Sample Preparation Using Solid Phase Extraction Medtechnica Dr. Shulamit Levin
Transcription
Sample Preparation Using Solid Phase Extraction Medtechnica Dr. Shulamit Levin
Sample Preparation Using Solid Phase Extraction Dr. Shulamit Levin Analytical Department Medtechnica Email: [email protected] [email protected] Tel: 03-9254040 Cell: 052-448632 Fax: 03-9249977 Home page: http://shulalevin.tripod.com http://www.forumsci.co.il/HPLC Based Based on: on: Yung-Fong Yung-Fong (Henry) (Henry) Cheng Cheng Waters Corporation Waters Corporation 34 34 Maple Maple Street Street Milford, MA Milford, MA 01757 01757 Present Present at at EAS'98 EAS'98 Workshop Workshop Outline Troubleshooting of Sample Preparation Methods Using Solid-Phase Extraction Based Based on: on: Yung-Fong Yung-Fong (Henry) (Henry) Cheng Cheng Waters Waters Corporation Corporation 34 34 Maple Maple Street Street Milford, Milford, MA MA 01757 01757 Importance of Sample Preparation Principle of Solid-Phase Extraction (SPE) Typical Problems in SPE detail steps of SPE examples Summary Present Present at at EAS'98 EAS'98 Workshop Workshop ©Waters 1998 Sample Preparation Why Sample Preparation? • Typically the most time-consuming step Analyte in matrix • Typically the most difficult • Typically the least amount of effort spent developing a rugged sample preparation method Extraction Magical Method Analysis ©Waters 1998 ©Waters 1998 Dr. Shulamit Levin, Medtechnica 1 Wouldn't It Be Nice -- Why Perform Sample Preparation? If We didn't have to Prepare Samples Remove interferences e.g. Analysis of drug and metabolite in plasma. Need to remove protein interferences Before Injection into the Instrument 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 ©Waters 1998 ©Waters 1998 Sample Prep Techniques Method Liquid-Liquid Extraction (LLE) Basis for Selectivity Chemical Technique • Precipitation Solubility • Liquid-Liquid Extraction Partitioning in one of two liquid phases Adsorption/partitioning onto solid sorbent Molecular weight/size Charge Boiling point/vapor pressure • Solid-Liquid Extraction (SPE) • Dialysis / Ultrafiltration • Electrophoresis • Distillation/Evaporation • Supercritical Fluid Extraction Partitioning into supercritical fluid ©Waters 1998 Dr. Shulamit Levin, Medtechnica 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) 2 Disadvantages of LLE Advantages of SPE vs. Other Extraction Techniques 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 Cleaner extracts Easier to automate Higher recoveries for polar compounds ©Waters 1998 ©Waters 1998 Solid Phase Extraction (SPE) - Formats and Configurations Solid Phase Extraction (SPE) - Chromatographic Particles - Packed-Bed Column Cartridges - 1st Commercialized In 1978 - Well Established Technology - Many Thousands of Literature References Cartridge Bed Bed Disk Disk Coated Coated Fiber 96 Well Plate Empore™ Disk SPME ©Waters 1998 ©Waters 1998 Dr. Shulamit Levin, Medtechnica 3 Differences Between HPLC and SPE Solid Phase Extraction (SPE) Technology Comparison of Efficiency - HPLC vs. SPE HPLC SPE ~5 µm high low 5-30 cm ~10,000 40-80 µm low high ~1 cm < 50 1 Normalized concentration Particle size Packed bed efficiency Extra-column volume Column length Number of plates (N) 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. HPLC: higher efficiency 0.8 0.6 0.4 0.2 0 0 5 10 15 20 SPE: poor efficiency 1 0.8 0.6 0.4 0.2 0 0 ©Waters 1998 5 10 Elution volume (mL) 15 20 ©Waters 1998 Solid Phase Extraction (SPE) Technology Solid Phase Extraction (SPE) Technology Sample Must be in Liquid State Driving Forces ^ Gravity ^ Pressure Vacuum Manifolds ^ Vacuum ©Waters 1998 ©Waters 1998 Dr. Shulamit Levin, Medtechnica 4 Solid Phase Extraction (SPE) Technology Manufacturer Brand Name Waters Varian SEP-Pak OASIS BondElute Baker BakerBond International Sorbent Technology 3M Isolute Supelco Supelclean Solid Phase Extraction (SPE) Technology 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 Empore * Concentrate product of interest + Many Others ©Waters 1998 ^ want k large for analyte / load large sample volume ^ elute concentrated analyte ^ enhanced sensitivity ©Waters 1998 Solid Phase Extraction (SPE) Technology Methods Development Approach Most Common TYPES OF CHROMATOGRAPHY - Normal Determine Nature of Analytes, and Sample Matrix Similar to Existing Method in Lab? Phase ^ The "Original" Type - Used By Tswett ^ Non-Polar Mobile Phase ^ Polar Stationary Phase - Reversed-Phase Most Common ^ Polar Mobile Phase ^ Non-Polar Stationary Phase - Ion Exchange ^ Buffer/Ionic Mobile Phase ^ Cationic/Anionic Exchanger Stationary Phase Try Conditions - Evaluate for Capacity/ Breakthrough, Recovery Reproducibility, Robustness and Ruggedness Meets Goals? Yes ©Waters 1998 Validate Method Dr. Shulamit Levin, Medtechnica No Yes No Yes Review SPE Bibliography, and Literature References for Exact or Similar Applications Any? No Determine Method Goals, and Strategy Call SPE Vendor Chromatography Mode Develop Method Conditions 5 Outline Solid Phase Extraction (SPE) Technology 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 Recovery of Analyte(s) • High Variability (RSDs) ©Waters 1998 ©Waters 1998 Solid Phase Extraction (SPE) Technology SPE Procedure Solid Phase Extraction (SPE) Technology Step 1 - Sample Preparation Sample ➀ Prepare: Homogenize, suspend, centrifuge, etc. ➁ Load onto conditioned cartridge ❶ Prepare: Homogenize, suspend, centrifuge, etc. ➂ Wash off weakly retained interferences with weak solvent ➁ Load onto conditioned cartridge ➃ Elute product with strong solvent ➂ Wash off weakly retained interferences with weak solvent ➃ Elute product with strong solvent Sample Analyze: HPLC, GC, etc. ©Waters 1998 Analyze: HPLC, GC, etc. ©Waters 1998 Dr. Shulamit Levin, Medtechnica 6 Sample Pretreatment: Effect of Acid on Recovery % Recovery Solid Phase Extraction (SPE) Technology Step 1 - 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 Phosphoric Acid, 2% No Acid No Acid Compounds Concentration [µg/mL] Saline Sample Serum Sample Serum Sample Naproxen 1.0 96 4 89 Ibuprofen 10.0 94 19 87 CH 3 OH H3CO O Naproxen CH3 CH3 H3C OH O Ibuprofen ©Waters 1998 Solid Phase Extraction (SPE) Technology Step 2 - Sample load Step 2 - Sample Load Sample ➀ Prepare: Homogenize, suspend, centrifuge, etc. ❷ Load onto conditioned cartridge ➂ Wash off weakly retained interferences with weak solvent ➃ Elute product with strong solvent Solid Phase Extraction (SPE) Technology Possible problems Solutions •Improper conditioning of cartridge •Condition cartridge as appropriate. Do not let dry, if silica based C18 •Poor analyte retention •Dilute with weaker solvent, use stronger sorbent, use larger cartridge Analyze: HPLC, GC, etc. ©Waters 1998 •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 ©Waters 1998 Dr. Shulamit Levin, Medtechnica 7 Incomplete Conditioning of Cartridges Effect on Recovery: Incomplete Conditioning of Cartridges 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) (1996), 405-417 C1818 vs. Oasis® HLB Cartridges C18 (1cc/100mg) 100 100 80 80 60 60 40 40 20 20 Percent recovery "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." 0 0 4 Drying Time (minutes) 8 0 HLB (1cc/30mg) * No Impact of Sorbent Drying * No Silanol Interaction * No Breakthrough of Polar Analytes 0 5 Drying Time (minutes) Procainamide Ranitidine Acetaminophen Propranolol 10 Doxepin ©Waters 1998 Effect of the Sample pH on Recovery Load at pH 7 Compounds Solid Phase Extraction (SPE) Technology Load at pH <2 Concentration [µg/mL] Recovery (%) Recovery (%) 10 62.5 101 Sample Loading COOH OH Salicylic Acid Normalized concentration 1 Salicylic Acid in Saline pKa 2.97, 13.4 k=10 0.8 k=20 k=30 0.6 higher k, less breakthrough (continuous loading) 0.4 Assume: N = 40 plates V0 = 1 mL 0.2 0 0 10 20 30 40 50 Load volume (mL) ©Waters 1998 Dr. Shulamit Levin, Medtechnica 8 Solid Phase Extraction (SPE) Technology Solid Phase Extraction (SPE) Technology Step 3 - Wash Sample Step 3 - Wash ➀ Prepare: Homogenize, suspend, centrifuge, etc. ➁ Load onto conditioned cartridge ❸ Wash off weakly retained interferences with weak solvent ➃ Elute product with strong solvent Possible Problems Solutions •Poor analyte retention •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 Analyze: HPLC, GC, etc. ©Waters 1998 ©Waters 1998 Effect of Incomplete Wash Washing Procedure: Effect of Wash Solvent on Recovery Interferences 96 inj. USP Tailing Factor: 1st wash: 40% MeOH, 2% NH4OH 1.67 111 inj. 6.00 5.00 4.00 3.00 Minutes 2.00 0.01 au 1st wash: 40% MeOH, 2% NH4OH USP Tailing Factor: 1.07 0.00 0.01 au Cheng 2 minutes 4 6 5% Methanol in Water Water Concentration [µg/mL] Recovery (%) Recovery (%) Theobromine 0.5 87 99 Paraxanthine 0.5 67 92 Theophylline 0.5 75 106 Caffeine 0.5 92 105 Compounds 2nd wash: 5% MeOH, 2% HAc. ©Waters 1998 Dr. Shulamit Levin, Medtechnica 9 Solid Phase Extraction (SPE) Technology Step 4 - Elute Solid Phase Extraction (SPE) Technology Step 4 - Elution Sample ➀ k=0 1 Prepare: Homogenize, suspend, centrifuge, etc. k=1 k=2 ➁ Load onto conditioned cartridge ➂ Wash off weakly retained interferences with weak solvent ❹ Elute product with strong solvent Normalized concentration 0.9 higher retention larger elution volume 0.8 0.7 0.6 0.5 0.4 Assume: N = 40 V0 = 1 mL 0.3 0.2 0.1 0 0 1 2 3 4 5 6 Elution volume (mL) Analyze: HPLC, GC, etc. ©Waters 1998 ©Waters 1998 Effect of Elution Solvent on Recovery and Reproducibility Methanol Compound Testosterone benzoate Evaporation and Reconstitution Methylene Chloride: Methanol 50:50 Concentration [µg/mL] Recovery (%) RSD (%) Recovery (%) RSD (%) 2.0 92 5.1 102 0.49 6.6 13.3 <0.50 First milliliter of elution solvent Second milliliter of elution solvent O Increase Assay Sensitivity Increase sample concentration Inject larger sample volume Improve HPLC Peak Shape Dissolve in mobile phase or weaker solvent Disadvantages O CH 3 Advantages Loss of more volatile analytes Poor solubility C CH3 Testosterone Benzoate O Dr. Shulamit Levin, Medtechnica ©Waters 1998 10 HPLC Analysis: Effect of Sample Solvent 0.006 Effect of Evaporation on Sample Recovery Sample in MeOH 0.005 Evaporation to Dryness 0.004 Compounds Minocycline AU 0.003 Demeclocycline Tetracycline 0.002 Concentration [µg/mL] 0.001 0.000 10.0 20.0 Minutes Evaporation to 100 µL Recovery (%) RSD (%) Recovery (%) RSD (%) 30.0 Benzoic Acid 5.0 62.8 9.1 87.6 3.0 Salicylic Acid 5.0 93.6 5.1 91.3 5.0 Sample in HPLC Mobile Phase 0.006 0.005 (0.1% TFA, 4%ACN and 5%MeOH in Water) Minocycline 0.004 Tetracycline AU 0.003 Demeclocycline 0.002 COOH 0.001 COOH OH 0.000 10.0 Minutes 20.0 30.0 Benzoic Acid Eliminating the Evaporation and Reconstitution Step: Effect of Sample Solvent 1 2 0.04 AU Sample in Water 3 Sample Identification 1.EDDP 2.Diphenhydramine(IS) 3. Methadone Strategy of Signal-to-Noise (S/N) Enrichment Comparison of S/N for Dilution (1:3 with water) of Urine Sample Solution after SPE Extraction 1:3 Dilution 25 uL injection Column: 5 10 15 Min. Sample in 80% MeOH, 2% HAc 0.04 AU 1 4 µg/ml Woods, Cheng Salicylic Acid 5 2 3 5 µg/ml 10 µg/ml 10 SymmetryShield™ RP18, 3.5 µm, 3.9 x 150 mm Guard Column: Sentry™ Guard Column SymmetryShield RP18, 5µm Temperature: 30°C Mobile Phase: 0.1% TFA:Methanol (60:40) Detection: UV at 210 nm Flow Rate: 1 mL/min Inj. Volume: 30 µL Extraction on Oasis® HLB, 96-well, 10 mg/well S/N=38 S/N=37 5 10 0.02 AU 15 Min. 1:3 Dilution S/N=88 S/N=99 50 uL injection 5 S/N=145 2-D SPE Method 10 S/N=164 S/N=89 0.02 AU 15 Min. S/N=147 1:3 Dilution 100 uL injection 15 Min. 0.02 AU At this dilution (1:3 with water); achieve - better peak shapes - higher S/N Extraction on Oasis® HLB, 96-well, 10 mg/well 2-D SPE Method ©Waters 1998 5 Dr. Shulamit Levin, Medtechnica S/N=42 10 15 Min. 11 Solid Phase Extraction (SPE) Technology Generic Reversed-Phase, 1-D, SPE Method (Oasis® HLB Sorbent) A One-Dimensional (1-D) Method- changing only the percent organic Prepare Sample Solution Impact On Today's Analytical Chemist Condition/Equilibrate 1 mL methanol/1 mL water 1 0% Load 1 mL spiked sample solution Faster Method Development More Sensitive Methods Shorter Processing Times Reduced Cost Per Analysis 3 2 5% % Organic Wash 1 mL 5% methanol in water 1 100% Load no organic to retain analytes Elute 1 mL methanol 2 Wash 3 Elute 5% MeOH to remove proteins in matrix high organic to elute the analytes Evaporate and Reconstitute ©Waters 1998 ©Waters 1998 Results: Tetracyclines Compound Concentration % Recovery % RSD Minocycline 2.5 µg/mL 94.8 1.4 Tetracycline 2.5 µg/mL 104 0.55 (CH 3 )2N 1 H H Comparison: Tetracyclines N(CH 3)2 OH Oasis® HLB Cartridge 0.020 2 3 0.016 HO O CONH O HO O H Minocycline CH 3 H HO 0.012 OH 0.008 sample 0.004 blank 0.000 10.0 Minutes 20.0 Tetracycline Cl HO H O H HO Compound Conc. [µg/mL] O O H CONH 2 N(CH 3)2 C18 Cartridge Recovery (%) RSD (%) n=6 Recovery (%) RSD (%) n=6 Minocycline 2.5 94.8 1.40 40.7 0.82 Tetracycline 104 0.55 67.4 0.44 N(CH 3 )2 AU HO 2 2.5 OH 30.0 HO O HO O O H CONH 2 Demeclocycline©Waters (IS)1998 Cheng et. al. Chromatographia 1997, 44 (3/4), p 187 ©Waters 1998 Dr. Shulamit Levin, Medtechnica 12 Results of 1-D SPE Method Acids Neutrals Bases Solid Phase Extraction (SPE) Technology 100 Successful Tips 80 Collect all Fractions (= Mass Balance) Load Wash(es) Elute 2nd Elute Doxepin (4 µg) Naltrexone (1 µg) Salbutamol (2 µg) Oxycodone (1 µg) Propranolol (4 µg) Ranitidine (0.5 µg) Caffeine (0.5 µg) Procainamide (0.5 µg) Theophylline (0.5 µg) Theobromine (0.5 µg) Paraxanthine (0.5 µg) Sulfadiazine (10 µg) Sulfamerazine (10 µg) Acetaminophen (0.5 µg) 0 Naproxen (2 µg) 20 Salicylic Acid (5 µg) 40 Ibuprofen (2.5 µg) % Recovery 60 Spiked Serum on 1 cc 30 mg Oasis™ HLB Cartridges RSD < 5.0% Capparella, Cheng, Phillips ©Waters 1998 ©Waters 1998 Solid Phase Extraction (SPE) Technology 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 ©Waters 1998 Solid-Phase Extraction (SPE) Technology Acknowledgments: Dr. Uwe Neue Dr. Edouard Bouvier Dr. Dorothy Phillips Dr. Patrick McDonald Dr. Tom Walter Dr. Michael Young Joe Arsenault Pamela Iraneta Mark Capparella Bonnie Alden We gratefully acknowledge all the Trademarks used in this presentation, which are the property of their respective owners. ©Waters 1998 Dr. Shulamit Levin, Medtechnica 13