Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Chief Toxicologist Document Type: GC & GC/MS Procedure No.: TOX07.2002 Title: Standard basic drug screen Rev.: 19 1.0 Purpose 1.1 This document describes procedures employed by the Forensic Toxicology Laboratory to screen blood or other biological specimens for the presence of alkaline-extractable (basic) drugs. 1.2 Biological specimens are basified to a moderately alkaline pH 9.3 at which basic drugs are soluble in organic solvent. The organic layer is separated and the basic drugs are back extracted into 2 N HCl. The acid layer is then made alkaline and basic drugs are re-extracted into an organic solvent. The extracts are analyzed by gas chromatography mass spectrometry (GC/MS) operating in full scan mode. 2.0 Scope 2.1 The assay is appropriate for qualitative detection of chromatographically stable alkaline extractable drugs in whole blood, serum, plasma, urine, bile, stomach contents or tissue homogenates. This methodd could Copyalso be applied to non- biological samples. 3.0 Definitions and Abbreviations 3.1 No method-specific or nonrolle-standard terms are used in this procedure. 4.0 Materials t 4.1 Instruments and Equipment 4.1.1 13 x 100 mm screw top test tubes 4.1.2 Teflon screw caps 4.1.3 Autosampler vials and caps Uncon4.1.4 100 µL Syringe 4.1.5 Vacuum Pump 4.1.6 GC/MS 4.1.7 Varian Factor 4 VF-5MS (15 m x 0.25 mm x 0.25 µm) GC column or equivalent 4.2 Reagents Page 1 of 15 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Chief Toxicologist Document Type: GC & GC/MS Procedure No.: TOX07.2002 Title: Standard basic drug screen Rev.: 19 4.2.1 Ammonium Hydroxide, Reagent Grade 4.2.2 n-Butyl Chloride (chlorobutanes), HPLC Grade 4.2.3 Saturated Sodium Borate Buffer, pH 9.3 A. To 1000 mL of deionized (DI) water, add sodium borate until no more sodium borate will dissolve. B. Stable for 6 months at room temperature. 4.2.4 Dichloromethane (methylene chloride), HPLC Grade 4.2.5 2 N HCl A. Add 166 mL of concentrated HCl to 500Copy mL deionized water and mix. B. Allow to cool to room temperature. QS to 1000 mL with DI water. C. Stir to mix. D. Stable at room temperature indefinitely. 4.2.6 Methanol, HPLC grade 4.3 Stock Standards and Controls Note: Certified reference materials shall be used within manufacturer’s expiration date and shall be stored at the recommended storage temperature. Note: Alternate vendors may be used as long as standards are equivalent. 4.3.1 Commercially prepared stock solutions Analyte Vendor Catalog Concentration Number Methamphetamine Cerilliant M-009 1.0 mg/mL Diphenhydramine Cerilliant D-015 1.0 mg/mL Chlorpheniramine Cerilliant C-036 1.0 mg/mL UncontrolledCodeine Cerilliant C-006 1.0 mg/mL Hydrocodone Cerilliant H-003 1.0 mg/mL Verapamil Cerilliant V-002 1.0 mg/mL Methadone Cerilliant M-007 1.0 mg/mL Lidocaine Cerilliant L-018 1.0 mg/mL Cocaine Cerilliant C-008 1.0 mg/mL Tramadol Cerilliant T-027 1.0 mg/mL Amitryptyline Cerilliant A-923 1.0 mg/mL Diazepam Cerilliant D-907 1.0 mg/mL Diltiazem Cerilliant D-035 1.0 mg/mL Page 2 of 15 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Chief Toxicologist Document Type: GC & GC/MS Procedure No.: TOX07.2002 Title: Standard basic drug screen Rev.: 19 Phencyclidine Cerilliant P-007 1.0 mg/mL Doxylamine Cerilliant D-045 1.0 mg/mL Venlafaxine Cerilliant V-004 1.0 mg/mL Dextromethorphan Cerilliant D-013 1.0 mg/mL Propoxyphene Cerilliant P-011 1.0 mg/mL Nordiazepam Cerilliant N-905 1.0 mg/mL Mirtazapine Cerilliant M-128 1.0 mg/mL Trazodone Cerilliant T-030 1.0 mg/mL Cyclobenzaprine Cerilliant C-060 1.0 mg/mL Oxycodone Cerilliant O-002 1.0 mg/mL Zolpidem Cerilliant Z-901 1.0 mg/mL Norsertraline Cerilliant N-049 0.10 mg/mL Nortriptyline Cerilliant N-907 1.0 mg/mL Sertraline Cerilliant CopyS-021 1.0 mg/mL Citalopram Cerilliant C-095 1.0 mg/mL Meprobamate Cerilliant M-039 1.0 mg/mL Carisoprodol Cerilliant C-077 1.0 mg/mL Alprazolam Cerilliant A-903 1.0 mg/mL Levamisole Cerilliant L-025 1.0 mg/mL Fentanyl Cerilliant F-002 0.10 mg/mL 4.3.2 Promethazine Stock Solution 1.0 mg/mL A. Weigh 11.3 mg of Promethazine HCl into a 10 mL volumetric flask. B. Adjust volume to 10 mL with methanol. C. Stable for 1 year at -10 to -20º C. D. Alternatively, a 1.0 mg/mL prepared promethazine solution may be substituted. (i.e., Cerilliant catalog #P-111 or equivalent) 4.3.3 Internal Standard Stock, SKF-525A 1.0 mg/mL A. Weigh 10 mg SKF-525A (proadifen hydrochloride) and dilute to 10 mL with methanol. B. Stable for 3 years at -10 to -20º C. Uncontrolled4.4 Working Standards and Controls 4.4.1 Working Positive Basic Control #1 0.01/0.004 mg/mL A. Pipette 100 µL of codeine, cyclobenzaprine, diphenhydramine, methadone, methamphetamine, verapamil, and zolpidem to a 10 mL volumetric flask. B. Pipette 40 µL of chlorpheniramine, hydrocodone, and oxycodone to the same volumetric flask. C. Dilute to 10 mL with methanol. Stable until the earliest manufacturer’s expiration date at -10 to -20º C. Page 3 of 15 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Chief Toxicologist Document Type: GC & GC/MS Procedure No.: TOX07.2002 Title: Standard basic drug screen Rev.: 19 4.4.2 Working Positive Basic Control #2 0.01 mg/mL A. Pipette 100 µL of amitriptyline, cocaine, diazepam, diltiazem, lidocaine, nortriptyline, promethazine, sertraline, and tramadol to a 10 mL volumetric flask. B. Dilute to 10 mL with methanol. C. Stable until the earliest manufacturer’s expiration date at -10 to -20º C. 4.4.3 Working Positive Basic Control #3 0.01/0.004 mg/mL A. Pipette 100 µL of nordiazepam, doxylamine, mirtazapine, phencyclidine, propoxyphene, trazodone, venlafaxine and citalopram to a 10 mL volumetric flask. B. Pipette 1 mL of norsertraline into the same volumetric flask. C. Pipette 40 µL of dextromethorphan into the same volumetric flask. D. Dilute to 10 mL with methanol. Copy E. Stable until the earliest manufacturer’s expiration date at -10 to -20º C. 4.4.4 Working Positive Basic Control #4 0.01/0.0008 mg/mL A. Pipette 100 µL of alprazolam and levamisole to a 10 mL volumetric flask. B. Pipette 80 µL of fentanyl into the same 10 mL volumetric flask. C. Dilute to 10 mL with methanol. D. Stable until the earliest manufacturer’s expiration date at -10 to -20º C. 4.4.5 Working Internal Standard SKF-525A 0.10 mg/mL A. Transfer 1 mL of stock internal standard to a 10 mL volumetric flask. B. Adjust the volume to 10 mL with methanol. C. Stable for 6 months at -10 to -20º C. 5.0 Procedure 5.1 Control and case sample preparation 5.1.1 Label sufficient 13 x 100 mm screw top test tubes for reagent blank, negative control, positive controls, and case samples. Uncontrolled5.1.2 Basic Blood Control #1 A. Add 50 µL of working positive basic control #1 to the appropriately labeled tube. Do not dry as methamphetamine will be lost. 5.1.3 Basic Blood Control #2 A. Add 50 µL of working positive basic control #2 to the appropriately labeled tube. Do not dry. 5.1.4 Basic Blood Control #3 A. Add 50 µL of working positive basic control #3 to the Page 4 of 15 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Chief Toxicologist Document Type: GC & GC/MS Procedure No.: TOX07.2002 Title: Standard basic drug screen Rev.: 19 appropriately labeled tube. Do not dry. 5.1.5 Basic Blood Control #4 A. Add 50 µL of working positive basic control #4 to the appropriately labeled tube. B. Add 50 µL of meprobamate stock control to the same test tube. C. Add 20 µL of carisoprodol stock control to the same test tube. D. Do not dry. Note: Other drugs may be used to prepare additional controls as necessary. Prepare using 0.01mg/mL concentration if available. 5.1.6 Pipette 2 mL of blank blood into reagent blank, negative control and positive control tubes. Copy 5.1.7 Pipette 2 mL of case specimen into the appropriately labeled tube. 5.1.8 Add 20 µL of 0.10 mg/mL SKF working internal standard into all tubes except reagent blank and vortex. 5.2 Extraction 5.2.1 Add 500 µL of saturated sodium borate and 5 mL of n-butyl chloride. A. In severely decomposed cases or tissues, 100 µL of NH4OH may replace saturated borate to perform a strong basic extraction. Add a set of positive controls and a negative control to be extracted with NH4OH replacement. 5.2.2 Cap and mix on rocker for 15 minutes. 5.2.3 Centrifuge tubes for 5 minutes at 3000 rpm. 5.2.4 Transfer upper organic layer into clean, labeled 13 x 100 mm screw top tube. Uncontrolled 5.2.5 Add 2 mL of 2N HCl. 5.2.6 Cap and mix on rocker for 15 minutes. 5.2.7 Centrifuge tubes for 5 minutes at 3000 rpm. 5.2.8 Discard upper organic layer.