Sample Preparation Using Solid Phase Extraction

Sample Preparation Using Solid Phase Extraction Dr. Shulamit Levin Based on: Yung-Fong (Henry) Cheng Waters Corporation 34 Maple Street Milford, MA 0757 Present at EAS'98 Workshop

Troubleshooting of Sample Preparation Methods Using Solid-Phase Extraction Based on: Yung-Fong (Henry) Cheng Waters Corporation 34 Maple Street Milford, MA 0757 Outline Importance of Sample Preparation Principle of Solid-Phase Extraction (SPE) Typical Problems in SPE detail steps of SPE examples Summary Present at EAS'98 Workshop Why Sample Preparation? Analyte in matrix Extraction Sample Preparation Typically the most time-consuming step Typically the most difficult Typically the least amount of effort spent developing a rugged sample preparation method Magical Method Analysis Dr. Shulamit Levin, Medtechnica

Wouldn't It Be Nice -- If We didn't have to Prepare Samples Before Injection into the Instrument Why Perform Sample Preparation? Remove interferences e.g. Analysis of drug and metabolite in plasma. Need to remove protein interferences Concentrate sample e.g. Pesticides in drinking water - Processing Steps needed to get Sample Ready Before Injecting into the Instrument ^ HPLC ^ GC ^ LC/MS ^ GC/MS ^ AA ^ Others Sample Prep Techniques Method Precipitation Basis for Selectivity Solubility Liquid-Liquid Extraction (LLE) Chemical Technique Liquid-Liquid Extraction Solid-Liquid Extraction (SPE) Dialysis / Ultrafiltration Electrophoresis Distillation/Evaporation Supercritical Fluid Extraction Partitioning in one of two liquid phases Adsorption/partitioning onto solid sorbent Molecular weight/size Charge Boiling point/vapor pressure Partitioning into supercritical fluid Where an Immiscible Solvent is Added to the Sample which then Separates into 2 Distinct Liquid Phases. Some Sample Analytes will go into the Bottom Phase (Aqueous), Some will Separate into the Top Phase, (Organic) Dr. Shulamit Levin, Medtechnica 2

Disadvantages of LLE Large solvent consumption Time/Labor intensive May require an evaporation step prior to analysis to remove excess solvent When one needs to assay for several analytes, it may be difficult to find proper solvent/conditions for all analytes, requiring more than one extraction per sample Problematic samples - emulsions Contamination issues Advantages of SPE vs. Other Extraction Techniques Cleaner extracts Easier to automate Higher recoveries for polar compounds Solid Phase Extraction (SPE) Solid Phase Extraction (SPE) - Chromatographic Particles - Packed-Bed Column Cartridges - st Commercialized In 978 - Well Established Technology - Many Thousands of Literature References - Formats and Configurations Cartridge Disk Coated Bed Fiber 96 Well Plate Empore Disk SPME Dr. Shulamit Levin, Medtechnica 3

Differences Between HPLC and SPE Comparison of Efficiency - HPLC vs. SPE HPLC SPE Particle size ~5 µm 40-80 µm Packed bed efficiency high low Extra-column volume low high Column length 5-30 cm ~ cm Number of plates (N) ~0,000 < 50 Bottom line: HPLC can separate similar compounds. SPE requires a significant selectivity difference between compounds for separation. Compounds not well resolved by HPLC cannot be separated by SPE with a similar retention mechanism. Normalized concentration 0.8 0.6 0.4 0.2 0 0.8 0.6 0.4 0.2 HPLC: higher efficiency 0 5 0 5 20 SPE: poor efficiency 0 0 5 0 5 20 Elution volume (ml) Sample Must be in Liquid State Driving Forces ^ Gravity ^ Pressure ^ Vacuum Waters Vacuum Manifolds 998 Dr. Shulamit Levin, Medtechnica 4

Manufacturer Waters Varian Baker Brand Name SEP-Pak OASIS BondElute BakerBond International Sorbent Isolute Technology 3 M Empore Supelco Supelclean + Many Others SPE Strategies Elute the product of interest, retain interferences want k 0 for analyte want k large for interferences Elute interferences, retain product want k 0 for interferences want k large for analyte * Concentrate product of interest ^ want k large for analyte / load large sample volume ^ elute concentrated analyte ^ enhanced sensitivity Methods Development Approach Most Common TYPES OF CHROMATOGRAPHY - Normal Phase ^ The "Original" Type - Used By Tswett ^ Non-Polar Mobile Phase ^ Polar Stationary Phase - Reversed-Phase ^ Polar Mobile Phase ^ Non-Polar Stationary Phase Most Common - Ion Exchange ^ Buffer/Ionic Mobile Phase ^ Cationic/Anionic Exchanger Stationary Phase Try Conditions - Evaluate for Capacity/ Breakthrough, Reproducibility, Robustness and Ruggedness Meets Goals? Yes Yes Determine Nature of Analytes, and Sample Matrix Similar to Existing Method in Lab? No Yes No Review SPE Bibliography, and Literature References for Exact or Similar Applications Any? No Determine Method Goals, and Strategy Call SPE Vendor Chromatography Mode Validate Method Develop Method Conditions Dr. Shulamit Levin, Medtechnica 5

Outline Importance of Sample Preparation Principle of Solid-Phase Extraction (SPE) Typical Problems in SPE detail steps of SPE examples Summary Common Problems in SPE Incomplete Removal of Interferences Low of Analyte(s) High Variability (RSDs) SPE Procedure ➀ ➁ ➂ ➃ Sample Prepare: Homogenize, suspend, centrifuge, etc. Load onto conditioned cartridge Wash off weakly retained interferences with weak solvent Elute product with strong solvent Analyze: HPLC, GC, etc. Step - Sample Preparation ❶ ➁ ➂ ➃ Sample Prepare: Homogenize, suspend, centrifuge, etc. Load onto conditioned cartridge Wash off weakly retained interferences with weak solvent Elute product with strong solvent Analyze: HPLC, GC, etc. Dr. Shulamit Levin, Medtechnica 6

Step - Sample Preparation Typical problems Analytes adsorpted to test tube walls adsorpted to or inclusion in matrix solids bound to proteins in matrix Possible solutions use silanized or plastic test tubes homogenize more completely add acid to sample solution Sample Pretreatment: Effect of Acid on Compounds % No Acid No Acid Phosphoric Acid, 2% Concentration [µg/ml] Saline Sample Serum Sample Serum Sample Naproxen.0 96 4 89 Ibuprofen 0.0 94 9 87 H 3 CO Naproxen CH 3 OH O H 3 C CH 3 Ibuprofen CH 3 OH O Step 2 - Sample load ➀ ❷ ➂ ➃ Sample Prepare: Homogenize, suspend, centrifuge, etc. Load onto conditioned cartridge Wash off weakly retained interferences with weak solvent Elute product with strong solvent Analyze: HPLC, GC, etc. Step 2 - Sample Load Possible problems Solutions Improper conditioning of Condition cartridge as appropriate. Do not cartridge let dry, if silica based C8 Poor analyte retention Dilute with weaker solvent, use stronger sorbent, use larger cartridge Matrix variability Buffer sample to constant ph, ionic strength Volume overload Decrease load volume, use larger cartridge Mass overload Decrease load volume, use larger cartridge Dr. Shulamit Levin, Medtechnica 7

Incomplete Conditioning of Cartridges "Note: Do not dry SPE cartridge between initial methanol conditioning wash and completion of addition of sample and sample wash. Monitor elutions closely to ensure that cartridges do not dry." J. D. MacNeil, V. K. Martz, G. O. Korsrud, C. D. C. Salisbury, H. Oka, R. L. Epstein, C. J. Barnes, J. AOAC Intl., 79(2) (996), 405-47 Percent recovery 00 80 60 40 20 Incomplete Conditioning of Cartridges Effect on : C88 vs. Oasis HLB Cartridges C8 (cc/00mg) Procainamide Acetaminophen 00 80 60 40 20 HLB (cc/30mg) 0 0 4 8 0 0 5 0 Drying Time Drying Time (minutes) (minutes) Ranitidine Propranolol * No Impact of Sorbent Drying * No Silanol Interaction * No Breakthrough of Polar Analytes Doxepin Effect of the Sample ph on Compounds Concentration [µg/ml] Load at ph 7 Load at ph <2 Sample Loading Salicylic Acid in Saline pka 2.97, 3.4 0 62.5 0 COOH OH Salicylic Acid Normalized concentration 0.8 0.6 0.4 0.2 k=0 k=20 k=30 0 0 0 20 30 40 50 Load volume (ml) higher k, less breakthrough (continuous loading) Assume: N = 40 plates V 0 = ml Dr. Shulamit Levin, Medtechnica 8

Step 3 - Wash ➀ ➁ ❸ ➃ Sample Prepare: Homogenize, suspend, centrifuge, etc. Load onto conditioned cartridge Wash off weakly retained interferences with weak solvent Elute product with strong solvent Possible Problems Poor analyte retention Matrix variability Volume overload Mass overload Step 3 - Wash Solutions Use stronger sorbent, use larger cartridge Buffer sample to constant ph, ionic strength Decrease load volume, use larger cartridge Decrease load volume, use larger cartridge Analyze: HPLC, GC, etc. Effect of Incomplete Wash 96 inj. 0.0 au Interferences USP Tailing Factor:.67 st wash: 40% MeOH, 2% NH4OH Washing Procedure: Effect of Wash Solvent on Compounds Concentration [µg/ml] 5% Methanol in Water Water inj. 2. 00 3. 00 Minutes 4. 00 USP Tailing Factor:.07 5.00 6.00 st wash: 40% MeOH, 2% NH4OH Theobromine 0.5 87 99 Paraxanthine 0.5 67 92 Theophylline 0.5 75 06 0.00 Cheng 0.0 au 2 4 6 minutes 2nd wash: 5% MeOH, 2% HAc. Caffeine 0.5 92 05 Dr. Shulamit Levin, Medtechnica 9

Dr. Shulamit Levin, Medtechnica 0 Step 4 - Elute Step 4 - Elution ➀ ➁ ➂ ❹ Sample Prepare: Homogenize, suspend, centrifuge, etc. Load onto conditioned cartridge Wash off weakly retained interferences with weak solvent Elute product with strong solvent Analyze: HPLC, GC, etc. Normalized concentration k=0 k= k=2 0.9 0.8 0.7 0.6 0.5 0.4 Assume: 0.3 N = 40 0.2 V 0 = ml 0. 0 0 2 3 4 5 6 Elution volume (ml) higher retention larger elution volume Effect of Elution Solvent on and Reproducibility Compound Testosterone benzoate Concentration [µg/ml] Methanol RSD Methylene Methanol Chloride: 50:50 RSD First milliliter of elution solvent 2.0 92 5. 02 0.49 Second milliliter of elution solvent 6.6 3.3 <0.50 O CH 3 CH 3 O O C Testosterone Benzoate Evaporation and Reconstitution Advantages Increase Assay Sensitivity Increase sample concentration Inject larger sample volume Improve HPLC Peak Shape Dissolve in mobile phase or weaker solvent Disadvantages Loss of more volatile analytes Poor solubility

0.006 0.005 0.004 AU 0.003 0.002 0.00 0.000 HPLC Analysis: Effect of Sample Solvent Minocycline Tetracycline Sample in MeOH Demeclocycline Compounds Effect of Evaporation on Sample Concentration [µg/ml] Evaporation to Dryness RSD Evaporation to 00 µl RSD 0.0 20.0 30.0 Minutes Benzoic Acid 5.0 62.8 9. 87.6 3.0 0.006 0.005 0.004 AU 0.003 0.002 0.00 0.000 Minocycline Sample in HPLC Mobile Phase (0.% TFA, 4%ACN and 5%MeOH in Water) Tetracycline Demeclocycline Salicylic Acid 5.0 93.6 5. 9.3 5.0 COOH COOH OH 0.0 20.0 30.0 Minutes Benzoic Acid Salicylic Acid Eliminating the Evaporation and Reconstitution Step: Effect of Sample Solvent Woods, Cheng 0.04 AU 0.04 AU 2 Sample in Water 5 0 5 Min. Sample in 80% MeOH, 2% HAc 2 5 µg/ml 3 4 µg/ml 0 µg/ml 5 0 5 Min. 3 Sample Identification.EDDP 2.Diphenhydramine(IS) 3. Methadone Column: SymmetryShield RP8, 3.5 µm, 3.9 x 50 mm Guard Column: Sentry Guard Column SymmetryShield RP8, 5µm Temperature: 30 C Mobile Phase: 0.% TFA:Methanol (60:40) Detection: UV at 20 nm Flow Rate: ml/min Inj. Volume: 30 µl Extraction on Oasis HLB, 96-well, 0 mg/well 2-D SPE Method Strategy of Signal-to-Noise (S/N) Enrichment Enrichment Comparison of S/N for Dilution (:3 with water) of Urine Sample Solution after SPE Extraction :3 Dilution 25 ul injection 0.02 S/N=38 S/N=42 S/N=37 AU S/N=88 5 0 5 Min. S/N=99 :3 Dilution 50 ul injection S/N=89 5 0 5 Min. S/N=45 S/N=64 S/N=47 :3 Dilution 00 ul injection 5 0 5 Min. 0.02 AU 0.02 AU At this dilution (:3 with water); achieve - better peak shapes - higher S/N Extraction on Oasis HLB, 96-well, 0 mg/well 2-D SPE Method Dr. Shulamit Levin, Medtechnica

Dr. Shulamit Levin, Medtechnica 2 Impact On Today's Analytical Chemist Faster Method Development More Sensitive Methods Shorter Processing Times Reduced Cost Per Analysis Generic Reversed-Phase, -D, SPE Method (Oasis HLB Sorbent) Prepare Sample Solution Condition/Equilibrate ml methanol/ ml water Load ml spiked sample solution Wash ml 5% methanol in water Elute ml methanol Evaporate and Reconstitute A One-Dimensional (-D) Method- changing only the percent organic 2 3 0% 5% 00% % Organic 2 3 Load no organic to retain analytes Wash 5% MeOH to remove proteins in matrix Elute high organic to elute the analytes Results: Tetracyclines Compound Concentration % % RSD Minocycline 2.5 µg/ml 94.8.4 Tetracycline 2.5 µg/ml 04 0.55 Comparison: Tetracyclines 0.020 0.06 0.02 AU 0.008 0.004 0.000 2 3 sample blank (CH H N(CH 3 ) 3 ) 2 N 2 H OH CONH O 2 HO O HO O H Minocycline HO CH H N(CH 3 ) 2 3 OH CONH O 2 HO O HO O H Tetracycline Cl HO H N(CH 3 ) 2 H OH Compound Minocycline Conc. [µg/ml] Oasis HLB Cartridge RSD n=6 C8 Cartridge RSD n=6 2.5 94.8.40 40.7 0.82 Tetracycline 2.5 04 0.55 67.4 0.44 0.0 20.0 30.0 Minutes O HO O HO O H CONH 2 Demeclocycline (IS) Cheng et. al. Chromatographia 997, 44 (3/4), p 87

Dr. Shulamit Levin, Medtechnica 3 Results of -D SPE Method 00 80 60 % 40 20 0 Acids Neutrals Bases Ibuprofen (2.5 µg) Naproxen (2 µg) Salicylic Acid (5 µg) Sulfadiazine (0 µg) Sulfamerazine (0 µg) Acetaminophen (0.5 µg) Theobromine (0.5 µg) Paraxanthine (0.5 µg) Theophylline (0.5 µg) Caffeine (0.5 µg) Procainamide (0.5 µg) Ranitidine (0.5 µg) Oxycodone ( µg) Propranolol (4 µg) Naltrexone ( µg) Salbutamol (2 µg) Doxepin (4 µg) Spiked Serum on cc 30 mg Oasis HLB Cartridges RSD < 5.0% Capparella, Cheng, Phillips Successful Tips Collect all Fractions (= Mass Balance) Load Wash(es) Elute 2nd Elute Summary Sample preparation is a necessary step prior to the analysis perception was/is time consuming and tedious Solid-Phase Extraction (SPE) provides cleaner extracts simpler protocol Successful Tips perform mass balance Ideal SPE Method one method, one good result for a wide range of compounds Solid-Phase Extraction (SPE) Technology Acknowledgments: Dr. Uwe Neue Dr. Edouard Bouvier Dr. Dorothy Phillips Dr. Patrick McDonald Dr. Tom Walter We gratefully acknowledge all the Trademarks used in this presentation, which are the property of their respective owners. Dr. Michael Young Joe Arsenault Pamela Iraneta Mark Capparella Bonnie Alden