Basic Quant GCMS

Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.3005 Title: Quantitation of basic drugs by gas chromatography / mass spectrometry Rev.:15 1.0 Purpose 1.1 This document describes the procedures used by the Toxicology Laboratory to quantitate basic drugs using gas chromatography/mass spectrometry. 1.2 Biological specimens are basified to the moderately alkaline pH 9 at which basic compounds are in an unionized form and are soluble in an organic solvent. After liquid/liquid extraction, the organic layer is separated and the compound X is back extracted into 2 N HCl. The acid layer is then made alkaline and compound X is re-extracted into an organic solvent. 2.0 Scope 2.1 The assay is appropriate for quantitation of any basic compound (X) in blood including serum and plasma, urine, bile, stomach contents or tissue homogenates. 3.0 Definitions and Abbreviations 3.1 No method-specific or non-standard terms are used in this procedure. 4.0 Materials 4.1 Instruments and Equipment 4.1.1 13 x 100 mm screw top tubes and caps 4.1.2 Mixer 4.1.3 Rocker 4.1.4 Centrifuge 4.1.5 Transfer pipettes 4.1.6 Autosampler vials and rubber septum caps 4.1.7 Autosampler vial inserts 4.1.8 Vial crimper 4.1.9 100uL syringe 4.1.10 GC/MS Uncontrolled4.2 Reagents Copy 4.2.1 Ammonium Hydroxide Reagent Grade 4.2.2 n-Butyl chloride (chlorobutane) HPLC Grade 4.2.3 Saturated Sodium Borate Buffer, pH 9.3 A. To 2000 mL of deionized (DI) water continue to add sodium borate until no more sodium borate will dissolve. B. Transfer into two, 1 L bottles. Page 1 of 11 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.3005 Title: Quantitation of basic drugs by gas chromatography / mass spectrometry Rev.:15 C. Sufficient for 4,000 extractions D. Stable for 6 months at room temperature. 4.2.4 2 N HCl A. Add 165 mL of concentrated HCl to 500 mL deionized water and mix. Allow to cool to room temperature. QS to 1000 mL with DI water. Stir to mix. B. Stable at room temperature. 4.2.5 Methanol, HPLC grade 4.2.6 Dichloromethane (methylene chloride), HPLC grade 4.3 Stock Standards and Controls Copy 4.3.1 Weighing factors A. Most basic drugs are obtained as salts in the form XY where X is the basic drug and Y is the acid and XY is the drug salt. To calculate weighing factors to convert salts to free base equivalents perform the calculation: Factor (f) = molecular weight XY molecular weight X 4.3.2 X Stock Standard 1.0 mg/mL A. Authentic reference material (powder) a. Weigh f x 10 mg of XY into a 10 mL volumetric flask. b. Adjust volume to 10 mL with methanol. c. Store at –10 to -20º C. Stable for 1 year. B. Purchase from Cerilliant 1.0 mg/mL or equivalent a. Stable until manufacturer’s expiration date. Uncontrolledb. Store per manufacturer’s instructions. 4.3.3 X Stock Control 1.0 mg/mL . A. Authentic reference material (powder) a. Weigh f X 10 mg of XY into a 10 mL volumetric flask. b. Adjust volume to 10 mL with methanol. c. Store at –10 to -20º C. Stable for 1 year. B. Purchase from Cerilliant 1.0 mg/mL or equivalent Page 2 of 11 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.3005 Title: Quantitation of basic drugs by gas chromatography / mass spectrometry Rev.:15 a. Stable until manufacturer’s expiration date. b. Store per manufacturer’s instructions. 4.3.4 Internal Standard Stock SKF-525A 1.0 mg/mL A. Weigh out 10 mg SKF-525A (proadifen hydrochloride) and dilute to 10 mL with methanol. B. Store at –10 to -20º C. Stable 3 years. 4.3.5 Methadone-D9 stock internal standard 1.0 mg/mL A. Purchase from Cerilliant, Catalog #M-089 or equivalent. B. Store per manufacturer’s instructions. C. Stable until the manufacturer’s expiration date. 4.3.6 Ketamine-D4 stock internal standard Copy 0.10 mg/mL A. Purchase from Cerilliant, Catalog #K-003 or equivalent. B. Store per manufacturer’s instructions. C. Stable until the manufacturer’s expiration date. 4.3.7 Diphenhydramine-D3 stock internal standard 0.10 mg/mL A. Purchase from Cerilliant, Catalog #D-017 or equivalent B. Store per manufacturer’s instructions C. Stable until the manufacturer’s expiration date 4.4 Working Standards and Controls 4.4.1 X Working Standard A 0.10 mg/mL A. Transfer 1.0 mL X Stock Standard into a 10 mL volumetric flask. B. Adjust volume to 10 mL with methanol. C. Store at –10 to -20º C. Stable for 6 months when prepared from a powder stock or until the manufacturer’s expiration date when prepared from a Cerilliant stock. 4.4.2 X Working Standard B 0.01 mg/mL A. Transfer 1.0 mL of X Working Standard A into a 10 mL volumetric Uncontrolledflask. B. Adjust volume to 10 mL with methanol. C. Store at -10 to -20 degrees C. Stable for 6 months when prepared from a powder stock or until the manufacturer’s expiration date when prepared from a Cerilliant stock. 4.4.3 Brompheniramine, Chlorpheniramine, Dextromethorphan Working Standard 0.01 mg/mL A. Transfer 100 uL of stock control into a 10 mL volumetric flask. Page 3 of 11 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.3005 Title: Quantitation of basic drugs by gas chromatography / mass spectrometry Rev.:15 B. Adjust volume to 10 mL with methanol. C. Store at -10 to -20 degrees Celcius. Stable for 6 months when prepared from a powder stock or until the manufacturer’s expiration date when prepared from a Cerilliant stock. 4.4.4 X Working Control A 0.10 mg/mL A. Transfer 1.0 mL X Stock Control into a 10 mL volumetric flask. B. Adjust volume to 10 mL with methanol. C. Store at –10 to -20º C. Stable for 6 months when prepared from a powder stock or until the manufacturer’s expiration date when prepared from a Cerilliant stock. 4.4.5 X Working Control B 0.01 mg/L A. Transfer 1.0 mL of X Working Control ACopy into a 10 mL volumetric flask. B. Adjust volume to 10 mL with methanol. C. Store at –10 to -20º C. Stable for 6 months when prepared from a powder stock or until the manufacturer’s expiration date when prepared from a Cerilliant stock. 4.4.6 Brompheniramine,Chlorpheniramine,DextromethorphanWorking Control 0.01 mg/mL A. Transfer 100 uL of stock control into a 10 mL volumetric flask. B. Adjust volume to 10 mL with methanol. C. Store at –10 to -20º C. Stable 6 months when prepared from a powder stock or until the manufacturer’s expiration date when prepared from a Cerilliant stock. 4.4.7 Working Internal Standard SKF-525A 0.10 mg/mL A. Pipette 5 mL of stock SKF-525A into a 50 mL volumetric flask. B. QS to 50 mL with methanol. C. Store at –10 to -20º C. Stable 6 months. Uncontrolled4.4.8 Working Internal Standard SKF-525A 0.01 mg/mL for Brompheniramine, Chlorpheniramine, Dextromethorphan A. Pipette 100 uL of stock SKF-525A into a 10 mL volumetric flask. B. QS to 10 mL with methanol. C. Store at –10 to -20º C. Stable 6 months. 4.4.9 Methadone-D9 Working Internal Standard 0.01 mg/mL A. Pipette 100 µL of the stock internal standard into a 10 mL volumetric flask. B. Dilute with methanol Page 4 of 11 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.3005 Title: Quantitation of basic drugs by gas chromatography / mass spectrometry Rev.:15 C. Stable at –10 to -20º C. Stable until the manufacturer’s expiration date. 4.4.10 Ketamine-D4 Working Internal Standard 0.01 mg/mL A. Pipette 1mL of the stock internal standard into a 10 mL volumetric flask. B. Dilute with methanol. C. Stable at -10 to -20º C. Stable until the manufacturer’s expiration date. 4.4.11 Diphenhydramine-D3 Working Internal Standard 0.01 mg/mL A. Pipette 1mL of the stock internal standard into a 10 mL volumetric Flask B. Dilute with methanol C. Stable at -10 to -20º C. Stable until the manufacturer’s expiration date. Copy Note: An analyst who prepared the stock and working standards should not prepare the stock and working controls. Note: Prepared working standards/QC may not have an expiration date that exceeds the expiration date of the stock standard used to prepare it. 5.0 Procedure 5.1 Standards and Controls 5.1.1 Label 13 X 100 mm screw cap tubes. 5.1.2 Add the indicated volumes of Working or Working Controls. (Table 1) 5.1.3 Table 1: Blanks, Standards, and Controls Tube Target Standard/ Standard/ Number Concentration Control B Control A (ng/mL) (0.01mg/mL) (0.10mg/mL) ( µL) (µL) 1 Reagent 0 0 UncontrolledBlank 2 Negative 0 0 Control 3 100 std 20 0 4 250 std 50 0 5 500 std 100 0 6 1000 std 0 20 Page 5 of 11 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.3005 Title: Quantitation of basic drugs by gas chromatography / mass spectrometry Rev.:15 7 2500 std 0 50 8 250 QC 50 0 9 500 QC 100 0 10 1000 QC 0 20 Note: Use the 0.10 mg/mL concentration of SKF internal standard for all basic drugs excluding citalopram, ketamine, doxylamine, and extractions with low concentration curves for brompheniramine, chlorpheniramine, and dextromethorphan.

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