The Secrets of Solid Phase Extraction (SPE) for Sample Preparation Agilent AccuBONDII, AccuBONDII ENV and EVIDEXII SPE What Is Solid-Phase Extraction (SPE)? • Sample preparation technique with principles similar to those of HPLC for selective adsorption of analytes or interferences from complex matrices • Used for sample cleanup and analyte concentration preceding LC, GC, ion chromatography and other techniques • Cost-effective alternative to/replacement for liquid-liquid extraction (productivity, solvent, waste) CI0126C 2 Typical Application: Naproxen from Serum H C 3 mAU 50 Proteins and Naproxen 40 CH 3 O A HO O SPE Method C18 Cartridge •PRETREAT: LC Instrument: -2.0 mL 1% acetic acid in Agilent 1100 MeOH Column: Zorbax SB-C8 150 x 4.6mm,-1.0 mL Deionized H2O •LOAD: 3.5µm -Sample (see sample Eluent: preparation) 50% MeCN in 25mM ammonium acetate •WASH: -1.0 mL 4% IPA in min UV: 240 nm 100mM formic acid Flow: 1.0 mL/min. -0.5 mL Deionized H2O Ambient Temp. •ELUTE: Inj. Vol.: 10 µL -1.0 mL 50% ACN in 40mM ammonium acetate Sample Prep Analyte Recoveries -500 µL Serum -500 µL 4% IPA in •Naproxen (2µg): 100mM formic acid HPLC Method Before 30 20 Nitrobenzene (Internal Standard) 10 0 0 1 mAU 50 2 3 4 5 6 7 B Proteins 40 30 Naproxen After 20 Nitrobenzen e 10 0 0 1 2 3 4 5 6 7 min X = 102.2%, n = 3 •Naproxen (10µg): X = 99.25%, n = 3 CI0126C 3 SPE Replaces Time Consuming and Laborious Liquid-Liquid Extraction Manual labor– vigorous shaking Typical Separatory Funnels Time—Waiting for layers to separate CI0126C 4 Advantages of SPE vs. Liquid-Liquid Extraction • Improved throughput (parallel vs. serial processing) • Decreased organic solvent usage and waste generation • Higher and more reproducible recoveries • Cleaner extracts (contamination, solvent impurities) • No emulsions • Tunable selectivity ( SPE phase choices, solvent mixtures) • Readily automated CI0126C 5 Typical Applications of SPE • Sample Cleanup – Combinatorial reaction cleanup before LC-MS or LC – Pharmacokinetic studies, dissolution testing – Isolate analytes from complex matrices – urine, plasma – Remove “column killers” or major interferences – Eliminate late-eluters to allow isocratic analysis • Trace Enrichment – Environmental analysis – Pharmaceutical and Agrochemical applications • Desalting • Solvent Exchange • Sample Preservation and Storage CI0126C 6 Application and Importance of SPE is Growing …Rapidly • Applied in cartridge format for 20+ years —Critically important for sample cleanup and trace enrichment (esp. environmental and pharmaceutical labs) • SPE usage is accelerating —Combinatorial synthesis and high-throughput LC-MS analysis —Overall LC-MS usage increases and greater instrument uptime —Smaller samples requiring smaller bed masses (tubes and plates) —Minimization of organic solvents and waste —Ease of automation (cost, reproducibility, throughput) • Liquid handling and SPE workstations • 96-well plate autosamplers CI0126C 7 Typical SPE Formats SPE Cartridge Schematic Diagram of a 96-Well SPE Schematic Diagram of a Extraction Plate System* 96-Well Plate Extraction System (LC-GC, S9, May, 1998) Single Extraction Disk Manifold System R. E. Majors, LC-GC, 15(4), 318 (1997) * 3M Corp. CI0126C 8 SPE Modes—”Digital Chromatography” Analyte Adsorption • Analyte(s) retained (k >> 1) • Matrix unretained and/or strongly retained (k ~ 0) (k >> 1) • Preconcentration factor • Cleaner extracts • Load at 1-3 drops/sec (recovery ∝ 1/flow) • Capacity issues may be more important CI0126C 9 Matrix Adsorption • Analyte(s) unretained • Matrix retained • • • • (k ~ 0) (k >> 1) No preconcentration advantage Eluates may not be as clean Sample loading often gravity fed Used less often than analyte adsorption Fundamental Steps for SPE Adsorption Mode Prewash* Remove contaminants that could elute with analyte Precondition Load Wash Elute Prepare cartridge to accept sample Load sample and rinse reservoir(s) Wash with solvent that won’t elute analyte Elute analyte in smallest volume possible Weakly retained matrix compds elute Analyte and other matrix compds retained Elute analyte leaving highly retained compds 1 2 aryE s est SP c neilen t o * N r Ag fo If elution solvent will be stronger than precond. solvent CI0126C 10 1. MeOH or ACN 2. Weak solvent (water, buffer) How is SPE performed in the laboratory? CI0126C 11 General SPE Method Development Strategy SPE Adsorption Mode: Background • Research the Problem —Previous SPE and analysis conditions for the analyte and matrix? • Characterize the Analyte —Structure, pKa, polarity, functional groups —Solvent solubility and stability • Any restrictions on final solvent and concentration (technique or instrument)? • Characterize the Sample Matrix —Possible interferences — similar functional groups, pKa, etc. —pH, ionic strength —Solvent solubility and stability —Qualitative and quantitative variability CI0126C 12 General SPE Method Development Strategy SPE Adsorption Mode: Experimental • Develop or apply effective HPLC or GC conditions to monitor progress — Assess recovery and eluate cleanliness • Select and test sorbents —Determine which sorbents provide maximum analyte retention —Determine which eluent solvents yield highest recoveries • Identify optimum wash solvent —Assess eluate cleanliness under conditions of maximum analyte retention —Determine strongest wash solvent that will not elute analyte • Test blank and fortified matrix —Assess eluate cleanliness and recovery using optimum wash and eluent solvents • Test real samples and fortified samples —Assess eluate cleanliness and recovery for different unfortified and fortified samples CI0126C 13 SPE Method Validation Variables to Consider • Validate SPE procedure —Sorbent (sorbent weight, different cartridges, different lots) —Preconditioning (strong solvent, weak solvent) —Loading solvent (% organic, pH, ionic strength, volume) —Wash solvent (% organic, pH, ionic strength, volume) • Eluent (volume, % organic …) —Flow rates (loading, wash, elution) —Linearity and range (different analyte concs. and matrix loadings) —Analyte stability (loading solvent, eluent) —Matrix stability (loading solvent) CI0126C 14 Design Characteristics for SPE Family • Provide a variety of popular chemistries to meet most application needs • Provide silica and PS-DVB base materials • Provide most popular sizes of cartridges and bulk material for “self-packers” • Pre-clean packing, tubes and frits – low organic extractables for high sensitivity work • Economical for routine applications • Excellent quality assurance program to ensure consistent results from batch-to-batch and year-to-year • Specialty phase for drugs of abuse testing CI0126C 15 SPE Products from Agilent AccuBONDII and EVIDEXII • Improved AccuBONDII SPE Products —Solid Phase Extraction Cartridges —Bulk SPE Adsorbents and Accessories —Method Development Kits —Applications • EVIDEXII SPE Specialty Cartridges —Drugs of Abuse —General Pharmaceutical Extractions —Applications CI0126C 16 AccuBONDII SPE Phases SPE Mode Reversed Phase Normal Phase (polar) Phases Available C2, C8, C18, Phenyl, ENV PS-DVB* Silica, -CN, Diol, Amino, Alumina (acidic, basic, and neutral), Florisil Strong Anion Exchange (SAX) Quaternary amine Strong Cation Exchange (SCX) Sulfonic acid * Newest phase CI0126C 17 AccuBONDII SPE Cartridges • Silica and new PS-DVB cartridges available • Wide variety of cartridge sizes —100, 200, 500 and 1000 mg —1, 3, and 6 mL tubes —96 well plates in final development —Tabless cartridges for automation (i.e. Gilson) • Excellent quality assurance program —Random testing of cartridges • surface characteristics • packing parameters —Performance certificate in each box • Low extractables —Pre-washed packings, frits, and tubes • Bulk packings available – 25 g bottles CI0126C 18 AccuBONDII SPE – Low Levels of Extractables Tube and Frit Programmed Temperature GC-FID Analysis Splitless Injection Waters’ Sep-Pak C18 Tube/Frit Only Total = 50 ppm 0 4 8 Internal Standard 12 16 min Agilent SPE AccuBONDII Pre-washed Tube/Frit Only Internal Standard Total = 5 ppm 0 CI0126C 19 4 8 12 16 min AccuBONDII SPE Method Development Kits (kits contain 10 cartridges each of 100mg/1-mL size) • SPE Method Development Kits – groups of phases that separate using the same mechanism —Kit A (Reversed Phase) • C2, CN, Phenyl, C8 and C18 —Kit B (Normal Phase) • Si, Amino, Diol and CN —Kit C (Ion Exchange) • SCX, SAX and Amino • Multiple kits provide a wide variety of options for determining the best mechanism and phase type for your analyte and matrix CI0126C 20 AccuBONDII SPE Application Notes • Application Notes for AccuBONDII —Environmental • Extraction of Chlorinated Pesticides in Water • Extraction of Polynuclear Aromatic Hydrocarbons in Water • Extraction of PCB’s in Water • Extraction of Triazines Using SPE Cartridges • SPE and GC/MS Analysis of Selected Phenols —Pharmaceutical • Anabolic Steroids in Urine and Serum • Benzodiazepines in Serum • Caffeine and Metabolites in Serum • Barbiturates in Serum —Available online: www.agilent.com/chem CI0126C 21 AccuBONDII Example Applications Triazines in Different Matrices Cl N • Characterize the Analyte • Structure, pKa, polarity, functional groups • • Solvent solubility and stability • Any restrictions on final solvent and concentration due to technique or instrument? Characterize the Sample Matrix • Solvent solubility and stability • pH, ionic strength • Possible interferences—similar functional groups, pKa, etc. • Qualitative and quantitative variability CI0126C 22 Triazines N NH NH R1 • • • • • • N R2 Three major species – simazine, atrazine and propazine – all structurally similar Mode of Action: Herbicides Practically insoluble in water Soil – large number of charged species-adjust pH to retain triazines and do ion-exchange Muscle tissue – large amounts of nonpolar lipids – retain these and elute triazines using C18 Corn oil – non-polar glycerides and fatty acids –weakly retained on diol while triazines are strongly retained SPE Methods for Triazines in Complex Matrices Soil Muscle Tissue Corn Oil CARTRIDGE SCX ODS Diol EXTRACTION Shaken in acetonitrile Homogenized in methanol None PRE-TREAT Acetic acid Methanol Methanol, hexane LOAD WASH ELUTE CI0126C 23 Diluted with acetic acid Acetic acid, acetonitrile, water, 0.1 M K2HPO4 Acetonitrile/K2HPO4 Diluted with water Diluted with hexane Water Hexane Methanol Methanol HPLC Analysis of Triazines Extracted from Different Matrices Triazines from Soil with SCX Triazines from Muscle Tissue with ODS Triazines from Corn Oil with Diol Column: C18, 4.6 x 150 mm, 5 mm Mobile Phase: 50% methanol:50% 0.01M K2HPO4 Flow Rate: 2 mL/min Detection: UV 254 nm Sample: Triazines 1. Simazine 2. Atrazine 3. Propazine CI0126C 24 AccuBONDII Example Applications Barbiturates in Serum O • Characterize the Analyte • Structure, pKa, polarity, functional groups • • Solvent solubility and stability • Any restrictions on final solvent and concentration due to technique or instrument? Characterize the Sample Matrix • Solvent solubility and stability • pH, ionic strength • Possible interferences—similar functional groups, pKa, etc. • Qualitative and quantitative variability CI0126C 25 Barbiturat es • R1 N O HN R2 R3 O Non-polar side groups make retention on C18 possible – but retention not strong Mid pH will eliminate charge and improve retention Soluble in water Final solvent easy to evaporate or compatible with HPLC • • • • • Serum has many components competing for retention Serum will vary from person to person Analysis of Barbiturates in Serum AccuBONDII SPE C18 Cartridge Solid Phase Extraction Method Load: CARTRIDGE: 5 mL Sample (see sample preparation above) AccuBONDII ODS (C18) 6 mL/500 mg (P/N 188-1356 30/box) Wash Sample Preparation: 2.0 mL 95% water:5% acetonitrile Add 1 mL 0.5M K2HPO4 to 4 mL of serum Elute: Precondition: 5 mL Acetone 5 mL Deionized Water CI0126C 26 3.0 mL Acetone Evaporate and Reconstitute: Evaporate Acetone in a nitrogen stream at <45°C Add 200 µL of acetonitrile:water (20/80) HPLC Analysis of Barbiturates Extracted from Human Serum Column: C18, 4.6 x 150 mm, 5 mm Mobile Phase: Time ACN Water 0 20 80 5 20 80 15 40 60 20 40 60 Flow Rate: 1 mL/min Detection: UV 235 nm Sample: Barbiturates 1. Barbital 2. Allobarbital 3. Aprobarbital 4. Phenobarbital 5. Butabarbital 6. Alphenal 7. Hexobarbital 8. Amobarbital 9. Mephobarbital 10. Secobarbital CI0126C 27 Recovery Data for Barbiturates in Serum Compound Barbital Allobarbital Aprobarbital Phenobarbital Butabarbital Butethal Butalbital Alphenal Hexobarbital Pentobarbital Amobarbital Mephobarbital Secobarbital RRT 1.00 1.99 2.47 2.87 3.06 3.29 3.45 3.46 4.09 4.12 4.20 4.36 4.59 Serum spiked at 1mg/mL n= 4 RRT = retention time relative to barbital CI0126C 28 Mean 70 82 92 78 89 88 88 87 92 102 93 93 95 Std. Dev. 11 11 9 8 7 6 14 8 6 5 3 5 8 New AccuBONDII ENV for Environmental Analytes • New PS-DVB SPE material for environmental applications — High surface area – 600 m2/g – provides good retention — Large particle size for high flow – 75 – 150 mm — High capacity for good retention – Capacity of caffeine 320 mg/g • Provides high recovery of phenols in water – wide range of polarities and solubilities — Higher recovery than with silica (phenol> 70%, other phenols>90%) — Optimized for the analysis of phenols — High sample delivery rate for short extraction time • Follow with GC or GC/MS analysis • Being evaluated for other environmental analytes - pesticides and PAHs CI0126C 29 SPE Procedure for Extraction of Phenols Solid Phase Extraction Method Load: CARTRIDGE: 1.0 L water sample at 20 – 25 mL/min (see sample preparation above) AccuBONDII ENV PS-DVB, 1000 mg*, 6 mL (P/N 188-3060, 30/box) Elute: *1000 mg is required for optimal recovery 9.0 mL Dichloromethane (dried with anhydrous sodium sulfate) Sample and Recovery Standard Preparation: 1L water Deuterated phenol, 2,4-dibromophenol, and 2,4,6-tribromophenol recovery standards added at 10 ppb level Sample and standards mixed and pH lowered to <2 with 5N HCl Evaporate and Reconstitute: Precondition: 9 – 12 mL Dichloromethane 9 – 12 mL Methanol 9 – 12 mL 0.05N HCl Do not allow to run dry at any time CI0126C 30 Evaporate Dichloromethane in nitrogen stream at room temperature and transferred to silanized amber vial Bring to 900 µL of Dichloromethane + 100 µL of a solution containing 2,5-dibromotolune and 2,2’,5,5’-tetrabromobiphenyl at 0.05 mg/mL in Dichloromethane as internal standards Publication Number of phenol application: 5988–5255EN Available on the Agilent web site: www.agilent.com/chem GC Analysis of Extractable Phenols CI0126C 31 Recoveries of Phenols on AccuBONDII ENV PD-DVB Compound Phenol 2-Chloro 3-Methyl 3- & 4-Methyl 2.4.-Dimethyl 2-Nitro 2,4-Dichloro 2,6-Dichloro 4-Chloro-3-methylphenol Recovery % RSD% 78% 98 99 98 99 100 99 99 101 100% 4% 2,3,4,5-Tetrachloro 97 3 2,3,5,6-tetrachloro 99 2 2,3,4,6-tetrachloro 98 2 2,4-Dinitro 106 5 2,4,6-Tribromo 97 2 2-Me-4.6-Dinitro 101 5 Pentachloro 98 4 Dinoseb 102 5 2-Cyclohexyl-4,6-dinitro 112 12 2,2’,5,5’- 105 5 3% 3 3 3 1 4 3 2 3 2,4-Dibromophenol 105 3 2,4,6-Trichlorophenol 97 3 2,4,5-Trichlorophenol 98 2 CI0126C 32 4-Nitrophenol Tetrabromobiphenyl Extraction and Separation of PAHs from Water II ODS AccuBOND Solid Phase Extraction Method CARTRIDGE: AccuBONDII , 500 mg, 6 mL (P/N 188-1356, 30/box) Sample Preparation: Cl Add 2 mL Isopropanol to 20 mL Water Sample Mix thoroughly Precondition: 5 mL Methylene Chloride 5 mL Methanol 5 mL Deionized Water Add ~1 mL Deionized Water to top of bed Load: Attach 24-mL sample reservoir to cartridge Add sample to reservoir. Ensure flow into cartridge with glass pipet. Apply vacuum and keep flow rate <10 mL/min. Slower flow gives better results. Wash: 3 mL 50:50 Acetonitrile/Deionized Water Apply vacuum until 30 sec. after wash has passed through cartridge Centrifuge cartridge at 1000-1500 rpm for 5 min. (removes excess water) Elute: 3 mL Methylene Chloride and collect eluent. Evaporate to 50-200 µL. Do not apply heat or smaller PAHs will be volatilized. Add Methylene Chloride to bring sample to final volume of 200 µL. Inject 2 µL. CI0126C 33 Water Spiked at 50 ppb Std. Avg. Compound Recovery Dev. (%) (%) Naphthalene 80 12 2-Chloronaphthalene 78 8 Acenaphthylene 81 9 Fluorene 99 9 Phenanthrene 98 9 Fluoranthene 91 8 Chrysene 97 12 Benzo(b)-fluoranthene 60 8 Benzo(a)pyrene 55 8 Indene(1,2,3-c,d)pyrene 60 10 Benzo(g,h,i)perylene 59 10 AccuBONDII SPE Product Features COA, Packaging and Labeling • Certificate of analysis with each box —Mass consistency —Flow rate consistency —Trace metal analysis —Extract cleanliness —QC chromatogram • Packaged in trilayer laminated bag —Excludes air and water —Ensures clean product • Individually printed tubes and plates —Easy identification —Versatility for method development CI0126C 34 Certificate of Performance for AccuBONDII – page 1 Description: AccuBONDII C8 100 mg 1 mL cartridge Catalog No: 188-0310 Lot No.: AMC18-0X Run No.: XXXXXXX This AccuBOND product and sorbent have been manufactured, tested and assembled under the control of an ISO 9001 registered quality system. This AccuBOND SPE product has been subjected to the following QC tests: Base Silica Characteristics Surface Area: Average Pore Size: Surface pH: Metal Analysis: 546 m2/g 60Å 7 Pass Particle Size Data Particle Shape: Irregular Average Particle Size (µm): 56 Percentage of Particles <10 µm: 0.00 Percentage of Particles <25 µm: 1.36 Percentage of Particles <90 µm: 4.95 CI0126C 35 Bonded Silica Tests Carbon Loading: Surface Coverage: Extraction Residue: Exchange Capacity: 13.0% 2.5 µmoles/m2 Pass N/A Packed Cartridge Test Cartridge Flow Resistance: Frit Purity Test (GC): Material Weight Check: Pass Pass Pass Certificate of Performance for AccuBONDII — page 2 T e s t C o n d i t io n a n d R e s u lt s C o n d itio n s : C o lu m n : M o b ile P h a s e : F lo w R a te : S p e c i f i c a t i o n s : QA K ’ b ip h e n y l K ’ to lu e n e K ’ a c e to p h e n o n e T f u r a c il 4 .6 x 2 5 0 m m 7 0 /3 0 M e O H /H 2 O 2 .0 m l / m i n = = = = 3 .5 1 .5 0 .6 0 .3 to to to to 6 .5 3 .5 1 .4 1 .5 R e s u l t s f o r L o t A M C 8 X - X X QC K ’ to lu e n e = 4 .5 K ’ d im e th y la n ilin e = 2 .1 K ’ n itr o b e n z e n e = 1 .0 T f d im e th y la n ilin e = 1 .4 P eak P eak P eak P eak 1 2 3 4 = = = = U r a c il A c e to p h e n o n e T o lu e n e B ip h e n y l N o tic e : M a te ria l S a fe t y D a ta S h e e ts (M S D S ) a re a v a ila b le a t: h t t p : / / w w w .c h e m .a g i l e n t .c o m / s c r i p t s / c a g _ m s d s s e a r c h .a s p T h is p ro d u c t h a s p a s s e d a ll A g ile n t T e c h n o lo g ie s , in c . q u a lity c o n tro l s p e c ific a tio n s . CI0126C 36 EVIDEXII SPE Cartridges for Drugs of Abuse in Urine • Proprietary bonding chemistry — Mixed RP and cation-exchange bonded phase CH3 CH3 Amphetamine • Designed for NIDA-5 Drug Classes N CH3 — Amphetamine/Methamphetamine — Codeine and Morphine N CH3 Methamphetamine HO O O O CH3 HO O Benzoylecgonine N N CH3 CH3 Codeine O Morphine OH OH H H3C O H3C — Ensures high recoveries — Produces clean extracts with excellent S/N ratios in GC-MS • Good general purpose pharmaceutical mixed mode phase CI0126C 37 OH H H — Ensures lot-to-lot reproducibility O H — THC-COOH (Marijuana metabolite) • Tested with actual drugs of abuse PCP O OH — PCP (“Angeldust”) — Benzoylecgonine (Cocaine metabolite) NH NH2 THC-COOH CH3 EVIDEXII Drugs of Abuse (DOA) Methods • EVIDEXII Methods for NIDA drug classes — One SPE cartridge type for all analytes and methods — Two cartridge configurations 200 mg/3mL and 400 mg/6 mL for different sample sizes Effect of Varying Method Conditions on Benzoylecgonine SPE Recovery — Step-by-step instructions — Robust procedures • minor changes in reagent volumes and concentration do not affect results — GC-MS analysis using column specific for DOA (DB-5 MS EVDX GC) — Accurate, reproducible results (<5% RSD) • Bottom Line: Results are defensible in court CI0126C 38 EVIDEXII methods are robust! EVIDEXII SPE Cartridges for Drugs of Abuse Opiates in Urine Solid Phase Extraction Method CARTRIDGE: EVIDEXII 400 mg, 6 mL (P/N 188-2946, Box of 30) Precondition: 6 mL methanol 6 mL 0.1 M potassium phosphate (pH 6.0) Do not let the phase go dry Load: Add 3 mL 0.1 M potassium phosphate (pH 6.0) to the cartridge Attach an 8 mL reservoir Add the urine sample Rinse: Remove reservoir 3 mL water 3 mL 0.1 M sodium acetate (pH 4.5) 3 mL methanol Elute: Place a collection tube beneath cartridge 3 mL methylene chloride/isopropyl alcohol/ NH4OH (78/20/2) Collect the eluate CI0126C 39 Codeine Morphine Codeine 30 ng Morphin e 30 ng Clean extracts from a very dirty matrix Column: Carrier: Oven: Injector: Detector: DB-5MS EVDX Helium at 40 cm/sec 65 degrees for 1 min, 65-325 degrees at 20 degrees/min Splitless, 250°C MSD, 300°C transfer line DOA Recoveries are Reproducible Using EVIDEXII SPE Cartridges Recovery Levels (x±%, n = 8) CI0126C 40 Level in ng/mL Amphetamine Methamphetamine 1000 76 ±4 85 ±1 1750 74 ±2 71 ±3 2500 96 ±3 98 ±3 Level in ng/mL PCP 50 90 ±3 87.5 91 ±2 125 88 ±3 Level in ng/mL Benzoylecgonine 300 98 ±8 525 93 ±4 750 97 ±3 Level in ng/mL Codeine Morphine 600 99 ±1 96 ±2 1050 97 ±3 96 ±3 1500 98 ±1 93 ±4 Level in ng/mL THC-COOH 30 92 ±4 52.5 96 ±8 75 94 ±3 Use of Multimodal SPE for Cleanup of Complex Samples Cartridge (Stationary Phase #1) Adapter Cartridge (Stationary Phase #2) Stopcock Port Plug CI0126C 41 Summary • Solid Phase Extraction —Probably the most important technique for sample cleanup and concentration today —Increases productivity, column lifetime and instrument uptime • AccuBONDII —Available with both silica and PS-DVB base materials —Many bonded phases for every sample type —Low extractable levels—prewashed tubes, frits, and packing —High quality SPE products for any sample type —Low extractables from packing, tubes and frits are compatible with sensitive detectors like MS • EVIDEXII —Accurate, reproducible, robust methods for drugs of abuse and other pharmaceutical applications CI0126C 42 Appendix SPE Tips For Improving Recovery and Precision • General —Keep cartridges in sealed bags until use. —Store in zipper-locked bags or in desiccator once opened. —When using empty reservoirs attached to cartridges, use long disposable pipets to ensure proper flow from reservoir to cartridge. —Use stopcocks to adjust/control flow through individual cartridges. • Use mass balance for all fractions to determine fate of analyte (adsorption to surfaces, loading effluent, washes, eluate, etc.) during method development. —Residual water can be removed effectively by centrifugation (5000 rpm, 5 min.) compared to drying with vacuum or nitrogen. —Cartridge capacity for analytes and matrix is typically about 1-3% of cartridge bed weight (ion-exchange not included). —NEVER allow the cartridge to dry out until the elution step. CI0126C 44 SPE Tips For Improving Recovery and Precision • Prewash —Remove all strong prewash solvent for GC (e.g., dichloromethane, hexane, ethyl acetate) before preconditioning and loading. • Precondition —Make sure pH is correct for ion-suppression (acids) or minimal silanol interactions (bases). —Leave ~1-2 mm of preconditioning solvent above sorbent bed to prevent bed from drying. —Leave ~1/4 to 1/2 of tube volume above sorbent bed when using empty reservoir above cartridge. CI0126C 45 SPE Tips For Improving Recovery and Precision • Load —Leave ~1/4 to 1/2 tube volume above sorbent bed when using tube reservoir above cartridge. —Use drop wise solvent flow when time/throughput is not a major concern. • Wash —Wipe needles of manifold before elute step to minimize contamination of eluate. • Elute —When choosing eluent, consider ease of evaporation if reconstitution is needed. —Allow cartridge/plate to soak with eluent for 0.5 - 1 min. (Àrecovery). —Sometimes several smaller eluent aliquots can improve recovery. CI0126C 46