2-10 Use Oasis MCX For Strong Acids pka <1.0 Use Oasis WAX For Strong Bases pka >10 Use Oasis WCX For Acids pka 2-8 Use Oasis MAX Protocol 1 Protocol 2 Prepare Sample Prepare Sample Condition/Equilibrate Load Sample Condition/Equilibrate Load Sample Reversed- Phase Backbone Wash: 2% Formic acid Elute 1: 100% MeOH Elute 2: 5% NH 4 OH in MeOH Neutrals Wash: 5% NH 4 OH Elute 1: 100% MeOH Elute 2: 2% Formic Acid in MeOH Bases Strong Acids Strong Bases Acids 2013 Waters Corporation 73

SPE Strategy #3: Fractionation of Analytes Capture and Fractionation: Load: k = high for analytes (max retention) Elution 1: k = 0 for one analyte class only [slightly stronger solvent] (remaining k s = high) Elution 2: k = 0 for second analyte class only [slightly stronger solvent] (remaining k s = high for remaining) Elution 3: etc Advantages: separates classes of compounds, if they have different polarities and hydrophobicities 2013 Waters Corporation 74

Capture and Fractionation Dry Powder Fruit Flavored Mix add Water Grape Kool-Aid Sample Matrix Solution LOOKS Purple Polarity Elution Solvent Polar Compounds Polar Water Red Dye # 40 8%IPA Blue Dye # 1 35%IPA Non-Polar Oils Non-Polar 70%IPA {Reversed-Phase Conditions} 2013 Waters Corporation 75

Capture and Fractionation: Load 2013 Waters Corporation 76

Capture and Fractionation: Elute #1 2013 Waters Corporation 77

Capture and Fractionation: Elute #2 2013 Waters Corporation 78

Capture and Fractionation: Elute #3 2013 Waters Corporation 79

Capture and Fractionation: Elute #4 2013 Waters Corporation 80

Capture and Fractionate: Summary Polars 2013 Waters Corporation Red Dye Blue Dye Non- Polars 81

SPE Strategy #4: Trace Enrichment Trace Concentration/Enrichment: Load: k = high for analytes (max retention) and load large sample volume Elution: k = 0 for analytes Advantages: concentration of very low level analytes 2013 Waters Corporation 82

Trace Enrichment: Load 2013 Waters Corporation 83

Trace Enrichment: Continue Loading 2013 Waters Corporation 84

Trace Enrichment: Elution Elute the analtyes, which now have been concentrated, by using a stronger solvent to release them from the device (k = 0) Determine the concentration of THIS NEW sample -- and then calculate the ORIGINAL Sample Concentration by dividing by the volume of ORIGINAL sample processed by the SPE Device 2013 Waters Corporation 85

Trace Enrichment: Elution (Reverse Flow Direction) Reverse Flow Direction Adapter Tube Elute Band in Less Solvent More Concentrated 2013 Waters Corporation 86

Trace Enrichment: Elution (Reverse Cartridge) Reverse Flow Direction Adapter Tube Elute Band in Less Solvent More Concentrated Normal Direction Greatly Enhanced Sensitivity 2013 Waters Corporation 87

Determining % Recovery: Calculation Analytical Result = 9.5 ng/ml 2013 Waters Corporation 89

Determining % Matrix Effect: Calculation Analytical Result =10 ng/ml Analytical Result =10 ng/ml 2013 Waters Corporation 90

Importance of % Recovery and % Matrix Effect % Analyte Recovery in plasma 100 90 80 70 60 50 40 30 20 10 0 PPT PLR Plate LLE RP SPE Oxycodone Oxycodone d6 Both provide Good % Recovery PPT maybe satisfactory 2013 Waters Corporation 91

Importance of % Recovery and % Matrix Effect % Analyte Recovery in plasma 100 90 80 70 60 50 40 30 20 10 0 PPT PLR Plate LLE RP SPE Oxycodone Oxycodone d6 SPE prepared sample much cleaner % Matrix Effects in plasma 40 20 Ion-Suppression with PPT prepared sample 0-20 -40-60 Oxycodone Oxycodone d6-80 -100 PPT PLR Plate LLE RP SPE 2013 Waters Corporation 92

Conclusions SPE is a powerful sample preparation technique that utilizes extraction selectivity with the tangible benefits of: Clean-up: the simplification of a complex sample matrix Reduce ion suppression (or enhancements) for MS applications Fractionate to analyze distinct classes of compounds Trace concentration (enrichment) of samples with very low concentration levels Solvent switch 2013 Waters Corporation 93

Additional SPE Seminar Topics SPE Troubleshooting Techniques Tuesday March 19 th 1:30 2:30 PM http://www.waters.com/spepart2 Food Compound Specific Analysis of Food Food Safety Screening Environmental Environmental Sample Preparation BioAnalytical Small Molecule Fit for Purpose Bioanalytical Method Development 2x4 Troubleshooting and Optimization BioAnalytical Large Molecule Intro to peptide Bioanalysis 2013 Waters Corporation 94

Thank You! Questions? Landing Page http://www.waters.com/spepart1 30% Promotional Offer on SPE Products Free Beginners Guide to SPE Text Book with SPE Purchase PDF Slide Deck Full Webinar Recording of Today s Session Compilation of Literature, White Papers, Brochures General Questions email: mychemrep@waters.com 2013 Waters Corporation 95

Appendix 2013 Waters Corporation 97

Technology Capability Literature: Primers 2013 Waters Corporation 98

www.waters.com 2013 Waters Corporation 99

Search Applications 2013 Waters Corporation 100

Applications 65,993 Results 2013 Waters Corporation 101

Refine Search 2013 Waters Corporation 102

Specific Topic 2013 Waters Corporation 103

Amyloid Beta Peptides 2013 Waters Corporation 104

Application Note 2013 Waters Corporation 105

SPE Protocol Analytical UPLC Method 2013 Waters Corporation 106

Pesticides in Fruit Juice 2013 Waters Corporation 107

Journal Listings 2013 Waters Corporation 108

SPE Mosquito Heads Sign up 2013 Waters Corporation 109

2013 Waters Corporation 110

Download 2013 Waters Corporation 111

SPE Brands Ostro Plate Videos on-line 2013 Waters Corporation 112