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

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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.

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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

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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.

Note: Use the 0.01 mg/mL methadone-D9 for internal standard in citalopram extractions.

Note: Use the 0.01 mg/mL ketamine-D4 for internal standard in ketamine extractions. Copy Note: Use the 0.01 mg/mL diphenhydramine-D3 for internal standard in doxylamine extractions.

5.1.4 Table 2: Blanks, Standards, and Controls for Brompheniramine, Chlorpheniramine, Dextromethorphan low curves

Tube Target Concentration Volume of Working Number (ng/mL) Solution 1 Reagent Blank 2 Negative Control 3 10 std 2 4 20 std 4 5 50 std 10 6 100 std 20 7 250 std 50

8 50 QC 10

9 100 QC 20

Note: Use the 0.01 mg/mL concentration of SKF internal standard. Uncontrolled 5.1.5 Pipette 2ml of negative blood to screw cap tubes and vortex.

5.2 Sample Preparation

5.2.1 Pipette 2 mL of specimen into labeled 13 x 100 mm screw top tubes.

5.2.2 To each tube except reagent blank add 20 µL of working SKF internal standard or 100uL of working methadone-d9 or 100uL of working ketamine-D4 or 100uL of working diphenhydramine-D3 internal

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standard.

5.3 Extraction

5.3.1 Add 500 µL of saturated sodium borate and 5 mL of n-butyl chloride.

Note: In severely decomposed cases replace saturated sodium borate with 100 µL of NH4OH. Add a set of positive controls to be extracted with NH4OH replacement.

5.3.2 Cap and mix on rocker for 15 minutes.

5.3.3 Centrifuge tubes for 5 minutes at 3000 rpm. Copy 5.3.4 Transfer upper organic layer into clean, labeled 13 x 100 mm screw top tube.

5.3.5 Add 2 mL of 2N HCL.

5.3.6 Cap and mix on rocker for 15 minutes.

5.3.7 Centrifuge tubes for 5 minutes at 3000 rpm.

5.3.8 Aspirate and discard upper organic layer with a vacuum pump. Evaporate any residual butyl chloride retained with the aqueous layer. Retain the lower aqueous layer.

5.3.9 Add 500 µL of concentrated NH4OH to the residual aqueous layers and mix well. Let cool for 2 minutes.

5.3.10 Add 200 µL of dichloromethane.

5.3.11 Cap and vortex 2 x 30 seconds.

Uncontrolled5.3.12 Centrifuge for 5 minutes at 3000 rpm.

5.3.13 Transfer the bottom organic layer to labeled autosampler vials and place into GCMS autosampler tray. If the sample will not be analyzed immediately, place them in the freezer for temporary storage.

5.4 GC/MS Acquisition Parameters

Table 3: GC/MS Parameters

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OVEN INLET COLUMN INJECTOR Initial temp 50ºC Pulsed Varian Factor 4 or equivalent Injection volume 1-2 splitless µL Maximum temp: Helium constant flow Syringe size 10 µL 310 ºC Temp 250ºC MSD 280°C Acquisition Run time: up to 30 MS source 230°C Mode:SCAN min MS Quad 150°C Method: SCANQ.m

5.4.1 Table 4: Target and Qualifier Ions

ANALYTES TARGET ION QUALIFIER IONS Brompheniramine 247 Copy167, 180 Bupivacaine 84 98, 120 Chlorpheniramine 203 167, 72 Chlorpromazine 58 86, 318 Citalopram 238 324, 208 Clomipramine 268 58. 85 Clozapine 243 192, 256 Desipramine 208 193, 234 Dextromethorphan* 271 214, 150 Diltiazem 58 71, 121 Diphenhydramine-D3 61 76 Doxylamine 58 71, 167 Imipramine 234 193, 280 Ketamine-D4 184 213, 156 Ketamine 180 152, 209 Meperidine 247 218, 172 Metoclopromide 86 99, 184 Midazolam 310 325, 297 Mirtazapine 195 208, 167 Normeperidine 158 232, 276 UncontrolledNorsertraline 290 119, 145 Sertraline 304 159, 239 SKF-525-IS 86 99 and/or 165 Verapamil 303 151, 260 Lidocaine 86 72, 234 Buspirone 177 277, 385 Pheniramine 169 58, 72 Methadone-D9 78 165

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*Case samples are reported as Methorphan

5.4.2 Table 5: Sample Injection log

Position Sample Number First Reagent Blank Second Negative control Third Standard 1 Fourth Standard 2 Fifth Standard 3 Sixth Standard 4 Seventh Standard 5 Eighth Negative Control Copy Ninth Reagent Blank Tenth QC-1 Eleventh QC-2 Twelfth QC-3 Continue with samples - alternating a negative QC every 5th specimen and a positive QC every 10th specimen, ending with a QC.

6.0 Data Analysis / Interpretation/ Documentation

6.1 Collect the following data for each chromatogram:

6.1.1 Total SIM chromatogram

6.1.2 Quantitation results Uncontrolled 6.1.3 Detailed Quantitation Report including determined analyte concentrations, target and qualifier ion chromatographic responses or ratios and SIM ion profiles and target ion integration baselines.

7.0 Acceptance Criteria

7.1 As described in the most recent version of the Standard Operating Procedure “GC/MS Analyses: Quality Control, Acceptance and Reporting Criteria”

8.0 References Page 9 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

8.1 Garriott, James C.; Foerster, Eberhard H.; Hatchett, Don: "A Rapid, Comprehensive Screening Procedure for Basic Drugs in Blood or Tissues by Gas Chromatography", .Journal of Analytical Toxicology. Vol. 2, March/April 1978, p. 50-5.

9.0 Revision History

Reviewed Revision Description of Change By Date Section 5.4.2 added new target and qualifying ions for both Sertraline and Norsertraline; Updated revision number Added section 4.3.6, describing new Ketamine-D4: Added section 4.4.13, detailing how toCopy prepare Ketamine-D4 working internal standard; Added note under Table 1 in section 5.1.3 to use Ketamine-D4 in ketamine extractions; Added ions for Ketamine-D4 and Methadone-D9 in Table 4 of Section 0713, 11 5.4.2 D.Mike 0813 Updated revision number to 12; Corrected the spelling of “Mirtazapine”. Deleted analytes that have moved to LC/MS/MS from the Target and Qualifier Ion table, removed reference to crimping standards and controls with Teflon caps, removed chlordiazepoxide working standards, controls, and from the standard/QC D.Mike/ 12 preparation chart A. Beard 0913 Updated instrument parameters, changed 13 dextromethorphan reporting to methorphan F. Guale 1013 Changed “Approved by” to Toxicology Manager and revision number to 14; Section 2.1: Included plasma and serum; Section 4.3: Changed storage to state “per manufacturer’s instructions” and added “or equivalent” to manufacturer catalog numbers: Section 5.1 Tables 1&2: changed the target concentration for standard 14Uncontrolled curves to reflect new levels in ng/mL. D. Mike 1213 Revised Sections 4.3.2 B & 4.3.3 B to include “or equivalent”; Revised steps to make working Std/QC B; Added Meperidine, Normeperidine, and chlorpromazine to list of Analytes in Section 5.4.1 Table 4. Changed revision number to 15. Added D. Mike/ 15 diphenhydramine-D3 for doxylamine quantitations A. Beard 0914

10.0 Attachment(s) or Appendices

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10.1 Not Applicable

Copy

Uncontrolled

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