FAST, EASY EFFICIENT SAMPLE PREPARATION Supra-Clean® and Supra-Poly® Solid Phase Extraction (SPE) A Reference Notebook of SPE Applications 2 INTRODUCTION AND OVERVIEW At PerkinElmer, we understand that sample preparation is one of the most critical steps in the analytical process. Often accounting for 60% of your timetable, it has a fundamental impact on a wide range of operational parameters. Any errors within this process undermine the quality of your data at all subsequent stages of your analysis. Solid Phase Extraction (SPE) helps avoid potential errors in sample preparation, reducing re-runs and dramatically increasing productivity. As one of the most cost-effective and flexible tools within the laboratory environment, SPE also provides efficient sample concentration and purification prior to many of today’s most popular analytical techniques, including HPLC, LC/MS, GC and GC/MS. This document is intended to provide you with the tools you need to quickly and efficiently develop extraction methods for your sampling analysis. PerkinElmer SPE Solutions are ideal for a broad array of analytes and matrices. We offer a variety of formats including Large Reservoir Capacity (LRC) columns, Polypropylene (PP) columns, cartridges 96 well plate format, and glass columns. Each technology is offered with a wide selection of polymer and silica sorbents. Varied column capacities and bed weights allow you to perform scalable analyses depending upon your sample size and required detection limits. PerkinElmer® Supra-Clean® silica-based and Supra-Poly® polymer-based SPE cartridges and columns. Answering your needs. Empowering Your Lab. www.perkinelmer.com/spe 3 TABLE OF CONTENTS ENVIRONMENTAL • 2,4-Dichlorophenol in Water................................................................................................................................................ 12 • 2,4-Dichlorophenoxyacetic Acid in Water............................................................................................................................. 13 • 4'4-Methylenedianaline in Serum......................................................................................................................................... 14 • Acephate in Water............................................................................................................................................................... 15 • Aniline in Solution................................................................................................................................................................ 16 • Atrazine in Water................................................................................................................................................................. 17 • Bentazone in Water............................................................................................................................................................. 18 • Bentazone in Water, Derivitized .......................................................................................................................................... 19 • Chlorophenol in Water.........................................................................................................................................................20 • Chlorophenoxy Acid Herbicides in Water............................................................................................................................. 21 • N,N-Dimethylaniline in Solution...........................................................................................................................................22 • Nitroanalines in Solution......................................................................................................................................................23 • Nitrobenzene in Water..................................................................................................................................................24 - 25 FORENSIC • 6-Acetylmorphine in Urine...................................................................................................................................................26 • Amphetamines in Urine, Oxidation with Periodate for GC or GC/MS Confirmations.............................................................27 • Anabolic Steroids in Urine....................................................................................................................................................28 • Antidepressants/Painkillers in Urine and Blood.....................................................................................................................29 • Benzoylecgonine in Urine.....................................................................................................................................................30 • Carboxy-delta-9-THC, Delta-9-THC, Delta-9-Hydroxy THC in Whole Blood.......................................................................... 31 • Delta-9-THC, Delta-9-Hydroxy THC, Carboxy-Delta-9-THC in Whole Blood.........................................................................32 • Gamma-Hydroxybutyrate (GHB) in Blood, Urine, and Tissue.................................................................................................33 • Gamma-Hydroxybutyrate (GHB) in Urine without conversion to Gamma-Butrylactone (GBL)................................................34 • Opiates in Urine...................................................................................................................................................................35 • Sympathomimetic Amines in Urine.......................................................................................................................................36 PHARMACEUTICAL • Acetominophen in Serum....................................................................................................................................................37 • Acetominophen in Solution..................................................................................................................................................38 • Benzodiazepine Screen in Blood, Serum, and Tissue by GC/MS............................................................................................39 • Benzodiazepine Screen in Urine by GC/MS...........................................................................................................................40 4 TABLE OF CONTENTS PHARMACEUTICAL • Beta Agonists in Urine......................................................................................................................................................... 41 • Beta Blockers in Blood and Urine.........................................................................................................................................42 • Caffeine, Theophylline and Theobromine in Blood, Plasma/Serum, and Urine......................................................................43 • Cefaperazone in Water........................................................................................................................................................44 • Clonazepam and 7-Aminoclonazepam in Urine....................................................................................................................45 • DHEA, Testosterone, and Epitestosterone in Urine...............................................................................................................46 • Doxepin in Rat Serum..........................................................................................................................................................47 • Gabapentin in Blood, Plasma/Serum............................................................................................................................ 48 - 50 • Ketotifen Fumarate in Solution............................................................................................................................................. 51 • Lovastatin in Water..............................................................................................................................................................52 • Methylmalonic Acid from Serum/Plasma..............................................................................................................................53 • Olanzapine in Whole Blood..................................................................................................................................................54 • Paroxetine in Blood, Plasma/Serum and Urine......................................................................................................................55 • Propranolol in Rat Serum.....................................................................................................................................................56 • Tacrolimus, Cyclosporin and Rapamycin in Whole Blood.......................................................................................................57 • Tricyclic Antidepressants in Plasma/Serum............................................................................................................................58 www.perkinelmer.com/spe 5 Supra-Clean and Supra-Poly Solid Phase Extraction (SPE) Columns with Precise Bed Technology™ Supra-Clean and Supra-Poly SPE products are designed to offer you a sample preparation solution that decreases your sample preparation time all while maintaining a high level of reproducibility. Supra-Clean uses a silica based media and Supra-Poly uses a polymer based media. Both formats are packed using Precise Bed Technology and spherical particles, which allow columns to be evenly and consistently filled resulting in optimum bed density. Optimum bed density delivers a level of performance unattainable with the irregularly shaped silica used in competitive solutions. The homogeneous filling and monodispersed spherical media not only provide unparalleled reproducibility, it also delivers superior performance. Homogeneous filling and spherical media yield a +/- 1% variation in bed volume precision, a significant improvement from the industry average. This level of quality control allows customers to experience consistent sample preparation performance column to column, batch to batch, and lot to lot. Sample diffusion through Supra-Clean/Supra-Poly spherical media is also better than in other systems, improving flow efficiency and avoiding channeling and clogging due to fines. This enables smaller sample sizes to be used without compromising recovery levels, dramatically reducing solvent volumes, analytical costs and processing times. This allows you to work faster and more cost effectively, all while experiencing complete recovery, reproducibility and reliability. Precise Bed Technology Weighing technology with +/- 1% accuracy Guarantee of reproducibility from batch to batch & column to column Bottom Polyethylene frit Zero extractable Polypropylene HMW tube Zero extractable Top Polyethylene frit a special cut-off ensure a perfect sample diffusion without clogging Optimum bed density Zero extractable All columns are thoroughly quality control tested in-house to guarantee tracability. Products are supplied with an individual certificate detailing the specific production number and sorbent batch. 6 Choose the Best SPE Solution For Your Method To find the ideal SPE column for your particular application, simply follow the flow diagram below, selecting your path based on the options at each junction. Your Sample Type of Matrix Aqueous Analyte Solubility Water Analyte Analyte Polarity Recommended Polar Ionic Neutral Neutral Anionic Acids Ion Exchange Chromatography Non-Polar Reversed Phase Chromatography Non-Polar Polar Normal Phase Chromatography Supra-Clean Florisil (FL-S) Supra-Clean Silica (SI-S) Supra-Clean Amino (NH2-S) Supra-Clean Polyamine (P6) Supra-Clean Amino (NH2-S) Supra-Poly 1200 m2/g (XWP)* Supra-Poly (ATH, ATL, AEV)* Supra-Poly HLB Supra-Clean C18 Supra-Clean 300Å C4 Supra-Clean Phenyl (PH-S) Supra-Clean Cyano (CN-S) Supra-Poly Extreme Capacity 1500 m2/g (XC)* Supra-Poly HLB Supra-Clean Strong Anion Exchange (SAX) Supra-Clean Amino (NH2-S) Supra-Clean Mixed-Mode (MM1) Supra-Clean Mixed-Mode (MM2) SPE Strong Cation Exchange (SCX) SPE Weak Cation Exchange (WCX) Supra-Clean Mixed-Mode (MM3) Supra-Clean REC18 Supra-Clean REC18 Supra-Clean C18 Supra-Clean 300Å C4 Supra-Clean Phenyl (PH-S) Supra-Clean LCC www.perkinelmer.com/spe Organic Solvents Cationic Bases Reversed Phase Chromatography *AEV = Advanced Environmental ATH = Hydrophobic/Hydrophilic ATL = Lipophilic Water path as Ionic analyte) Moderately Polar Non-Polar Follow same path as Organic Ionic (Follow same Neutral Chromatography Recommended SPE Products Organic HLB = Hydrophilic Lipophylic Balanced XC = Extreme Capacity XWP = Extra Wide Particle 7 A Wide Array of Columns for a Broad Range of Applications SUPRA-CLEAN SPHERICAL SILICA Phase Mechanism Interaction mode Compounds Matrix C18-S Hydrophobic Reversed Phase Polar to Non-Polar compounds Biological fluids, aqueous samples High Recovery REC18 Hydrophobic Reversed Phase Non-polar and mid-polar compounds Biological fluids, aqueous samples including 100% water solvents Phenyl (PH-S) Hydrophobic Reversed Phase Non-polar to mid-polar Biological fluids aromatic compounds Silica (SI-S) Hydrophilic Normal Phase Polar compounds Non-polar organics, oils, lipids Amino (NH2-S) Hydrophilic Normal Phase Polar to Mid-Polar aromatic compounds Biological fluids, aqueous samples, buffered organics Strong Cation Ion Exchange Ion Exchange Basic compounds Biological fluids, aqueous samples, Exchange (SCX) buffered organics Weak cation Ion Exchange Ion Exchange Strong basic compounds Biological fluids, aqueous samples Exchange (WCX) Strong Anion Ion Exchange Ion Exchange Acidic compounds Biological fluids, aqueous samples Exchange (SAX) Cyano (CN-S) Hydrophilic Normal Phase Polar to Mid-Polar compounds Non-polar organics, oils, lipids Florisil (FL-S) Hydrophilic Normal Phase Polar compounds Ideal for polar compounds in non-polar matrix Florisil Pesticide (FL-S) Hydrophilic Normal Phase Polar compounds Ideal for polar compounds in non-polar matrix Polyamine (P6) Hydrophilic Reversed Phase Carboxylic acids, phenolics Aqueous and mid-polar matrices and nitroaromatics 300 A (C4) Hydrophobic Reversed Phase Non-polar to mid-polar compounds Biological Samples LCC Hydrophobic Reversed Phase Non-polar to mid-polar compounds Biological fluids, aqueous samples Mixed mode (MM1) Ion Exchange/ Hydrophobic Reversed Phase / SCX Basic compounds Biological samples Mixed mode (MM2) Ion Exchange/ Hydrophobic Reversed Phase / WCX Very basic compounds Biological samples Mixed mode (MM3) Ion Exchange/ Hydrophobic Reversed Phase / SAX Acidic compounds Biological samples SUPRA-POLY SPHERICAL POLYMER Extreme Capacity (XC) Hydrophobic Reversed Phase Polar and non-polar Aqueous or organic Extreme Capacity Hydrophobic Reversed Phase Polar and non-polar Biological and viscous samples Wide Pore (XWP) Hydrophilic (ATH) Hydrophilic Reversed Phase Mid to non-polar compounds Aqueous or organic Lipophilic (ATL) Lipophilic Reversed Phase Mid to non-polar compounds Crude samples Environmental (AEV) Hydrophilic/ Hydrophobic Reversed Phase Mid to non-polar compounds Aqueous or Organic HLB Hydrophilic/lipophilic balanced Reversed Phase Mid to non-polar compounds Aqueous or organic 8 Typical Applications Drugs and drug metabolites in biological matrices, trace organic material in water, toxins in food pH Range End- capping Pore Size (A) Surface Area (m2/g) Particle Size (um) 2-8 Yes 60 500 50 Comments 18% Carbon Load (CL) Drugs and drug metabolites in biological 2-8 Yes NA NA 50 matrices, trace organic material in water, toxins in food High capacity and better recovery especially for high acqueous conditions. 15% CL Benzodiazepines in biological matrices, extraction of aromatic compounds 2-8 No 60 500 50 9% CL Aldehydes, amines, pesticides, herbicides, carotenoids, fat soluable vitamins, baflatoxins, fatty acids, and phospholipids 2-8 No 60 500 50 Bare Silica Basic compounds, polar amine compounds, carbohydrates 2-8 No 60 500 50 5% CL Cations, antibiotics, drugs, amino acids, 2-8 No 60 450 60 catecholamines, herbicides, nucleic acid bases, nucleosides, and surfactants Strong Acid - Sulfonic acid; Exchange capacity 0.70 meq/g Cations, amines, antibiotics, drugs, amino acids, 2-8 No 60 450 60 Weak Acid - Carboxylic acid; Exchange catecholamines, nucleic acid bases, nucleosides, capacity 0.22 meq/g and surfactants Acidic food pigments, organic acids, phenol 2-8 No 60 450 60 compounds, nucleic acids, nucleotides, surfactants Strong Base - quaternary amine; Exchange capacity 0.30 meq/g Polar compounds in hexane and oil 50 8% CL; Mid-range polarity between silica and C18 Pesticides, Polychlorinated Biphenyls (PCB) 2-8 No NA NA 200 Standard grade. Alternative to silica for viscous matrices due to large particle size. Granular shape Pesticides 2-8 No NA NA 200 High purity pesticide grade. Alternative to silica for viscous matrices. Granular shape 2-8 Yes 60 500 Aromatic and naturalproducts; Flavones, Chalkones, Anthraquinones 2-8 No NA NA 100 Nylon 6 Hydrophobic peptides and polypeptides 2-8 No 300 - - Non-polar compounds in aqueous solution 2-8 Yes 60 500 50 10% CL; Lower carbon load than C18-S and REC18 Drugs and drug metabolites 2-8 No 60 450 60 Exchange capacity 0.09 meq/g Drugs and drug metabolites 2-8 No 60 450 60 Exchange capacity 0.10 meq/g Drugs and drug metabolites 2-8 No 60 450 60 Exchange capacity 0.14 meq/g Large pore size for isolation of large biomolecules Drugs and drug metabolites biological fluids 0-14 No NA 1500 70 Drugs and drug metabolites biological fluids 0-14 No Wide Pore 1200 90 Mid-polar and non-polar compounds in aqueous and organic solvents 1-13 No 70 800 75 Lipids 0-14 No 70 800 100 High capacity polystyrene-divinylbenzene (PSDVB) High capacity PSDVB for large biomolecules and viscous matrices Mixed hydrophilic/hydrophobic interactions PSDVB; Alternative to high flow silica for mid-polar to non-polar compounds (<3000D) in crude samples Aqueous environmental compounds that are 1-12 No 70 800 75 not retained on C18 Advanced environmental; Polystyrene-co-2hydroxyethyl methacrylate (PSHEMA) Mid-polar and non-polar compounds in 0-14 No 80 850 30 & 60 aqueous and organic solvents Hydrophilic-lipophilic-balanced reversedphase sorbent for acids, bases and neutrals www.perkinelmer.com/spe 9 Six Steps for Clean Extract 1. Pretreat Sample Some samples require initial pretreatment to obtain the optimal extraction. This pretreatment can include homoginization of solid samples, adjustment of the matrix to the proper ionic strength and pH, and removal of particulates via filtration or centrifugation. This step may also include addition of an internal standard. 2. Condition Column Sorbent activation and functional group activation are achieved by passing a volume of an appropriate solvent or a mixture of solvent, through the column. Column frits are simultaneously conditioned as well. Methanol or acetonitrile are commonly used for activating hydrophobic sorbents, while hexane or dichloromethane activate hydrophilic sorbents. 2 to 4 bed volumes are typically recommended. See SPE Solvent Selection Guide (below). Note: Once the conditioning is done never let the column dry unless it is specified in the method. Load Sample 3. Load Sample Apply your sample onto the upper part of the sorbent bed. Matrix contaminants may pass through the column unretained while other matrix components may be more or less strongly retained on the sorbent surface. To get a maximum purification efficiency, the sample flow needs to be controlled via vacuum or pressure. To achieve faster flow of viscous sample through a column, 90 µm sorbents or larger particle size can be used. The exchange capacity and selectivity are unaffected. Note: It is necessary to analyze the unretained fraction to check if all compounds of interest have been retained. 4. Wash Column Passing solvents through columns washes away interfering compounds while leaving the analyte of interest undisturbed on the sorbent bed. Different solvents or solvent mixtures may be used to improve the rinsing efficiency. See SPE Solvent Selection Guide (below). 5. Dry Column (optional) A drying step may be required. Solvent traces are evaporated by circulating air through the column over a 2 to 10 minute time period. This improves the extraction yield for certain methods and compounds. 6. Elute Compounds of Interest An appropriate solvent is passed through the column to disrupt the analyte-sorbent interaction and to elute 100% of compounds of interest. The appropriate solvent must have maximum interaction with the compound of interest and a minimal interaction with the remaining impurities, leaving them undisturbed on the sorbent bed. See SPE Solvent Selection Guide (below). In addition, the volume of the elution solvent needs to be as small as possible to maximize the concentration factor. Note: Sorbent with low particle size (e.g 30, 50 µm) gives a lower elution volume than larger sorbent particle size (e.g 90, 140 µm). NONPOLAR Acetic Acid Water Methanol Ethanol Isopropanol Pyridine Acetonitrile Acetone Ethyl Acetate Diethyl Ether Tetrahydrofuran Methylene Chloride Chloroform Toluene Carbon tetrachloride Cyclohexane Petroleum Ether Solvent Hexane The choice of solvent is dependent upon the sample matrix and the retention mechanism used. This table shows the differing polarities of solvents commonly used in SPE. Isooctane SPE Solvent Selection Guide POLAR Water Miscible No No No No No No No NoYes NoPoorYesYes YesYes YesYes YesYes 10 Method Development for SPE Imagine how much faster, easier and more efficient your method development would be if your Solid Phase Extraction (SPE) column selection kits were designed to streamline your process. PerkinElmer has created column specific kits for some of the more common analyses performed in today’s laboratories. These kits dramatically reduce set-up time for these methods and make it easy to select the appropriate solution based on your specific matrices. PerkinElmer SPE Column Selection Kits Media Weight Volume Qty. Part No. Pre-Concentration of Hydrophobic Compounds from Aqueous Matrix 200 mg 6 mL 50 N9306594 200 mg 3 mL 50 N9306595 Type of matrix: aqueous Principle: concentration of non-polar, mid-polar compounds Includes 10 each: Supra-Clean C18 with cartridge, REC18; Supra-Poly Lipophilic (ATL), extreme capacity (XC), hydrophobic/hydrophilic (ATH) Extraction of Hydrophobic Compounds from Aqueous Matrix 500 mg 6 mL 50 500 mg 3 mL 50 Type of matrix: aqueous or (organics and aqueous) mixtures Principle: extraction of non-polar and mid-polar compounds Includes 10 each: Supra-Clean C18 (un-end-capped), C18 with cartridge, REC18, LCC, Phenyl N9306596 N9306597 Pre-Concentration of Hydrophilic Compounds 500 mg 6 mL 500 mg 3 mL Type of matrix: aqueous or (organics and aqueous) mixtures Principle: extraction of non-polar and mid-polar compounds Includes 10 each: Supra-Clean Amino, Cyano, Silica with cartridge 30 30 N9306598 N9306599 Removal of Polar Compounds from Aqueous and Organic Matrix 500 mg 6 mL 500 mg 3 mL Type of matrix: aqueous or organics Principle: Extraction and concentration of acidic, basic and neutral compounds Includes 10 each: Supra-Clean Amino, Florisil, Silica with cartridge 30 30 N9306600 N9306601 Extraction of Acidic Basic and Neutral Compounds from Aqueous or Organic Matrix 100 mg 3 mL 50 N9306602 Type of matrix: aqueous or organics Principle: Extraction and concentration of acidic, basic and neutral compounds Includes 10 each: Supra-Poly Lipophilic (ATL), extra wide particle (XWP), extreme capacity (XC), advanced environmental (AEV), hydrophobic/hydrophilic (ATH) Extraction of Carboxylic Acids and Strong Bases from Aqueous Matrix 500 mg 6 mL 40 Type of matrix: aqueous Principle: selective extraction of weak acids and strong bases with the same protocol Includes 10 each: Supra-Clean Strong Anion Exchange, Weak Cation Exchange, Mixed-Mode 2, 3 N9306603 Extraction of Weak Bases from Aqueous Matrix Type of matrix: aqueous Principle: extraction of weak bases Includes 15 each: Supra-Clean Strong Cation Exchange, Mixed-Mode 1 N9306604 www.perkinelmer.com/spe 500 mg 6 mL 30 11 ENVIRONMENTAL 2,4-Dichlorophenol in Water PerkinElmer SPE Supra-Poly XC 500 mg/6 mL, Plastic Tube (Straight) Part No. N9306405 SAMPLE PRETREATMENT ELUTION Adjust pH to 2 with H2SO4 3 mL tetrahydrofuran COLUMN CONDITIONING CONCENTRATION 5 mL methanol 5 mL water Concentrate sample to 3 mL under nitrogen ANALYSIS RECOMMENDATIONS SAMPLE LOADING HPLC Load 500 mL water sample Elute at 5 mL/minute RECOMMENDED COLUMN COLUMN WASHING PerkinElmer Brownlee Validated PFP 250 mm x 4.6 mm x 5 um Part No. N9303600 5 mL water 1 mL methanol DRYING Dry column (20 minutes under nitrogen) 12 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 ENVIRONMENTAL 2,4-Dichlorophenoxyacetic Acid in Water PerkinElmer SPE Supra-Poly XC 500 mg/6 mL, Plastic Tube (Straight) Part No. N9306405 SAMPLE PRETREATMENT ELUTION Adjust the pH of 500 mL of water to 1.5 to 2 using 0.5 M H2SO4 Use 0.8 mL of methanol to replace the residual water in the XC packing COLUMN CONDITIONING 3 mL of tetrahydrofuran 5 mL of methanol 5 mL of DI water SAMPLE LOADING Load the 500 mL sample at a rate no greater than 5 mL/min COLUMN WASHING 5 mL of DI water Discard the water which passes through the SPE column Let methanol infiltrate the packing material for 2 minutes Elute the column with 3 mL of THF Collect the eluate and reconstitute to 3 mL usingmobile phase RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 DRYING Dry column (20 minutes at >10"Hg/full flow for positive pressure manifold) www.perkinelmer.com/spe 13 ENVIRONMENTAL 4'4-Methylenedianaline in Serum PerkinElmer SPE Supra-Clean C18-S 100 mg/1 mL, Plastic Tube (Straight) Part No. N9306478 COLUMN CONDITIONING ANALYSIS RECOMMENDATIONS 3 mL of methanol 3 mL of DI water Inject 10 μL onto HPLC system Extracted 100 μg/mL sample Mobile phase: methanol/water (50:50) Flow rate: 1.2 mL/min Injection volume: 10 μL Wavelength: UV 254 nm SAMPLE LOADING Load sample at 1 mL/minute COLUMN WASHING RECOMMENDED COLUMN 1 mL of DI water PerkinElmer Brownlee Validated C18 250 mm x 4.6 mm x 5 um Part No. N9303561 ELUTION 0.25 mL of methanol containing 1 M ammonium hydroxide RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 14 ENVIRONMENTAL Acephate in Water PerkinElmer SPE Supra-Clean C18-S 100 mg/1 mL, Plastic Tube (Straight) Part No. N9306478 SAMPLE PRETREATMENT CONCENTRATION Prepare a 10 mL, 10 ppm sample of acephate in ammonium sulphate aqueous solution (w/w 20%) Evaporate to full dryness under a stream of nitrogen Reconstitute the sample to 0.5 mL with water ANALYSIS RECOMMENDATIONS COLUMN CONDITIONING 2 mL of methanol 2 mL of DI water SAMPLE LOADING Load 4 mL samples at 1 to 2 mL/minute COLUMN WASHING Mobile phase: ACN/water (40:60, v/v) Flow: 1 mL/minute Temperature: 30°C Detection: UV 214 nm RECOMMENDED COLUMN PerkinElmer Brownlee Validated C18 250 mm x 4.6 mm x 5 um Part No. N9303561 1 mL of DI water RELATED ITEMS ELUTION 5 mL of methanol www.perkinelmer.com/spe Vacuum Manifold -12 position; Part No. N9306619 Vacuum Manifold -24 position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 15 ENVIRONMENTAL Aniline in Solution PerkinElmer SPE Supra-Poly XC 60 mg/3 mL, Plastic Tube (Straight) Part No. N9306502 SAMPLE PRETREATMENT CONCENTRATION Prepare solutions of aniline in DI water at concentrations of 200, 20 and 2 ppm Evaporate to full dryness at room temperature under a stream of nitrogen Reconstitute the sample to 1 mL with methanol COLUMN CONDITIONING 3 mL of methanol 3 mL of DI water Mobile phase: methanol/water (50:50, v/v) SAMPLE LOADING RECOMMENDED COLUMN Load 3 mL sample at 1 to 2 mL/minute PerkinElmer Brownlee Validated Aqueous C18 250 mm x 4.6 mm x 5 um Part No. N9303549 COLUMN WASHING RELATED ITEMS 1 mL of DI water Vacuum Manifold -12 position; Part No. N9306619 Vacuum Manifold -24 position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 ELUTION 3 mL of methanol/water (95:5, v/v) 16 ANALYSIS RECOMMENDATIONS ENVIRONMENTAL Atrazine in Water PerkinElmer SPE Supra-Poly XC 60 mg/3 mL, Plastic Tube (Straight) Part No. N9306502 SAMPLE PRETREATMENT CONCENTRATION To 10 mL of water containing 10 ppm atrazine, add 20 μL of acetic acid Evaporate to full dryness under a stream of nitrogen Reconstitute the sample to 0.5 mL with water COLUMN CONDITIONING ANALYSIS RECOMMENDATIONS 2 mL of methanol 2 mL of DI water Mobile phase: ACN/0.2% acetic acid (10:90, v/v) Flow: 1mL/minute Temperature: 30°C Detection: UV 214nm SAMPLE LOADING Load 4 mL samples at 1 to 2 mL/minute RECOMMENDED COLUMN COLUMN WASHING 1 mL of DI water ELUTION 5 mL of methanol www.perkinelmer.com/spe PerkinElmer Brownlee Validated C18 250 mm x 4.6 mm x 5 um Part No. N9303561 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 17 ENVIRONMENTAL Bentazone in Water PerkinElmer SPE Supra-Poly XC 500 mg/6 mL, Plastic Tube (Straight) Part No. N9306405 SAMPLE PRETREATMENT ELUTION Adjust the pH of 500 mL of water to ≤ 3 using 0.5 M H2SO4 Use 0.8 mL of methanol to replace the residual water in the XC packing COLUMN CONDITIONING 3 mL of tetrahydrofuran 5 mL of methanol 5 mL of DI water SAMPLE LOADING Wait for 2 minutes to make sure the methanol infiltrates the packing material Elute the column with 3 mL of tetrahydrofuran Load the 500 mL sample at a rate no greater than 5 mL/min Evaporate the tetrahydrofuran and reconstitute to 3 mL using mobile phase COLUMN WASHING RELATED ITEMS 5 mL of DI water Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 DRYING Dry column (20 minutes at >10""Hg/full flow for positive pressure manifold) 18 Discard the water which passes through the SPE column ENVIRONMENTAL Bentazone in Water, Derivitized PerkinElmer SPE Supra-Poly XC 500 mg/6 mL, Plastic Tube (Straight) Part No. N9306405 SAMPLE PRETREATMENT DRYING Adjust the pH of 500 mL of water to 3 using 0.5 M H2SO4 Dry column (20 minutes under nitrogen) COLUMN CONDITIONING ELUTION 3 mL of tetrahydrofuran 5 mL of methanol 5 mL of DI water 3 mL tetrahydrofuran Concentrate sample to 3 mL under N2 RELATED ITEMS SAMPLE LOADING Load the 500 mL sample at a rate no greater than 5 mL/min COLUMN WASHING Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 5 mL of DI water www.perkinelmer.com/spe 19 ENVIRONMENTAL Chlorophenol in Water PerkinElmer SPE Supra-Poly XC 500 mg/6 mL, Plastic Tube (Straight) Part No. N9306405 SAMPLE PRETREATMENT CONCENTRATION Collect 500 mL of water Adjust pH to 2 with H2SO4 Concentrate sample to 3 mL under nitrogen ANALYSIS RECOMMENDATIONS COLUMN CONDITIONING HPLC 5 mL methanol 5 mL water RECOMMENDED COLUMN SAMPLE LOADING PerkinElmer Brownlee Validated PFP 250 mm x 4.6 mm x 5 um Part No. N9303600 Load 500 mL water sample Elute at 5 mL/minute RELATED ITEMS COLUMN WASHING 5 mL water 1 mL methanol ELUTION 3 mL tetrahydrofuran 20 Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 ENVIRONMENTAL Chlorophenoxy Acid Herbicides in Water PerkinElmer SPE Supra-Clean C18 1 g /6 ml, Plastic Tube (Straight) Part No. N9306422 SAMPLE PRETREATMENT ELUTION Adjust pH of 1 L of water sample to pH 1.0 with hydrochloric acid 10 mL of hexane/acetone (50:50) COLUMN CONDITIONING CONCENTRATION 10 mL of hexane/acetone (50:50) 10 mL of acidified methanol (5% HCl in methanol) 10 mL of DI water Add 500 μL of a keeper solvent (methanol, DMF, other) Evaporate to 500 μL under a nitrogen stream at room temperature ANALYSIS RECOMMENDATIONS SAMPLE LOADING Inject 1 to 2 μL onto GC Load 1 liter of sample at a rate of 8 to 10 mL/minute RELATED ITEMS COLUMN WASHING 10 mL of DI water adjusted to pH 1.0 with HCl DRYING Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 Dry column using maximum vacuum pressure for 15 to 30 minutes www.perkinelmer.com/spe 21 ENVIRONMENTAL N,N-Dimethylaniline in Solution PerkinElmer SPE Supra-Poly XC 60 mg/3 mL, Plastic Tube (Straight) Part No. N9306502 SAMPLE PRETREATMENT CONCENTRATION Prepare solutions of N,N-dimethylaniline in DI water at concentrations of 200, 20 and 2 ppm Evaporate to full dryness at room temperature under a stream of nitrogen Reconstitute the sample to 1 mL with methanol COLUMN CONDITIONING 3 mL of methanol 3 mL of DI water Mobile phase: methanol/water (50:50, v/v) SAMPLE LOADING RECOMMENDED COLUMN Load 3 mL sample at 1 to 2 mL/minute PerkinElmer Brownlee Validated Aqueous C18 250 mm x 4.6 mm x 5 um Part No. N9303549 COLUMN WASHING 1 mL of DI water ELUTION 3 mL of methanol/water (95:5, v/v) 22 ANALYSIS RECOMMENDATIONS RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 ENVIRONMENTAL Nitroanalines in Solution PerkinElmer SPE Supra-Poly XC 60 mg/3 mL, Plastic Tube (Straight) Part No. N9306502 SAMPLE PRETREATMENT CONCENTRATION Prepare solutions of 2-nitroaniline and 4-nitroaniline in 0.1% ammonia aqueous at concentrations of 20, 2 and 0.2 ppm Evaporate to full dryness at room temperature under a stream of nitrogen COLUMN CONDITIONING Reconstitute the sample to 1 mL with methanol 3 mL of methanol 3 mL of DI water ANALYSIS RECOMMENDATIONS Mobile phase: methanol/1% formic acid (10:90, v/v) SAMPLE LOADING Load 3 mL samples at 1 to 2 mL/minute COLUMN WASHING 1 mL of 0.1% ammonia aqueous solution ELUTION 3 mL of methanol/1% formic acid (95:5, v/v) www.perkinelmer.com/spe RECOMMENDED COLUMN PerkinElmer Brownlee Validated Aqueous C18 250 mm x 4.6 mm x 5 um Part No. N9303549 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 23 ENVIRONMENTAL Nitrobenzene in Water PerkinElmer SPE Supra-Poly XC 500 mg/6 mL, Plastic Tube (Straight) Part No. N9306405 SAMPLE PRETREATMENT ELUTION Ensure that a 497.5 mL sample of water is pH neutral Use 0.8 mL of acetone to replace the residual water in the XC packing To this sample, add 2.5 mL of methanol COLUMN CONDITIONING 3 mL of hexane 5 mL of methanol 5 mL of DI water SAMPLE LOADING Load the 500 mL sample at a rate no greater than 5 mL/min COLUMN WASHING 5 mL of DI water 24 Discard the water which passes through the SPE column Wait for 2 minutes to make sure the acetone infiltrates the packing material Connect the Supra-Poly XC column to a column packed with 5 g of anhydrous sodium sulphate which has been washed with 3 mL of acetone, 3 mL of hexane and 3 mL acetone Elute the column series with 10 mL of hexane/acetone (90:10, v/v) Collect the eluate and concentrate to 1 mL with nitrogen at 40 °C DRYING RELATED ITEMS Dry column (20 minutes at >10"Hg/full flow for positive pressure manifold) Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 ENVIRONMENTAL Nitrobenzene in Water PerkinElmer SPE Supra-Poly XC 500 mg/6 mL, Plastic Tube (Straight) Part No. N9306405 SAMPLE PRETREATMENT DRYING Add methanol to water sample to provide a 0.5% solution Adjust pH to 7 Dry column (20 minutes at >10"Hg) ELUTION COLUMN CONDITIONING 3 mL n-hexane 5 mL methanol 10 mL n-hexane/acetone (90:10, V/V) Concentrate sample to 1 mL RECOMMENDED COLUMN SAMPLE LOADING Load 10 mL water sample Elute at 5 mL/minute PerkinElmer Brownlee Validated C18 250 mm x 4.6 mm x 5 um Part No. N9303561 RELATED ITEMS COLUMN WASHING 10 mL of DI water www.perkinelmer.com/spe Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 25 FORENSIC 6-Acetylmorphine in Urine PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/15 ml, Plastic Tube (Straight) Part No. N9306713 SAMPLE PRETREATMENT ELUTION To 2 mL of 100 mM phosphate buffer (pH= 6.0) add internal standard* 3 mL CH2Cl2/IPA/NH4OH (78:20:2) at a flow rate of 1-2 mL/min Mix/vortex CONCENTRATION Add 4 mL of urine Centrifuge for 10 minutes at 2,000 rpm Discard pellet Adjust pH to 6 with 100 mM monobasic or dibasic sodium phosphate * Suggested internal standard D6-6-AM-TMS COLUMN CONDITIONING 3 mL methanol Evaporate to dryness at < 40 °C POST TREATMENT Add 50 μL ethyl acetate Mix/vortex Add 50 μL BSTFA (with 1% TMCS) Overlay with N2 and cap Mix/vortex React 45 minutes at 70 °C; cool. ANALYSIS RECOMMENDATIONS Inject 2 µl to a GC/MS 3 mL DI water RESULTS 1 mL 100 mM phosphate buffer (pH 6) Ions to monitor (Mass Spectrometry) SAMPLE LOADING Load the previously prepared sample onto the SPE column at a flow rate of 1 mL/min Compound Primary Ion Secondary Tertiary D6-6-AM-TMS405 406 343 6-AM-TMS 399 400340 RECOMMENDED COLUMN COLUMN WASHING 3 mL DI water 2 mL 100 mM acetate buffer (pH 4.5) 3 mL methanol DRYING Dry the column for 10 minutes under vacuum 26 PerkinElmer Elite-5 MS 30 m x 0.25 mm x 0.25 um Part No. N9316282 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 FORENSIC Amphetamines in Urine, Oxidation with Periodate for GC or GC/MS Confirmations PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200mg/15ml, Plastic Tube (Straight) Part No. N9306713 SAMPLE PRETREATMENT CONCENTRATION To 2 mL of urine add internal standard(s)*, 1 mL of 100 mM phosphate buffer (pH 6.0) and 1 mL of 0.35 M sodium periodate Add 30 μL silylation grade DMF to eluate Evaporate to 30 μL at < 40°C Mix/vortex POST TREATMENT Incubate at room temperature for 20 minutes Add 50 μL PFPA (PFAA) Overlay with N2 and cap Improve derivatization by addition of 50 μL PFPOH React for 20 minutes at 70 °C Evaporate to dryness at < 40 °C Adjust pH to 6.0±0.5 with 100 mM monobasic or dibasic sodium phosphate * Suggested internal standards D5-amphetamine and D5-methamphetamine ANALYSIS RECOMMENDATIONS Inject 2 µl to a GC/MS COLUMN CONDITIONING 3 mL methanol 3 mL DI water 1 mL 100 mM phosphate buffer (pH 6) SAMPLE LOADING Load the previously prepared sample onto the SPE column at a flow rate of 1-2 mL/min RESULTS Ions to monitor (Mass Spectrometry) Compound Primary Ion Secondary Tertiary D5-amphetamine194 92 123 Amphetamine190 91 118 D5-methamphetamine208 92 163 Methamphetamine204 91 160 COLUMN WASHING 3 mL DI water 1 mL 100 mM acetic acid 3 mL methanol RECOMMENDED COLUMN DRYING RELATED ITEMS Dry the column for 5 minutes Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 ELUTION 3 mL CH2Cl2/IPA/NH4OH (78:20:2) at a flow rate of 1-2 mL/min www.perkinelmer.com/spe PerkinElmer Elite-5 MS 30 m x 0.25 mm x 0.25 um Part No. N9316282 27 FORENSIC Anabolic Steroids in Urine PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/15 ml, Plastic Tube (Straight) Part No. N9306713 SAMPLE PRETREATMENT CONCENTRATION ß-Glucuronidase hydrolysis: To 5 mL of urine add suitable internal standards and 2 mL of ß-Glucuronidase ß-Glucuronidase: 5,000 F units/mL Patella vulgate in 100 mM acetate buffer (pH 5.0) Evaporate to dryness at < 40 °C Mix/vortex Hydrolyze for 3 hours at 65 °C; cool. Centrifuge for 10 minutes at 2,000 rpm and discard pellet Adjust sample pH to 6.0±0.5 with approximately 700 μL of 1.0 N NaOH Add 50 μL ethyl acetate and 50 μL MSTFA (with 3% trimethylsilyliodide) Overlay with N2 and cap Mix/vortex React for 20 minutes at 70 °C Remove from heat source to cool ANALYSIS RECOMMENDATIONS Inject 2 µl to a GC/MS COLUMN CONDITIONING RESULTS 3 mL methanol then aspirate 3 mL DI water then aspirate 1 mL 100 mM phosphate buffer (pH 6) then aspirate Ions to monitor (Mass Spectrometry) SAMPLE LOADING Load the previously prepared sample onto the SPE column at 1-2 mL/min flow rate COLUMN WASHING 3 mL of 10% (v/v) methanol in DI water Dry the column for 5 minutes 1 mL hexane or hexane/ethyl acetate (50:50) DRYING Dry the column for 5 minutes ELUTION Option 1: Elute with 3 mL of CH2Cl2/IPA/NH4OH (78:20:2) at 1-2 mL/min Option 2: 3 mL CH2Cl2/IPA (80:20) Option 3: 3 mL ethyl acetate Option 4: 3 mL methanol 28 POST TREATMENT Compound Primary Ion Secondary Testosterone-TMS 432 19-noretiocholanone-TMS405 Oxymethalone 640 Dehydroepiandosteronw-2TMS432 10-nortestosterone-2TMS418 Oxymethalone metabolite #1640 Oxymethalone metabolite #2625 11-ß-hydroxyandosterone522 Methandienone 409 19-norandosterone-2TMS405 Alpha-hydroxyetiocholanone504 17-a-epitestosterone-TMS432 Stanazolol 472 Tertiary Other 301209 315 225 52 462 370, 143 327 297 287 194 52 462 143 462 370 143 417 158 313281 315 225 417 341 327 209 381 342149 RECOMMENDED COLUMN PerkinElmer Elite-5 MS 15 m x 0.25 mm x 0.25 um Part No. N9316279 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 FORENSIC Antidepressants/Painkillers in Urine and Blood PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/15 ml, Plastic Tube (Straight) Part No. N9306713 SAMPLE PRETREATMENT CONCENTRATION To 1 mL of 100 mM phosphate buffer (pH 6.0) add internal standard* Evaporate to dryness at < 40 °C Add 1 mL of urine or blood POST TREATMENT Reconstitute sample in 100 μL of methanol Add 2 mL of 100 mM phosphate buffer (pH 6.0) ANALYSIS RECOMMENDATIONS Mix/vortex Adjust pH 6.0±0.5 with 100 mM of monobasic or dibasic sodium phosphate Inject 2 µl onto a LC RESULTS Ions to monitor (Mass Spectrometry) Mix/vortex Centrifuge * Suggested internal standards Amitriptyline-D3, Diphenhydramine-D3, Methadone-D9, Norpropoxyphene-D5, Propoxyphene-D11, Tramadol-D3 COLUMN CONDITIONING 3 mL methanol 3 mL DI water 1 mL 100 mM phosphate buffer (pH 6) SAMPLE LOADING Load the previously prepared sample onto the SPE column at 1-2 mL/min COLUMN WASHING 3 mL DI water 3 mL of 1% acetic acid 3 mL methanol DRYING Compound Amitriptyline Amitriptyline-D3 Diphenhydramine Diphenhydramine-D3 Doxepin EDDP EDDP-D3 Methadone Methadone-D9 Nortriptyline Norpropoxyphene Norpropoxyphene-D5 Propoxyphene Propoxyphene-D11 Sertraline Tramadol Tramadol-D3 Venlafaxine Zolpidem Detection Ions 278.8 / 91.1 281.2 / 91.2 256.2 / 167.1 259.2 / 167.1 280.2 / 107.1 278.2 / 234.2 281.4 / 234.3 310.2 / 105.1 319.2 / 268.3 264.2 / 91.1 326.2 / 44.1 331.1 / 267.1 340.2 / 58.1 351.3 / 64.0 308.1 / 161.0 264.2 / 58.1 268.2 / 58.0 278.2 / 58.2 308.2 / 235 RECOMMENDED COLUMN PerkinElmer Brownlee Validated C 18 150 mm x 2.1 mm x 5 um Part No. N9303556 Dry the column for 5 minutes RELATED ITEMS ELUTION Elute with 3 mL of ethyl acetate: acetonitrile: ammonia (78:20:2, v/v/v) at 1-2m L/min www.perkinelmer.com/spe Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 29 FORENSIC Benzoylecgonine in Urine PerkinElmer SPE Supra-Clean C18-S 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306462 SAMPLE PRETREATMENT Add deuterated internal standard (ISTD) to 1-2 mL of urine 2 mL 100 mM phosphate buffer (pH 6) COLUMN CONDITIONING BSTFA Ions: Compound Primary Ion Secondary Tertiary Benzoylecgonine240 361 256 d3-benzoylecgonine243 364 CHROMATOGRAM / SPECTRUM 3 mL methanol 3 mL DI water 1 mL 100 mM phosphate buffer (pH 6) SAMPLE LOADING Load the previously prepared sample at 1-2 mL/min COLUMN WASHING 3 mL DI water 100 mM HCL 1 mL of methanol ELUTION 3 mL Methylene chloride: Isopropanol:Ammonium Hydroxide (78:20:2) into conical tube CONCENTRATION Evaporate to dryness < 50 °C RECOMMENDED COLUMN POST TREATMENT Reconstitute in 50 uL PFPA Add 25 uL PFPOH Cover with N2, cap, mix, heat to 70 °C and hold for 20 min. Evaporate to dryness < 50 °C. Reconstitute in 100 μL ethyl acetate Transfer final treated sample to low volume autosampler vial insert ANALYSIS RECOMMENDATIONS Inject 1 μL into GC/MS RESULTS Three ion ratio chromatograms must all apex within ±2 scans of standard retention time. Ion ratios must fall within ±20% of standard ratios. Deuterated internal standards may use only 2 ions, a primary ion and only 1 confirmation ion. PerkinElmer Elite-5 12 m x 0.20 mm x 0.33 μm Part No. N9316110 Helium carrier – 2 mL/min RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 NOTES Acetic Acid, 100 mM = 2.86 mL glacial acetic acid diluted to 500 mL DI water. Phosphate buffer, 100 mM pH6 = 1.7 g Na2HPO4 + 12.14 g Na2HPO4 dilute to 1000 mL with DI water, adjust to pH6 with 100 mM Na2HPO4 (raises pH) or 100 mM Na2HPO4 (lowers pH). Methylene Chloride/Isopropanol/Ammonium Hydroxide(78:20:2) extraction solvent = 40 mL IP-OH + 4 mL concentrated NH4OH + 156 mL MeCl2. Make fresh daily. PFPA SIM Ions: Compound Primary Ion Secondary Tertiary Benzoylecgonine300 421 316 d3-benzoylecgonine303 424 30 REFERENCES Bezoylecgonine in Urine by SAMHSA GC/MS, Timothy D. Ruppel, PerkinElmer Application Note, Project I.D. 008424A_01Timothy D. Ruppel, PerkinElmer Application Note, Order No. 009784_01 FORENSIC Carboxy-delta-9-THC, Delta-9-THC, Delta-9-Hydroxy THC in Whole Blood PerkinElmer SPE Supra-Clean Mixed-Mode MM3 500 mg/6 mL, Plastic Tube (Straight) Part No. N9306649 SAMPLE PRETREATMENT CONCENTRATION Add internal standards* to 1 to 2 mlL of whole blood Evaporate to dryness < 40 °C Mix/vortex POST TREATMENT Vortex and add dropwise 1 mL of cold acetronitrile Add 50 µL of ethly acetate and 50µL of BSTFA (with 1% TMCS) Centrifuge and transfer acetronitrile to a clean tube Mix/vortex Adjust sample pH to 3.0±0.5 with approximately 2.0 mL of 100 mM sodium acetate buffer React for 20 minutes ot 70 °C * Suggested internal standards D3-Carboxy-delta-9-THC; D3-delta-9-THC Cool ANALYSIS RECOMMENDATIONS Inject 1 to 2 µL to a GC/MS COLUMN CONDITIONING 5 mL methanol 5 mL DI water 2 mL acetate buffer (pH 3.0) then aspirate SAMPLE LOADING Load sample at 1 to 2 mL/min COLUMN WASHING 5 mL DI water 4 mL 100 mM HCl/acetronitrile (95:5) Dry the column (5 to 10 minutes at >10"Hg/full flow for positive pressure manifold) 400 µL hexane then aspirate DRYING Optional: Dry the column for 5 minutes ELUTION 4 mL hexane 5 mL hexane/ethyl acetate (50:50) www.perkinelmer.com/spe RESULTS Ions to monitor (Mass Spectrometry) Compound Primary Ion Secondary Tertiary D3-Carboxy-delta-9-THC374 476 491 D9-Carboxy-delta-9-THC380 479 497 Carboxy-delta-9-THC371 473 488 D3-Hydroxy-delta-9-THC374 462 477 Hydroxy-delta-9-THC371 459 474 D3-delta-9-THC374 389 Delta-9-THC371 386 RECOMMENDED COLUMN PerkinElmer Elite-5 MS 15 m x 0.25 mm x 0.25 um Part No. N9316279 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 31 FORENSIC Delta-9-THC, Delta-9-Hydroxy THC, Carboxy-Delta-9-THC in Whole Blood PerkinElmer SPE Supra-Clean Mixed-Mode MM3 500 mg/6 mL, Plastic Tube (Straight) Part No. N9306649 SAMPLE PRETREATMENT CONCENTRATION Add internal standards* to 1 to 2 mL of whole blood Evaporate to dryness with air or nitrogen at < 40 °C Vortex and add dropwise 2 mL of cold acetronitrile Mix and centrifuge; transfer acetronitrile to a clean tube Add 50 µl of ethly acetate Evaporate acetronitrile under air or nitrogen to 200 µL Mix/vortex Add 2 mL DI water (pH=6.0 to 7.0) Add 50 µl BSTFA (with 1% TMCS) * Suggested internal standards D3-Carboxy-delta-9-THC; D3-delta-9THC; D3-Hydroxy-delta-9-THC React for 20 minutes ot 70 °C SAMPLE LOADING Load sample directly onto column (no preconditioning) Cool ANALYSIS RECOMMENDATIONS Inject 1 to 2 µL to a GC/MS COLUMN WASHING RECOMMENDED COLUMN Wash the column with 2.5 mL DI water/acetronitrile/ammonium hydroxide (84:15:1) PerkinElmer Elite-35 MS 30 m x 0.25 mm x 0.25 um Part No. N9316440 DRYING RELATED ITEMS Dry the column for 5 minutes Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 ELUTION 5 mL hexane/ethyl acetate/glacial Acetic acid (49:29:2) at a flow rate of 1 to 2 mL/min 32 POST TREATMENT FORENSIC Gamma-Hydroxybutyrate (GHB) in Blood, Urine, and Tissue PerkinElmer SPE Supra-Clean Mixed-Mode MM3 500 mg/6 mL, Plastic Tube (Straight) Part No. N9306649 SAMPLE PRETREATMENT CONCENTRATION Pipet 200 µL sample into 1.5 mL plastic tubes Evaporate to dryness at 70 °C under nitrogen Add 25 µL internal standard* Add 1 mL of acetone Centrifuge Transfer acetone layer to culture tube POST TREATMENT Add 100 µL ethyl acetate and 100µL BSTFA with 1% TCMS Mix/vortex Heat at at 70 °C for 30 minutes ANALYSIS RECOMMENDATIONS Evaporate extract with nitrogen at 70 °C Inject 1 to 2 µL to a GC/MS Reconstitute extracts with 200 µL of 100 mM phosphate buffer (pH 6.0) RESULTS Ions to monitor (Mass Spectrometry) Mix/vortex * Suggested internal standard D6-GHB Compound Primary Ion Secondary Tertiary GHB-D6-di-TMS239 240 241 GHB-di-TMS 233 234235 COLUMN CONDITIONING 5 mL methanol 5 mL DI water 2 mL 100 mM phosphate buffer (pH 6.0) SAMPLE LOADING Load the previously prepared sample onto the SPE column ELUTION RECOMMENDED COLUMN PerkinElmer Elite-35 MS 30 m x 0.25 mm x 0.25 um Part No. N9316440 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 2 mL methanol/ammonium hydroxide (99:1) in the original sample test tube Mix/vortex Pour into column and collect extract Aspirate at ~1"Hg www.perkinelmer.com/spe 33 FORENSIC Gamma-Hydroxybutyrate (GHB) in Urine without conversion to Gamma-Butrylactone (GBL) PerkinElmer SPE Supra-Clean Mixed-Mode MM3 500 mg/6 mL, Plastic Tube (Straight) Part No. N9306649 SAMPLE PRETREATMENT CLEANUP Pipet 200 µL sample into a test tubes Add 200 µL of dimethylformamide Add 1 mL of hexane saturated with dimethylformamide Mix by inversion for 5 minutes Centrifuge at 3000 rpm for 5 minutes. Transfer lower dimethylformamide layer to a clean test tube Evaporate to dryness at < 50 °C under air or nitrogen Add internal standard* and 100 µL 100mM phosphate buffer (pH 6.0) Mix/vortex * Suggested internal standard D6-GHB POST TREATMENT COLUMN CONDITIONING Add 100 µL ethyl acetate and 100 µL BSTFA with 1% TCMS 5 mL methanol 5 mL DI water ANALYSIS RECOMMENDATIONS 2 mL 100 mM phosphate buffer (pH 6.0) Inject 1 to 2 µL onto a GC/MS RESULTS SAMPLE LOADING Load the previously prepared sample onto the SPE column and aspirate at ~1"Hg Sample load and wash are both collected Ions to monitor (Mass Spectrometry) Compound Primary Ion Secondary Tertiary GHB-D6-di-TMS239 240 241 GHB-di-TMS 233 234235 COLUMN WASHING Add 2 mL methanol/sodium hydroxide (99:1) to the original s ample test Mix/vortex thoroughly Decant wash onto column and combine eluate with sample load collected in previous step RECOMMENDED COLUMN DRYING Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 Evaporate to dryness at 60 °C under air or nitrogen 34 PerkinElmer Elite-35 MS 30 m x 0.25 mm x 0.25 um Part No. N9316440 RELATED ITEMS FORENSIC Opiates in Urine PerkinElmer SPE Supra-Clean C18-S 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306462 SAMPLE PRETREATMENT CHROMATOGRAM / SPECTRUM Acid Hydrolysis: Combine 3 mL urine sample with ISTD and 500 mL conc. HCl, vortex, heat 30 min at 120 °C Cool, centrifuge, decant and keep top layer. Add 1 mL of 7.4 N ammonium hydroxide, vortex. Adjust pH to 6.0 with 1-3 mL of 500 mM phosphoric acid COLUMN CONDITIONING 3 mL methanol 3 mL DI water 1 mL 100 mM phosphate buffer (pH6) SAMPLE LOADING Load the prepared sample onto the SPE column at 1-2 mL/min COLUMN WASHING 3 mL DI water 1 mL 100 mM acetic acid 1 mL of methanol ELUTION 3 mL Methylene chloride:Isopropanol:Ammonium Hydroxide (78:20:2) into conical tube RECOMMENDED COLUMN PerkinElmer Elite-5 12 m x 0.20 mm x 0.33 μm Part No. N9316010 Helium carrier – 2 mL/min. RELATED ITEMS Evaporate to dryness < 50 °C Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 POST TREATMENT NOTES Reconstitute and derivatize dried extract with 50 mL ethyl acetate, 50 mL BSTFA with 1% TWCS Cover with nitrogen, cap, mix, heat 70 °C (20 min), cool, do not evaporate Acetic Acid, 100 mM = 2.86 mL glacial acetic acid diluted to 500 mL DI water. Phosphate buffer, 100 mM pH6 = 1.7 g Na2HPO4 + 12.14 g Na2HPO4 dilute to 1000 mL with DI water, adjust to pH6 with 100 mM Na2HPO4 (raises pH) or 100 mM Na2HPO4 (lowers pH). Methylene hloride/Isopropanol/Ammonium Hydroxide (78:20:2) extraction solvent = 40 mL IP-OH + 4 mL concentrated NH4OH + 156 mL MeCl2. Make fresh daily. CONCENTRATION ANALYSIS RECOMMENDATIONS Transfer final treated sample to low volume autosampler vial insert, inject 1 μL into GC/MS RESULTS BSTFA SIM Ions: REFERENCES Opiates in Urine by SAMHSA GC/MS, Timothy D. Ruppel, PerkinElmer Application Note, Project I.D. 009816_01 Drug Primary Ion** Secondary Tertiary Codeine 371 234343 d3-Codeine374237 Morphine 429 287324 d3-Morphine432 290 www.perkinelmer.com/spe 35 FORENSIC Sympathomimetic Amines in Urine PerkinElmer SPE Supra-Clean C18-S 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306462 SAMPLE PRETREATMENT Add deuterated internal standard (ISTD) to 2 mL of urine Add 2 mL 100 mM phosphate buffer (pH 6) COLUMN CONDITIONING 3 mL methanol 3 mL DI water 1 mL 100 mM phosphate buffer (pH 6) SAMPLE LOADING Load previously prepared sample at 1 to 2 mL/min COLUMN WASHING 3 mL DI water 1ml of 100 mM acetic acid 1 mL of methanol HFBA SIMS Ions: Drug Primary Ion Secondary Tertiary Amphetamine118 240 91 d5-amphetamine244 123 d8-amphetamine243 126 Methamphetamine118 210 254 d5-methamphetamine258 213 d8-methamphetamine261 213 TFAA SIMS Ions: Drug Primary Ion Secondary Tertiary Amphetamine118 140 91 d5-amphetamine144 123 d8-amphetamine143 126 Methamphetamine154 118 110 d5-methamphetamine158 113 d8-methamphetamine161 113 CHROMATOGRAM ELUTION 3 mL Methylene chloride: Isopropanol:Ammonium Hydroxide (78:20:2) into conical tube CONCENTRATION Evaporate to dryness < 50 °C POST TREATMENT Reconstitute in 50 uL derivatization reagent (PFPA, HFBA or TFAA) Cover with N2, cap, mix, heat 70 °C (20 min) Evaporate todryness < 50 °C. Reconstitute in 100 μL ethyl acetate RECOMMENDED COLUMN PerkinElmer Elite-5 12 m x 0.20 mm x 0.33 μm Part No. N9316010 Helium carrier – 2 mL/min. ANALYSIS RECOMMENDATIONS RELATED ITEMS Transfer final treated sample to low volume autosampler vial insert, inject 1 μL into GC/MS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 RESULTS PFPA SIMS Ions: Drug Primary Ion Secondary Tertiary Amphetamine190 118 91 d5-amphetamine194 123 d8-amphetamine193 126 Methamphetamine204 160 118 d5-methamphetamine208 163 d8-methamphetamine211 163 NOTES Acetic Acid, 100 mM = 2.86 mL glacial acetic acid diluted to 500 mL DI water. Phosphate buffer, 100 mM pH6 = 1.7 g Na2HPO4 + 12.14 g Na2HPO4 dilute to 1000 mL with DI water, adjust to pH6 with 100 mM Na2HPO4 (raises pH) or 100 mM Na2HPO4 (lowers pH). Methylene hloride/Isopropanol/Ammonium Hydroxide(78:20:2) extraction solvent = 40 mL IP-OH + 4 mL concentrated NH4OH + 156 mL MeCl2. Make fresh daily. REFERENCES Sympathomimetic Amines in Urine by SAMHSA GC/MS, Timothy D. Ruppel, PerkinElmer Application Note, Project I.D. 009784_01 36 PHARMACEUTICAL Acetominophen in Serum PerkinElmer SPE Supra-Poly XC 60 mg/3 mL, Plastic Tube (Straight) Part No. N9306502 SAMPLE PRETREATMENT COLUMN WASHING Dilute 10 mg of acetaminophen in 100 mL of water to give a 100 ppm solution 2 mL of methanol/DI water (5/95, v/v) Mix 25 mL of acetaminophen (100 ppm) solution in a 50 mL flask and dilute to volume with water (standard solution 50 ppm) Mix 25 mL of acetaminophen (100 ppm) solution in a 50 mL flask and dilute to volume with serum and 1% H3PO4 (sample 50% serum and 1% H3PO4) DRYING Dry column (5 to 10 minutes ) ELUTION 2 mL of methanol CONCENTRATION COLUMN CONDITIONING 2 mL of methanol 2 mL of DI water Evaporate to dryness at < 50 °C using nitrogen Reconstitute sample using 1 mL of mobile phase RELATED ITEMS SAMPLE LOADING Load 2 mL of prepared sample www.perkinelmer.com/spe Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 37 PHARMACEUTICAL Acetominophen in Solution PerkinElmer SPE Supra-Poly XC 60 mg/3 mL, Plastic Tube (Straight) Part No. N9306502 SAMPLE PRETREATMENT CONCENTRATION Prepare a 5 ppm sample in 0.2% ammonium acetate Evaporate to full dryness under a stream of nitrogen Reconstitute the sample to 1 mL with water Adjust to pH 5 COLUMN CONDITIONING 2 mL of methanol 2 mL of DI water SAMPLE LOADING Mobile phase: acetonitrile:1 mM KH2PO4 (30:70, v/v) Flow: 1 mL/minute Temperature: 30 °C Detection: UV 254 nm Load 4 mL samples at 1-2 mL/minute RECOMMENDED COLUMN COLUMN WASHING PerkinElmer Brownlee Validated C18, 5 um, 250 x 4.6 mm Part No. N9303561 1 mL of DI water ELUTION 5 mL of methanol 38 ANALYSIS RECOMMENDATIONS RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 PHARMACEUTICAL Benzodiazepine Screen in Blood, Serum, and Tissue by GC/MS PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306543 SAMPLE PRETREATMENT CONCENTRATION To 1 ml of 100 mM phosphate buffer (pH=6) add internal standards* Evaporate to dryness at < 40 °C under nitrogen Add 1 mL blood or 1 g of (1:4) tissue homogenate POST TREATMENT Mix/vortex Add 50 µl acetonitrile and 50 µl BSTFA with 1% TCMS Heat at 70 °C for 30 minutes Add 3 mL of 100 mM phosphate buffer (pH= 6) ANALYSIS RECOMMENDATIONS Sample pH should be 6.0 ±0.5 (Adjust pH accordingly with 100 mM monobasic or dibasic sodium phosphate) Inject 1 to 2 µl to a GC/MS Mix/vortex and centrifuge as appropriate * Suggested internal standards Alprazolam-D, Alphahydroxyalprazolam-D5, Diazepam-D5, Lorazepam-D4, Nordiazepam-D5, Oxazepam-D5, Temazepam-D5 COLUMN CONDITIONING 3 mL methanol 3 mL 100 mM phosphate buffer 9pH 6) SAMPLE LOADING Load previously prepared sample at 1 to 2 mL/min COLUMN WASHING RESULTS Ions to monitor (Mass Spectrometry) Compound Primary Ion Secondary Tertiary Alprazolam 308 279204 Alprazolam-D**513 284 Alphahydroxyalprazolam318 396 383 Alphahydroxyalprazolam-D5386 401 Diazepam 256 283 284 Diazepam-D5** 287 289 Lorazepam 429 430347 Lorazepam-D4**433 435 Nordiazepam 34 342343 Nordiazepam-D5**345 347 Oxazepam 429 313430 Oxazepam-D5**435 433 Temazepam 343 257283 Temazepam-D5**348 262 3 mL of 5% (v/v) acetonitrile in 100 mM phosphate buffer (pH=6.0) Dry the column for 5 minutes under Vacuum 3 mL hexane ** Suggested internal standards DRYING PerkinElmer Elite-5 MS 30 m x 0.25 mm x 0.25 um Part No. N9316282 Dry the column for 5 minutes under vacuum ELUTION 3 mL ethyl acetate/ammonia (98:2 v/v) www.perkinelmer.com/spe RECOMMENDED COLUMN RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 39 PHARMACEUTICAL Benzodiazepine Screen in Urine by GC/MS PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306543 SAMPLE PRETREATMENT CONCENTRATION To 1mL of acetate buffer (pH=5) containing 5,000 F units/mL of ß -Glucuronidase and add internal standards* Evaporate to dryness at < 40 °C under nitrogen Add 1 mL Urine Mix/vortex Add 50 µl acetonitrile and 50 µl BSTFA with 1% TCMS Heat at 70 °C for 30 minutes Hydrolyze for 3 hours at 65 °C then cool ANALYSIS RECOMMENDATIONS Centrifuge for 10 minutes at 2000 rpm and discard pellet Inject 1 to 2 µl to a GC/MS Add 3 mL of 100 mM phosphate buffer (pH 6.0) and mix RESULTS * Suggested internal standards Alprazolam-D, Alphahydroxyalprazolam-D5, Diazepam-D5, Lorazepam-D4, Nordiazepam-D5, Oxazepam-D5, Temazepam-D5 Ions to monitor (Mass Spectrometry) COLUMN CONDITIONING 3 mL methanol 3 mL 100 mM phosphate buffer 9pH 6) SAMPLE LOADING Load previously prepared sample at 1 to 2 mL/min COLUMN WASHING 3 mL of 5% (v/v) acetonitrile in 100 mM phosphate buffer (pH=6.0) Dry the column for 5 minutes under Vacuum 3 mL hexane DRYING 40 POST TREATMENT Compound Primary Ion Secondary Tertiary Alprazolam 308 279204 Alprazolam-D** 513 284 Alphahydroxyalprazolam318 396 383 Alphahydroxyalprazolam-D5386 401 Diazepam 256 283 284 Diazepam-D5** 287 289 Lorazepam 429 430347 Lorazepam-D4**433 435 Nordiazepam 34 342343 Nordiazepam-D5**345 347 Oxazepam 429 313430 Oxazepam-D5**435 433 Temazepam 343 257283 Temazepam-D5**348 262 ** Suggested internal standards RECOMMENDED COLUMN Dry the column for 5 minutes under vacuum PerkinElmer Elite-5 MS 30 m x 0.25 mm x 0.25 um Part No. N9316282 ELUTION RELATED ITEMS 3 mL ethyl acetate/ammonia (98:2 v/v) Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 PHARMACEUTICAL Beta Agonists in Urine PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306543 SAMPLE PRETREATMENT CONCENTRATION To 1 mL of 100 mM acetate buffer (pH 4.5) add 1 mL of urine Evaporate to dryness at < 40 °C Add 2 mL of 100 mM Acetate buffer (pH 4.5) POST TREATMENT Mix/vortex Prepare Derivatization solution: Methaneboronic acid at 5 mg/mL prepared in dry ethyl acetate (use molecular sieve). Store this solution at –20 °C (freezer conditions) until use Add 100 μL of the methaneboronic acid solution to eluate Mix/vortex React 15 minutes at 70 °C; cool Centrifuge COLUMN CONDITIONING 3 mL of methanol 3 mL of DI water 3 mL 100 mM acetate buffer (pH 4.7) ANALYSIS RECOMMENDATIONS Inject 1 to 2 µl to a GC/MS SAMPLE LOADING Load the previously prepared sample onto the column at 1-2 mL/min RECOMMENDED COLUMN PerkinElmer Elite-5 MS 30 m x 0.25 mm x 0.25 um Part No. N9316282 COLUMN WASHING 2 x 1 mL acetone/methanol (1:1) DRYING Dry the column for 5 minutes under vacuum RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 ELUTION Elute with 1 mL dichloromethane/isopropanol/ammonium hydroxide (78:20:2) at 1-2 mL/min www.perkinelmer.com/spe 41 PHARMACEUTICAL Beta Blockers in Blood and Urine PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306543 SAMPLE PRETREATMENT ELUTION To 1 mL of 100 mM acetate buffer (pH 4.5) add 1 mL of blood or urine 1 mL dichloromethane/isopropanol/ammonium hydroxide (78:20:2) Add 2 mL of 100 mM Acetate buffer (pH 4.5) CONCENTRATION Mix/vortex Centrifuge POST TREATMENT 3 mL of methanol 3 mL of DI water 3 mL of 100 mM acetate buffer (pH 4.5) Prepare Derivatization solution: Methaneboronic acid at 5 mg/mL prepared in dry ethyl acetate (use molecular sieve). Store this solution at –20 °C (freezer conditions) until use Add 100 μL of the methaneboronic acid solution to eluate Mix/vortex React 15 minutes at 70 °C; cool SAMPLE LOADING ANALYSIS RECOMMENDATIONS Load the previously prepared sample onto the SPE column at 1-2 mL/min Inject 1 to 2 µl to a GC/MS COLUMN CONDITIONING COLUMN WASHING 42 Evaporate to dryness at < 40 °C RECOMMENDED COLUMN 2 x 1 mL acetone/methanol (1:1) PerkinElmer Elite-5 MS 30 m x 0.25 mm x 0.25 um Part No. N9316282 DRYING RELATED ITEMS Dry the column for 5 minutes under vacuum Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 PHARMACEUTICAL Caffeine, Theophylline and Theobromine in Blood, Plasma/Serum, and Urine PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306543 SAMPLE PRETREATMENT DRYING To 1 mL of 100 mM Acetic Acid add internal standard* Dry the column for 5 minutes under vacuum Add 1 ml blood, plasma/serum, or urine ELUTION Add 2 mL of 100 mM Acetic Acid 3 mL ethyl acetate/methanol (90:10) Mix/vortex Centrifuge * Suggested internal standard 8-Chlorotheophylline CONCENTRATION Evaporate to dryness at < 40 °C POST TREATMENT COLUMN CONDITIONING Reconstitute sample in 1000 µl of 0.1% formic acid (aq) 3 mL of methanol 3 mL of DI water 1 mL of 100 mM acetic acid ANALYSIS RECOMMENDATIONS SAMPLE LOADING RELATED ITEMS Load the previously prepared sample onto the SPE column at 1-2 mL/min Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 COLUMN WASHING Inject 20 µl on to an LC 3 mL DI water 3 mL 100 mM acetic acid www.perkinelmer.com/spe 43 PHARMACEUTICAL Cefaperazone in Water PerkinElmer SPE Supra-Poly XC 60 mg/3 mL, Plastic Tube (Straight) Part No. N9306502 SAMPLE PRETREATMENT ANALYSIS RECOMMENDATIONS 5 ppm of cefoperazone in water 2 mL of methanol 2 mL of DI water Mobile phase: 0.005 M tetrabutyl ammonium phosphate (pH 3.63)/ACN (70:30, v/v) Flow: 1 mL/minute Temperature: 30 °C Detection: UV 254 nm SAMPLE LOADING RECOMMENDED COLUMN Load 10 mL samples at 1 to 2 mL/minute PerkinElmer Brownlee Validated Aqueous C18 250 mm x 4.6 mm x 5 um Part No. N9303549 COLUMN CONDITIONING COLUMN WASHING 1 mL of methanol/DI water (5:95, v/v) ELUTION 6 mL of methanol 44 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 PHARMACEUTICAL Clonazepam and 7-Aminoclonazepam in Urine PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306543 SAMPLE PRETREATMENT CONCENTRATION ß-Glucuronidase hydrolysis: To 2 mL of urine add internal standard and 1 mL ß-Glucuronidase solution* Evaporate to dryness at <40 °C under nitrogen ß-Glucuronidase contains 5,000 F units/mL Patella vulgata in 100 mM acetate buffer (pH 5.0) POST TREATMENT Mix/vortex Add 50 μL ethyl acetate and 50 μL MTBSTFA (with 1% TBDMCS) Mix/vortex React 20 minutes 90°C; cool Hydrolyze for 3 hours at 65 °C; cool ANALYSIS RECOMMENDATIONS * Suggested internal standard Clonazepam-D4, 7-aminoclonazepam-D4 Inject 1 to 2 µl to a GC/MS RESULTS COLUMN CONDITIONING 3 mL methanol 3 mL water Continue to condition with 1 ml 100 mM phosphate buffer (pH 6.0) SAMPLE LOADING Load the previously prepared sample onto the SPE column COLUMN WASHING 2 mL DI water Wash the column with 2 mL 20% acetronitrile in 100 mM phosphate buffer (pH 6.0) Dry the column for 5 minutes 2 mL hexane ELUTION Elute with 3 mL ethyl acetate with 2% ammonium hydroxide at 1-2 mL/min flow www.perkinelmer.com/spe Ions to monitor (Mass Spectrometry) Compound Primary Ion Clonazepam-TBDMS372 7-Aminoclonazepam-TBDMS342 Clonazepam-D4-TBDMS376 7-Aminoclonazepam-346 D4-TBDMS Secondary 374 344 378 348 Tertiary 326 399 377 403 RECOMMENDED COLUMN PerkinElmer Elite-5 MS 30 m x 0.25 mm x 0.25 um Part No. N9316282 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 45 PHARMACEUTICAL DHEA, Testosterone, and Epitestosterone in Urine PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306543 SAMPLE PRETREATMENT CONCENTRATION To 5 mL of urine, add internal standard* and adjust sample pH to 5.5-6.5 using concentrated sodium phosphate monobasic or dibasic Add 5 mL of n-butyl chloride to sample. Cap the tube and shake vigorously for 10 minutes and then centrifuge at 3,000 rpm for 5 minutes. Transfer the organic layer to clean test tube and dry under a stream of nitrogen. Place dried sample in a desiccator and further dry under Vacuum for 30 minutes. Mix Centrifuge samples at 3,000 rpm for 5 minutes * Suggested internal standard at 20ng/mL: 16 a Hydroxytestosterone COLUMN CONDITIONING 3 mL methanol 3 mL DI water 1 mL 100 mM phosphate buffer (pH 6.0) POST TREATMENT Add 50 μL of MSTFA/NH4l/dithioerythritol (1,000:2:5, V/W/W) and incubate at 70 °C for 20 minutes Centrifuge at 3,000 rpm for 1 minutes and transfer to GC vial ANALYSIS RECOMMENDATIONS Inject 1 to 2 µl to a GC/MS SAMPLE LOADING RESULTS Load the previously prepared sample onto the SPE columnand allow to flow via gravity Ions to monitor (Mass Spectrometry) COLUMN WASHING 3 mL DI water Cmopound Primary Ion Secondary Testosterone432 417 Epitestosterone432 417 DHEA 432417 16 a Hydroxytestosterone 520 259 DRYING Dry the column for 10 minutes under vacuum ELUTION 3 mL methanol at 1-2 mL/min flow rate CLEANUP Dry eluate under a stream of nitrogen. Add 2 mL of 200 mM phosphate buffer (pH 7.0) and 250 units of ß-glucuronidase Mix/vortex and allow to incubate at 50 °C for 1 hour. Cool sample, cap and adjust the pH to 10 to 11 using a 1:1 mixture of NaHCO3/Na2CO3 46 RECOMMENDED COLUMN PerkinElmer Elite-5 MS 30m x 0.25mm x 0.25um Part No. N9316282 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 PHARMACEUTICAL Doxepin in Rat Serum PerkinElmer SPE Supra-Poly XC 60 mg/3 mL, Plastic Tube (Straight) Part No. N9306502 SAMPLE PRETREATMENT CONCENTRATION Mix 10 mL of doxepin aqueous solution (20mg/L) and 30 mL of rat serum into a 100 mL vessel Evaporate to full dryness at room temperature under a stream of nitrogen Reconstitute the sample to 1 mL with ACN:20 mM sodium acetate (pH 4) (40:60, v/v) Dilute to 100 mL with 0.5% ammonia solution to give a 2 ppm solution COLUMN CONDITIONING 2 mL of methanol 2 mL of DI water SAMPLE LOADING ANALYSIS RECOMMENDATIONS Mobile phase: ACN:20 mM sodium acetate (pH 4) (40:60, v/v) Flow: 1.0 mL/minute Injection: 10 μL Temperature: 30 °C Detection: 290 nm Load 2mL samples RECOMMENDED COLUMN COLUMN WASHING 2 mL of 0.5% ammonia solution containing 5% methanol PerkinElmer Brownlee Validated C18 250 mm x 4.6 mm x 5 um Part No. N9303561 ELUTION RELATED ITEMS 2 mL of 1% acetic acid in methanol Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 www.perkinelmer.com/spe 47 PHARMACEUTICAL Gabapentin in Blood, Plasma/Serum PerkinElmer SPE Supra-Clean C18-S 100 mg/1 mL, Plastic Tube (Straight) Part No. N9306478 SAMPLE PRETREATMENT CONCENTRATION To 500 μL of 20% acetic acid add internal standard* Evaporate to dryness at < 40 °C Mix thoroughly POST TREATMENT Add 500 μL of blood, plasma/serum Derivitize by adding 50 μL of ethyl acetate and 50 μL of BSTFA (1% Mix thoroughly Centrifuge as appropriate TCMS) and 50 μL ethyl acetate Cap and heat at 70 °C for 30 minutes Remove and allow to cool COLUMN CONDITIONING ANALYSIS RECOMMENDATIONS 3 mL of methanol 3 mL of DI water 1 mL 100 mM HCl Inject 1 to 2 μL onto GC/MS SAMPLE LOADING Load at 1 to 2 mL/minute COLUMN WASHING PerkinElmer Elite-5MS 30 m x 0.25 mm x 0.25 μm Part No. N9316282 RELATED ITEMS 3 mL DI H2O 3 mL ethyl acetate 3 mL hexane Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 DRYING NOTES Dry column (5 minutes at >10"Hg) or until column is dry * Internal standard: 1-aminomethyl-1-cycloheptyl acetic acid (FID):Gabapentin-D10 (GC/MS). BSTFA + TCMA = N,Obis[Trimethylsilyl]trifluoroacetamide plus Trimethylchlorosilane ELUTION 1 mL 2% ammonium hydroxide in methanol 48 RECOMMENDED COLUMN PHARMACEUTICAL Gabapentin in Blood, Plasma/Serum PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306543 SAMPLE PRETREATMENT DRYING To 0.2 to 0.5 mL of sample add 1 mL of acetone (dropwise) while vortexing Dry the column for 10 minutes under vacuum Add internal standards* ELUTION Mix/Vortex and centrifuge 3 mL methanol containing 2% ammonium hydroxide at 1-2 mL/min flow rate Transfer organic phase to ctean test tube and evaporate to dryness CONCENTRATION Add 3 ml of 100 mM HCL Evaporate to dryness at < 40 °C Mix/Vortex and centrifuge POST TREATMENT * Suggested internal standards: Gabapentin-D10, Aminocyclohexane-propionic acid Dissolve residue in 100 µl methanol ANALYSIS RECOMMENDATIONS COLUMN CONDITIONING 3 mL methanol 3 mL water 1 mL 100mM HCl SAMPLE LOADING Load the previously prepared sample onto the SPE column at 1-2 mL/min COLUMN WASHING 3 mL DI water 3 mL ethyl acetate 3 mL hexane www.perkinelmer.com/spe Inject 5 µl on to an LC/MS RECOMMENDED COLUMN PerkinElmer Brownlee Validated C18 100 mm x 2.1 mm x 3 um Part No. N9303551 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 49 PHARMACEUTICAL Gabapentin in Blood, Plasma/Serum PerkinElmer SPE Supra-Clean C18-S 100 mg/1 mL, Plastic Tube (Straight) Part No. N9306478 SAMPLE PRETREATMENT POST TREATMENT Add internal standard to 500 mL of 20% Acetic acid and mix thoroughly Add 50 μL of ethyl acetate and 50 μL of BSTFA (1%TCMS) and 50 μL ethyl acetate Cap and heat at 70 °C for 30 minutes Remove and allow to cool Add 500 μL of blood, plasma/serum and mix thoroughly and centrifuge if necessary COLUMN CONDITIONING 3 mL methanol 3 mL DI water 1 mL 100 mM HCl SAMPLE LOADING Load at 1 to 2 mL/minute ANALYSIS RECOMMENDATIONS Transfer final treated sample to low volume autosampler vial insert, inject 1 -2 μL into GC/MS RECOMMENDED COLUMN PerkinElmer Elite-5MS 30 m x 0.25 mm x 0.25 μm Part No. N9316282 RELATED ITEMS COLUMN WASHING 3 mL DI water 3 mL ethyl acetate 3 mL hexane Dry column (5 minutes at >10"Hg) or until column is dry Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 NOTES ELUTION 1 mL 2% ammonium hydroxide in methanol CONCENTRATION Evaporate to dryness at < 40°C 50 Internal standard: 1-aminomethyl-1-cycloheptyl acetic acid (FID): Gabapentin-D10 (GC/MS). BSTFA + TCMA = N,O-bis[Trimethylsilyl] trifluoroacetamide plus Trimethylchlorosilane. PHARMACEUTICAL Ketotifen Fumarate in Solution PerkinElmer SPE Supra-Poly XC 60 mg/3 mL, Plastic Tube (Straight) Part No. N9306502 SAMPLE PRETREATMENT ELUTION Dilute 50 mg of ketotifen fumarate in 50mL of 0.5% ammonia aqueous solution 2mL of methanol POST TREATMENT COLUMN CONDITIONING 2 mL of methanol 2 mL of DI water Evaporate to full dryness at room temperature under a stream of nitrogen Reconstitute sample in methanol for analysis SAMPLE LOADING RELATED ITEMS Load 2mL sample Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 COLUMN WASHING 2mL of DI water www.perkinelmer.com/spe 51 PHARMACEUTICAL Lovastatin in Water PerkinElmer SPE Supra-Clean C18-S 100 mg/1 mL, Plastic Tube (Straight) Part No. N9306478 SAMPLE PRETREATMENT CONCENTRATION Sample 1: 2 ppm lovastatin in 10% acetonitrile aqueous solution Sample 2: 0.2 ppm lovastatin in 1% acetonitrile aqueous solution Evaporate to full dryness under a stream of nitrogen Reconstitute the sample to 1 mL with methanol:1% formic acid solution (85:15, v/v) COLUMN CONDITIONING ANALYSIS RECOMMENDATIONS 2 mL of methanol 2 mL of DI water Mobile phase: methanol:1% formic acid solution (85:15, v/v) Flow: 1 mL/minute Temperature: 30 °C Detection: UV 230 nm SAMPLE LOADING Load 10 mL samples at 1 to 2 mL/minute COLUMN WASHING 1 mL of DI water ELUTION 4 mL of acetonitrile 52 RECOMMENDED COLUMN PerkinElmer Brownlee Validated Aqueous C18 150 mm x 4.6 mm x 5 um Part No. N9303546 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 PHARMACEUTICAL Methylmalonic Acid from Serum/Plasma PerkinElmer SPE Supra-Clean C18-S 100 mg/1 mL, Plastic Tube (Straight) Part No. N9306478 SAMPLE PRETREATMENT CONCENTRATION Add 100 μL of internal standard D3-MMA and 1 mL of acetonitrile to 1 mL of sample Dry under a stream of nitrogen at < 35 °C Mix thoroughly Centrifuge for 5 minutes at 2,000 rpm POST TREATMENT Derivatize by Reconstituting with 25 μL of MSTFA + 1% TMCS and 25 μL ethyl acetate Heat for 20 minutes at 60 °C COLUMN CONDITIONING 3 mL methanol 3 mL DI water ANALYSIS RECOMMENDATIONS Transfer final treated sample to low volume autosampler vial insert, inject 1 -2 μL into GC/MS SAMPLE LOADING Decant supernatant onto SAX column RECOMMENDED COLUMN COLUMN WASHING PerkinElmer Elite-5MS 30 m x 0.25 mm x 0.2 5 μm Part No. N9316282 10 mL of DI water 5 mL of methanol 2 mL of MTBE RELATED ITEMS Dry with Vacuum for 3 minutes after each wash step Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 ELUTION NOTES 5 mL of 3% formic acid in MTBE, collect at 1 to 2 mL/min MTBE - Methyl tertiary-butyl ether DRYING www.perkinelmer.com/spe 53 PHARMACEUTICAL Olanzapine in Whole Blood PerkinElmer SPE Supra-Clean Cyano 500 mg/3 mL, Plastic Tube (Straight) Part No. N9306645 SAMPLE PRETREATMENT ELUTION Add internal standard* to 1 ml of DI water 5 mL 1% acetic acid in methanol at 1 to 2 mL/min Repeat the previous elution step Add 1 ml blood Add 8 ml of DI water CONCENTRATION Mix/vortex and centrifuge Evaporate to dryness at < 40 °C *Suggested internal standard Prazepam POST TREATMENT Reconstitute sample in 100 μL 0.1% aqueous trifluoroacetic acid COLUMN CONDITIONING 5 mL of methanol 5 mL of DI water ANALYSIS RECOMMENDATIONS Inject 50 μL into HPLC SAMPLE LOADING RECOMMENDED COLUMN Load at 1 to 2 mL/minute PerkinElmer Brownlee Choice C18 150 mm x 4.6 mm x 3 um Part No. N9303624 COLUMN WASHING 5 mL of 1% acetic acid in water DRYING Dry the column for 5 minutes 54 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 PHARMACEUTICAL Paroxetine in Blood, Plasma/Serum and Urine PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306543 SAMPLE PRETREATMENT CONCENTRATION To 1 mL of 100 mM phosphate buffer (pH= 6) add internal standards* Evaporate to dryness at <40 °C under nitrogen Add 1 mL whole blood, serum/plasma or urine POST TREATMENT Dissolve residue in 100 μL methanol Add 2 mL of 100 mM phosphate buffer (pH= 6) Mix/vortex and centrifuge as appropriate ANALYSIS RECOMMENDATIONS Inject 5 μL onto LC/MS * Suggested internal standard: Paroxetine-D6 RESULTS COLUMN CONDITIONING 3 mL methanol 3 mL water 3 mL 100 mM phosphate buffer (pH 6) SAMPLE LOADING Load the previously prepared sample onto the SPE column Drug MRM Transition Paroxetine330.0/190.1 Paroxetine-D6336.0/76.1 RECOMMENDED COLUMN PerkinElmer Brownlee Validated C 8 100 mm x 4.6 mm x 5 um Part No. N9303565 COLUMN WASHING 3 mL DI water 3 mL 100 mM acetic acid 3 mL methanol ELUTION 3 mL ethyl acetate:acetonitrile:ammonium hydroxide (78:20:2) at a flow rate of 1-2 mL/min www.perkinelmer.com/spe RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 55 PHARMACEUTICAL Propranolol in Rat Serum PerkinElmer SPE Supra-Poly XC 60 mg/3 mL, Plastic Tube (Straight) Part No. N9306502 SAMPLE PRETREATMENT CONCENTRATION Mix 10 mL of Propranolol aqueous solution (100 mg/L) and 30mL of rat serum in to a 100 mL vessel Evaporate to full dryness at room temperature under a stream of nitrogen Reconstitute the sample to 1 mL with ACN:20 mM sodium acetate (pH 4) (30:70, v/v) Dilute to 100 mL with 0.5% ammonia solution to give a 10 ppm solution ANALYSIS RECOMMENDATIONS COLUMN CONDITIONING 2 mL of methanol 2 mL of DI water SAMPLE LOADING Load 2mL samples COLUMN WASHING 2 mL of 0.5% ammonia solution containing 5% methanol ELUTION 2 mL of 1% acetic acid in methanol 56 Mobile phase: ACN:20 mM sodium acetate (pH 4) (40:60, v/v) Flow: 1.0 mL/minute Injection: 10 μL Temperature: 30 °C Detection: UV 290 nm RECOMMENDED COLUMN PerkinElmer Brownlee Validated Aqueous C18 250 mm x 4.6 mm x 5 um Part No. N9303549 RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 PHARMACEUTICAL Tacrolimus, Cyclosporin and Rapamycin in Whole Blood PerkinElmer SPE Supra-Poly XC 200 mg/6 mL, Plastic Tube (Straight) Part No. N9306635 SAMPLE PRETREATMENT DRYING Add 50 mL whole blood and 50mL of 0.1 M ZnSO4 to a centrifuge tube Dry column (20 minutes at >10"Hg) Thoroughly mix Add 500 mL methanol and internal standards* ELUTION Add 750 mL of ethyl acetate Collect eluate at 1 to 2 mL/minute Thoroughly mix Centrifuge for 2 min at 2000 rpm RECOMMENDED COLUMN Transfer supernate to a clean tube, add 500 mL DI H2O PerkinElmer Brownlee Analytical C18 150 mm x 4.6 mm x 3 um Part No. N9303508 Thoroughly mix COLUMN CONDITIONING 2 mL of methanol 2 mL of DI water SAMPLE LOADING Decant the sample onto the column Load at 1 to 2 mL/minute RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 NOTES * Internal standards: Cyclosporin Cyclosporin-D, Tacrolimus Ascomycin, and Rapamycin Desmethoxyrapamycin COLUMN WASHING 2 mL DI water www.perkinelmer.com/spe 57 PHARMACEUTICAL Tricyclic Antidepressants in Plasma/Serum PerkinElmer SPE Supra-Clean Mixed-Mode MM1 200 mg/3 mL, Plastic Tube (Straight) Part No. N9306543 SAMPLE PRETREATMENT DRYING To 2 mL of 100 mM phosphate buffer (pH= 6.0) add internal standard* Dry the column for 5 minutes under vacuum Add 1 mL of plasma/serum Mix/vortex Centrifuge for 10 minutes at 2,000 rpm and discard pellet Adjust pH to 6.0±0.5 with 100 mM monobasic or dibasic sodium phosphate * Suggested internal standards: Trimipramine, Protriptyline COLUMN CONDITIONING 3 mL methanol 3 mL water 3 mL 100 mM phosphate buffer (pH=6) SAMPLE LOADING Load at 1 mL/min flow rate COLUMN WASHING 3 mL DI water 1 mL of 100 mM acetic acid 3 mL methanol 58 ELUTION 3 mL CH2Cl2/IPA/NH4OH (78:20:2) at 1 mL/min or gravity flow CONCENTRATION Evaporate to dryness at < 40 °C POST TREATMENT Reconstitute with 200 μL ethyl acetate/DI H2O (1:3) Mix/vortex ANALYSIS RECOMMENDATIONS Inject 100 μL onto HPLC RELATED ITEMS Vacuum Manifold -12 Position; Part No. N9306619 Vacuum Manifold -24 Position; Part No. N9306626 Vacuum Pump 20 L/min 115V, Part No. N9308035 Vacuum Pump 17 L/min 230V, Part No. N9308331 ORDERING INFORMATION Supra-Clean Columns and 96 Well Plates PerkinElmer offers a comprehensive selection of SPE products that have been precisely manufactured for guaranteed reproducibility. Not just column to column. Not just batch to batch. But lot to lot — no matter how many days, weeks or years between production times. Supra-Clean and Supra-Poly SPE products are availabe in a range of media weights, volumes and bundles, including easy to order selection kits and application packs. SUPRA-CLEAN COLUMNS AND 96 WELL PLATES • Pure spherical silica • Pore size 60 - 120 Å • 20 chemistries with pH range 2-8 Supra-Clean C18 Supra-Clean REC18 Supra-Clean C18 Columns Supra-Clean REC18 Columns Media Weight Volume Part No. Quantity Media Weight Volume Part No. Quantity 50 mg 1 ml N9306476 50 50 mg 1 ml N9306519 50 100 mg 1 ml N9306478 100 100 mg 1 ml N9306520 100 100 mg 3 ml N9306523 50 100 mg 3 ml N9306455 50 3 ml N9306521 50 200 mg 3 ml N9306462 50 200 mg 500 mg 3 ml N9306438 50 500 mg 3 ml N9306522 50 6 ml N9306633 30 500 mg 3 ml* N9306642 50 200 mg 200 mg 6 ml N9306634 30 500 mg 6 ml N9306457 30 1g 6 ml N9306491 30 Part No. Quantity 500 mg 6 ml N9306448 30 500 mg 6 ml* N9306640 30 1g 6 ml N9306422 30 2g 6 ml N9306430 30 2g 15 ml N9306479 20 2g 25 ml N9306475 20 Supra-Clean REC18 96 Well Plates Media Weight Volume 25 mg 2 ml N9306563 1 50 mg 2 ml N9306564 1 100 mg 2 ml N9306565 1 Part No. Quantity * Not end-capped Supra-Clean 300Å C4 Supra-Clean C18 96 Well Plates Media Weight Volume Part No. Quantity Supra-Clean 300Å C4 Columns 25 mg 2 ml N9306566 1 50 mg 2 ml N9306567 1 50 mg 1 ml N9306590 50 100 mg 2 ml N9306568 1 100 mg 1 ml N9306591 100 100 mg 3 ml N9306592 50 200 mg 3 ml N9306593 50 Supra-Clean C18 Cartridges Media Weight Volume Part No. Quantity 390 mg -- N9306587 50 910 mg -- N9306588 50 1690 mg -- N9306589 50 www.perkinelmer.com/spe Media Weight Volume Supra-Clean Strong Anion Exchange (SAX) Supra-Clean Strong Anion Exchange (SAX) Columns Media Weight Volume Part No. Quantity 50 mg 1 ml N9306553 50 100 mg 1 ml N9306471 100 100 mg 3 ml N9306554 50 200 mg 3 ml N9306482 50 500 mg 3 ml N9306555 50 500 mg 6 ml N9306556 30 59 ORDERING INFORMATION Supra-Clean Columns and 96 Well Plates Supra-Clean Strong Anion Exchange (SAX) 96 Well Plates Supra-Clean Mixed-Mode (MM1) 96 Well Plates Media Weight Volume Part No. Quantity Media Weight Volume Part No. Quantity 25 mg 2 ml N9306581 1 25 mg 2 ml N9306578 1 50 mg 2 ml N9306582 1 50 mg 2 ml N9306579 1 100 mg 2 ml N9306583 1 100 mg 2 ml N9306580 1 Supra-Clean Strong Cation Exchange (SCX) Supra-Clean Mixed-Mode (MM2) Supra-Clean Strong Cation Exchange (SCX) Columns Supra-Clean Mixed-Mode (MM2) Columns Media Weight Volume Part No. Quantity Media Weight Volume Part No. 50 mg 1 ml N9306536 50 50 mg 1 ml N9306548 50 100 mg 1 ml N9306432 100 100 mg 1 ml N9306549 100 100 mg 3 ml N9306537 50 100 mg 3 ml N9306550 50 200 mg 3 ml N9306538 50 200 mg 3 ml N9306551 50 500 mg 3 ml N9306539 50 500 mg 3 ml N9306411 50 500 mg 6 ml N9306540 30 500 mg 6 ml N9306552 30 Supra-Clean Strong Cation Exchange (SCX) 96 Well Plates Supra-Clean Mixed-Mode (MM3) Media Weight Volume Part No. Quantity 25 mg 2 ml N9306575 1 50 mg 2 ml N9306576 1 Media Weight Volume Part No. Quantity 100 mg 2 ml N9306577 1 500 mg 6 ml N9306649 30 Volume Part No. Quantity Supra-Clean Mixed-Mode (MM3) Columns Supra-Clean Weak Cation Exchange (WCX) Supra-Clean Florisil (FL-S) Supra-Clean Weak Cation Exchange (WCX) Columns Supra-Clean Florisil (FL-S) Columns Media Weight Media Weight Volume Part No. Quantity 50 mg 1 ml N9306544 50 200 mg 3 ml N9306511 50 3 ml N9306512 50 100 mg 1 ml N9306545 100 500 mg 100 mg 3 ml N9306546 50 500 mg 6 ml N9306494 30 6 ml N9306413 30 200 mg 3 ml N9306547 50 1g 500 mg 3 ml N9306420 50 2g 6 ml N9306513 20 30 2g 15 ml N9306514 20 2g 25 ml N9306515 20 500 mg 6 ml N9306407 Supra-Clean Mixed-Mode (MM1) * Granular shape Supra-Clean Mixed-Mode (MM1) Columns Supra-Clean Florisil (FL-S) Pesticide Grade Media Weight Volume Part No. Quantity 50 mg 1 ml N9306541 50 100 mg 1 ml N9306542 100 100 mg 3 ml N9306419 50 200 mg 3 ml N9306543 50 500 mg 3 ml N9306481 50 500 mg 6 ml N9306416 30 200 mg 15 ml N9306713 20 Supra-Clean Florisil (FL-S) Pesticide Grade Columns Media Weight Volume Part No. Quantity 200 mg 3 ml N9306516 50 500 mg 3 ml N9306400 50 500 mg 6 ml N9306517 30 1g 6 ml N9306436 30 2g 6 ml N9306470 30 2g 15 ml N9306443 20 2g 25 ml N9306447 20 * Granular shape 60 Quantity ORDERING INFORMATION Supra-Clean Columns and 96 Well Plates Supra-Clean Silica (SI-S) Supra-Clean Polyamine (P6) Supra-Clean Silica (SI-S) Columns Supra-Clean Polyamine (P6) Columns Media Weight Volume Part No. Quantity Media Weight Volume Part No. Quantity 100 mg 3 ml N9306532 50 500 mg 3 ml N9306518 50 500 mg 6 ml N9306434 30 200 mg 3 ml N9306444 50 500 mg 3 ml N9306402 50 500 mg 6 ml N9306466 30 Supra-Clean Phenyl (PH-S) 1g 6 ml N9306404 30 Supra-Clean Phenyl (PH-S) Columns 2g 6 ml N9306533 20 2g 15 ml N9306534 20 2g 25 ml N9306535 20 Supra-Clean Silica (SI-S) Cartridges Media Weight Volume Part No. Quantity 50 mg 1 ml N9306401 50 100 mg 1 ml N9306524 100 100 mg 3 ml N9306525 50 200 mg 3 ml N9306490 50 500 mg 3 ml N9306421 50 Media Weight Volume Part No. Quantity 300 mg -- N9306584 50 700 mg -- N9306585 50 500 mg 6 ml N9306526 30 50 1g 6 ml N9306527 30 1300 mg -- N9306586 Supra-Clean Phenyl (PH-S) 96 Well Plates Supra-Clean Cyano (CN-S) Supra-Clean Cyano (CN-S) Columns Media Weight Volume Part No. Quantity 25 mg 2 ml N9306569 1 Part No. Quantity 50 mg 2 ml N9306570 1 3 ml N9306645 50 100 mg 2 ml N9306571 1 6 ml N9306644 30 Media Weight Volume 500 mg 500 mg Supra-Clean LCC Supra-Clean Amino (NH2-S) Supra-Clean LCC Columns Supra-Clean Amino (NH2-S) Columns Media Weight Volume Part No. Quantity Media Weight Volume Part No. Quantity 500 mg 3 ml N9306643 50 50 mg 1 ml N9306528 50 500 mg 6 ml N9306641 30 100 mg 1 ml N9306410 100 100 mg 3 ml N9306529 50 500 mg 3 ml N9306414 50 200 mg 6 ml N9306530 50 500 mg 6 ml N9306531 30 Supra-Clean Amino (NH2-S) 96 Well Plates Media Weight Volume Part No. Quantity 25 mg 2 ml N9306572 1 50 mg 2 ml N9306573 1 100 mg 2 ml N9306574 1 www.perkinelmer.com/spe 61 ORDERING INFORMATION Supra-Poly HLB Columns and 96 Well Plates SUPRA-POLY HLB COLUMNS AND 96 WELL PLATES • Contains macro-porous polymers with ultra-pure, pharmaceutical grade spherical particles • Shorter analysis times, greater load capacity and reduced solvent usage • Ideal for high throughput assays Supra-Poly HLB Supra-Poly HLB 30 µm Columns Media Weight Volume Part No. Quantity 30 mg 1 ml N9306650 50 50 mg 1 ml N9306655 50 60 mg 1 ml N9306656 50 100 mg 1 ml N9306657 50 30 mg 3 ml N9306651 50 60 mg 3 ml N9306658 50 100 mg 3 ml N9306659 50 200 mg 3 ml N9306660 50 500 mg 3 ml N9306661 30 100 mg 6 ml* N9306672 30 150 mg 6 ml N9306662 30 200 mg 6 ml N9306663 30 200 mg 6 ml* N9306673 30 500 mg 6 ml* N9306674 30 500 mg 6 ml N9306664 30 500 mg 15 ml N9306665 20 1g 15 ml N9306666 20 30 mg 15 ml** N9306668 50 60 mg 15 ml** N9306669 100 mg 15 ml** 200 mg 1g 62 Volume Part No. Quantity 200 mg 500 mg 6 ml* N9306693 30 6 ml N9306684 30 500 mg 15 ml N9306685 20 500 mg 6 ml* N9306694 30 1g 15 ml N9306686 20 30 mg 15 ml** N9306688 50 60 mg 15 ml** N9306689 50 100 mg 15 ml** N9306690 50 200 mg 15 ml** N9306691 50 1g 25 ml N9306687 20 * Glass columns ** LRC columns Supra-Poly HLB 30 µm 96 Well Plates Media Weight Volume Part No. Quantity 30 mg 2 ml N9306698 1 50 mg 2 ml N9306699 1 60 mg 2 ml N9306700 1 Supra-Poly HLB 60 µm 96 Well Plates Media Weight Volume Part No. Quantity 30 mg 2 ml N9306695 1 50 50 mg 2 ml N9306696 1 N9306670 50 60 mg 2 ml N9306697 1 15 ml** N9306671 50 25 ml N9306667 20 Part No. Quantity Supra-Poly HLB 60 µm Columns Media Weight Media Weight Volume 30 mg 1 ml N9306652 50 50 mg 1 ml N9306675 50 60 mg 1 ml N9306676 50 100 mg 1 ml N9306677 50 30 mg 3 ml N9306653 50 60 mg 3 ml N9306678 50 100 mg 3 ml N9306679 50 200 mg 3 ml N9306680 50 500 mg 3 ml N9306681 30 100 mg 6 ml* N9306692 30 150 mg 6 ml N9306682 30 200 mg 6 ml N9306683 30 ORDERING INFORMATION Supra-Poly Columns and 96 Well Plates SUPRA-POLY COLUMNS AND 96 WELL PLATES • Contains macro-porous polymers with ultra-pure, pharmaceutical grade spherical particles • Shorter analysis times, greater load capacity and reduced solvent usage • Ideal for high throughput assays Supra-Poly Extreme Capacity 1500 m2/g (XC) Supra-Poly Extreme Capacity 1500 m2/g (XC) Columns Supra-Poly Environmental (AEV) Media Weight Volume Part No. Quantity 30 mg 1 ml N9306441 50 50 mg 1 ml N9306500 50 Media Weight Volume Part No. Quantity 100 mg 3 ml N9306648 50 Supra-Poly AEV Columns 60 mg 1 ml N9306501 50 100 mg 1 ml N9306403 50 60 mg 3 ml N9306502 50 Supra-Poly Hydrophilic (ATH) 100 mg 3 ml N9306440 50 Supra-Poly ATH Columns 200 mg 3 ml N9306428 50 200 mg 6 ml N9306635 30 500 mg 6 ml N9306405 30 1g 15 ml N9306503 20 Media Weight Volume Part No. Quantity 100 mg 3 ml N9306646 50 200 mg 3 ml N9306638 50 200 mg 6 ml N9306636 30 Supra-Poly Extreme Capacity 1500 m2/g (XC) 96 Well Plates Media Weight Volume Part No. Quantity 30 mg 2 ml N9306557 1 50 mg 2 ml N9306558 1 60 mg 2 ml N9306559 1 Supra-Poly Extra Wide Particle 1200 m2/g (XWP) Supra-Poly Lipophilic (ATL) Supra-Poly ATL Columns Media Weight Volume Part No. Quantity 100 mg 3 ml N9306647 50 200 mg 3 ml N9306639 50 200 mg 6 ml N9306637 30 Supra-Poly Extra Wide Particle 1200 m2/g (XWP) Columns Media Weight Volume Part No. Quantity 30 mg 1 ml N9306504 50 50 mg 1 ml N9306427 50 60 mg 1 ml N9306505 50 100 mg 1 ml N9306506 50 60 mg 3 ml N9306507 50 100 mg 3 ml N9306508 50 200 mg 3 ml N9306509 50 500 mg 6 ml N9306418 30 1g 15 ml N9306510 20 Supra-Poly Extra Wide Particle 1200 m2/g (XWP) 96 Well Plates Media Weight Volume Part No. Quantity 30 mg 2 ml N9306560 1 50 mg 2 ml N9306561 1 60 mg 2 ml N9306562 1 www.perkinelmer.com/spe 63 ORDERING INFORMATION SPE Kits and Packs SPE SELECTION KITS Selection kits enable quick column selection for development of reproducible and repeatable SPE methods. SPE Selection Kits Description Media Weight Volume Part No. Quantity Pre-Concentration of Hydrophobic Compounds from Aqueous Matrix 200 mg 200 mg 6 ml 3 ml N9306594 N9306595 50 50 Extraction of Hydrophobic Compounds from Aqueous Matrix 500 mg 500 mg 6 ml 3 ml N9306596 N9306597 50 50 Pre-Concentration of Hydrophilic Compounds 500 mg 500 mg 6 ml 3 ml N9306598 N9306599 30 30 Removal of Polar Compounds from Aqueous and Organic Matrix 500 mg 500 mg 6 ml 3 ml N9306600 N9306601 30 30 Extraction of Acidic Basic and Neutral Compounds from Aqueous or Organic Matrix 100 mg 3 ml N9306602 50 Extraction of Carboxylic Acids and Strong Bases from Aqueous Matrix 500 mg 6 ml N9306603 40 Extraction of Weak Bases from Aqueous Matrix 500 mg 6 ml N9306604 30 SPE APPLICATION PACKS Ideal for extraction of known entities from a variety of matrices, our packs are expertly tailored to meet your application needs and are designed to support major EPA methods and applications. SPE Application Packs Description Extraction of Basic Drugs from Biological Fluids Extraction of Bisphenol A from Aqueous Matrix Volume Part No. Quantity 200 mg 3 ml N9306605 50 1g 6 ml N9306613 30 500 mg 1g 3 ml 6 ml N9306612 N9306611 50 30 Extraction of PAH from Soil and Oil 1.5 g 6 ml N9306609 30 Extraction of PAH from Soil and Oil (Glass Straight Column) 1.5 g 6 ml N9306610 30 Extraction of PAH from Water Containing Humic Acids Extraction of Oil and Grease from Aqueous Matrix-EPA 1664 1.5 g 6 ml N9306608 30 Extraction of PAH from Water or Soil 4g 6 ml N9306606 30 Extraction of PAH from Water or Soil (Glass Straight Column) 4g 6 ml N9306607 30 Extraction of PCB from Oil 1g 1g 6 ml 3 ml N9306617 N9306616 30 50 Extraction of Pesticides and Herbicides from Aqueous Matrix 500 mg 3 ml N9306614 50 Extraction of Steroids from Biological Fluids 500 mg 6 ml N9306615 30 1g 6 ml N9306618 30 Extraction of SVOC from Water-EPA 525 64 Media Weight ORDERING INFORMATION SPE Vacuum Pumps, Manifolds and Accessories SPE VACUUM PUMPS, MANIFOLDS AND ACCESSORIES PerkinElmer SPE Vacuum Manifolds allow multiple samples to be processed simultaneously by conveniently holding SPE media cartridges in place and allowing samples to pass through them. SPE Vacuum Manifolds are available to accommodate either 12 or 24 cartridges; 1, 3, 6, 15, and 25 mL cartridges can be used with the manifolds. Manifolds are equipped with a vacuum port where a standard laboratory vacuum pump can be connected. Vacuum pulls the sample through the stationary phase, metered by the stopcocks, to control the speed of the extraction process and the sample flow over the media bed in the SPE cartridge. Waste and wash solvents are discarded and analytes are collected in sample tubes below the manifold completing the extraction. SPE Manifolds and Accessories Description Part No. Quantity Vacuum Manifold - 12 Position N9306619 1 each Vacuum Manifold - 24 Position N9306626 1 each Replacement Chamber (Glass) - 12 Position N9306620 1 each Replacement Chamber (Glass) - 24 Position N9306627 1 each Cover Gasket - 12 Position N9306621 1 each Cover Gasket - 24 Position N9306628 1 each Stopcocks - 12 Position N9306624 12 pack Stopcocks - 24 Position N9306631 24 pack Needles - Polypropylene - 12 Position N9306622 12 pack Needles - Polypropylene - 24 Position N9306629 24 pack Needles - Stainless Steel - 12 Position N9306623 12 pack Needles - Stainless Steel - 24 Position N9306630 24 pack Drying Attachment - 12 Position N9306625 1 each Drying Attachment - 24 Position N9306632 1 each Vacuum Pumps Description Part No. Quantity Vacuum Pump 20 L/min 115V N9308065 1 each Vacuum Pump 60 L/min 115V N9308063 1 each Vacuum Pump 17 L/min 230V N9308331 1 each Vacuum Pump 58 L/min 230V N9308332 1 each www.perkinelmer.com/spe 65 At PerkinElmer, we understand that sample preparation is one of the most critical steps in the analytical process. Often accounting for 60% of your timetable, it has a fundamental impact on a wide range of operational parameters. Any errors within this process undermine the quality of your data at all subsequent stages of your analysis. Solid Phase Extraction helps avoid potential errors in sample preparation, reducing re-runs and dramatically increasing productivity. As one of the most cost-effective and flexible tools within the laboratory environment, SPE also provides efficient sample concentration and purification prior to many of today’s most popular analytical techniques, including HPLC, LC/MS, GC and GC/MS. The right accessories, consumables, methods and application support are as integral to the success of your laboratory as your instrumentation. That’s why we invest heavily in testing and validating our complete portfolio of solutions to ensure you receive accurate, repeatable results on time, every time. SPE columns are part of PerkinElmer’s complete offering of analytical solutions—an industry-leading portfolio that encompasses instruments, accessories, consumables, supplies, training and service. It’s a breadth of capabilities that enables us to offer the ease and convenience of having a single supplier for all your needs at every stage of your workflow. So you can benefit from greater responsiveness, superior reliability, and dramatic cost savings. Couple SPE With Leading Instrumentation For Ultimate Laboratory Efficiency Clarus® 680/580/480 GC Clarus SQ 8 GC/MS Flexar™ SQ 300 MS Flexar LC and UHPLC Our OneSource Laboratory Services division even takes it a step further. With more than 1,500 trained and certified field service engineers and service personnel around the world, OneSource offers the most comprehensive portfolio of professional laboratory services in the industry, including complete care programs for virtually every technology and manufacturer. So turn to PerkinElmer. For the experience. For the confidence. For the better. PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs Copyright ©2012, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 010097_01 Printed in USA