Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.2001 Title: Acidic-Neutral Drug Screens Rev.: 16

1.0 Purpose

1.1 This document describes procedures employed by the Toxicology Laboratory to screen blood or other biological specimens for the presence of acidic or neutral drugs.

1.2 Acidic or neutral drugs are extracted at an acidic pH into Toluene from blood or other biological specimens. The organic extract is evaporated to approximately 100 μL, and transferred to an autosampler vial. . Semi-quantitations are achieved by extraction of the ions indicated in Table 3. Compound identification is achieved by library match of the total ion mass spectra.

2.0 Scope

2.1 This procedure is applicable to screening blood and other biological specimens. 2.2 The analysis is appropriate to all acidic or neutralddrugs Copy including but not limited to, barbiturates, phenytoin, topiramate, carbamazepine, levetiracetam, valproic acid, naproxen, and ibuprofen. Qualitative and semi-quantitative analyses of these compounds plus other neutral substances such as meprobamate and carisoprodol may be performed by this procedure. 3.0 Definitions and Abbreviations rolle 3.1 No method-specific ort 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 test tubes

4.1.2 16 x 100 mm test tubes Uncon4.1.3 Mixer 4.1.4 Rocker

4.1.5 Nitrogen evaporator

4.1.6 Compressed nitrogen

4.1.7 Autosampler vials, inserts, and septum caps

Page 1 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.2001 Title: Acidic-Neutral Drug Screens Rev.: 16

4.1.8 GCMS

4.2 Reagents

4.2.1 Toluene HPLC Grade

4.2.2 2N HCl A. Slowly and with constant stirring, add 166 mL concentrated hydrochloric acid to 500 mL DI water. B. Allow the mixture to cool and adjust the volume to 1 L C. Stable at room temperature.

4.3 Stock Standards and Controls Copy Note: Certified reference materials should be used within manufacturer’s expiration date and should be stored at the recommended storage temperature.

4.3.1 Pentobarbital Stock Control 1.0 mg/mL A.Purchase from Cerilliant, catalog #P-010 or equivalent

4.3.2 Butalbital Stock Control 1.0 mg/mL A. Purchase from Cerilliant, catalog #B-006 or equivalent

4.3.3 Secobarbital Stock Control 1.0 mg/mL A. Purchase from Cerilliant, catalog #S-002 or equivalent

4.3.4 Phenobarbital Stock Control 1.0 mg/mL A. Purchase from Cerilliant, catalog #P-008 or equivalent

4.3.5 Valproic Acid Stock Control 1.0 mg/mL A. Weigh 11.5 mg Valproic acid sodium salt into a 10 mL volumetric flask. B. Adjust the volume to 10 mL with methanol. C. Store at –10 to –20º C. UncontrolledD. Stable 1 year.

4.3.6 Carbamazepine Stock Control 1.0 mg/mL A. Purchase from Cerilliant, catalog #C-053 or equivalent

4.3.7 Carisoprodol Stock Control 1.0 mg/mL A. Purchase from Cerilliant, catalog #C-039B or equivalent

4.3.8 Topiramate Stock Control 1.0 mg/mL A. Weigh out 10 mg Topiramate into a 10 mL flask.

Page 2 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.2001 Title: Acidic-Neutral Drug Screens Rev.: 16

B. Adjust the volume to 10 mL with methanol. C. Store at –10 to –20º C. D. Stable 1 year.

4.3.9 Phenytoin Stock Control 1.0 mg/mL A. Purchase from Cerilliant, catalog #P-063 or equivalent

4.3.10 Ibuprofen Stock Control 1.0 mg/mL A. Purchase from Cerilliant, catalog #I-009 or equivalent

4.3.11 Meprobamate Stock Control 1.0 mg/mL A. Purchased from Cerilliant, catalog #C-077 or equivalent

4.3.12 Naproxen Stock Control Copy 1.0 mg/mL A. Purchase from Cerilliant, catalog #N-042 or equivalent

4.3.13 Levetiracetam stock standard or control solution 1.0 mg/mL A. Purchase from Cerilliant, catalog #L-020 or equivalent

4.3.14 Hexobarbital Stock Internal Standard 1.0 mg/mL A. Purchase from Cerilliant, catalog #H-013 or equivalent

4.4 In-house Working Standards and Controls

4.4.1 Acidic Blood Control #1 A. Transfer 10 µL of butalbital, carisoprodol, pentobarbital, phenobarbital, secobarbital, topiramate, and valproic acid stock controls into a 13 x 100 mm screw cap tube. B. Transfer 2 uL of carbamazepine stock control into the same 13 X 100 screw cap tube. C. Add 1 mL of negative blood and mix.

4.4.2 Acidic Blood Control #2 A. Transfer 10 uL of phenytoin and levetiracetam stock control into a 13 Uncontrolledx 100 mm screw cap tube. B. Transfer 25 uL of meprobamate, naproxen, and ibuprofen stock controls into the same 13 x 100 mm screw cap tube. C. Add 1 mL of negative blood and mix.

4.5 Commercial Working Standards and Controls

4.5.1 Commercial Positive Blood Acidic Control #1 A. Quality Assurance Service Corp. control #1 contains butalbital, carisoprodol, pentobarbital, phenobarbital, secobarbital, topiramate,

Page 3 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.2001 Title: Acidic-Neutral Drug Screens Rev.: 16

carbamazepine, and valproic acid. Controls are stored at 2 to 8°C. Once reconstituted, the control is stable for 5 days B. Transfer 1 mL of the commercial control into a 13 X 100 screw cap tube.

4.5.2 Commercial Positive Blood Acidic Control #2 A. Quality Assurance Service Corp. control #2 contains ibuprofen , meprobamate, naproxen, and phenytoin. Controls are stored at 2 to 8°C. Once reconstituted, the control is stable for 5 days. B. Transfer 1 mL of the commercial control into a 13 X 100 screw cap tube. C. Transfer 10 uL of levetiracetam stock control into the same 13X 100 screw cap tube. Copy 4.6 Hexobarbital Working Internal Standard 0.10 mg/mL A. Transfer 10 mL Hexobarbital stock internal standard into a 100 mL volumetric flask. D. Adjust the volume to 100 mL with methanol. E. Divide between two 50 mL containers (Only one has to be validated) F. Store at –10 to –20º C. G. Stable until manufacturer’s expiration date.

Note: Use the commercial positive blood acidic control #1 and #2. If the commercial positive blood acidic controls are unavailable, prepare and use In-house acidic blood controls #1 and #2.

5.0 Procedure

Note: For quantitations, refer to the acid neutral drug quantitation procedure.

5.1 Sample Preparation

5.1.1 Label sufficient 13 x 100 mm screw cap test tubes.

Uncontrolled5.1.2 Pipette 1 mL of specimen.

5.1.3 Add 100 µL of hexobarbital internal standard, to all tubes except the reagent blank.

5.2 Extraction

5.2.1 Add 0.1 mL 2 N HCl and vortex.

5.2.2 Immediately add 5 mL Toluene and mix on a rocker for 10 minutes.

Page 4 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.2001 Title: Acidic-Neutral Drug Screens Rev.: 16

Note: Each tube should be vortexed after adding 2N HCL, and put on the rocker immediately after adding the toluene, as the blood will coagulate.

5.2.3 Centrifuge at 3000 rpm for 5 minutes.

5.2.4 Transfer the upper organic layer to evaporating tubes and concentrate it to 0.10 mL under a stream of nitrogen. Transfer the concentrated extract to an auto-sampler vial

5.2.5 Assay each sample by GCMS under full scan conditions (ACIDIC.M.).

5.2.6 Assess all major peaks for acid or neutral drugs. Copy 5.3 Table 1: Sample Injection Log

Tube Description Number (conc) 1 Reagent Blank 2 Negative QC

3, 4, 5….. Specimens

Pos QC 1

Negative QC

Last tube Pos QC 2

(end of run)

Note: Use the Positive QC #2 as the test mix. If there are more than 20 specimens, an additional re-injection of a positive control is required in the middle of the run.

Note: The instrument should be loaded with the blood samples first, followed by any urine and stomach contents with negative controls injected between to reduce carryover.

Uncontrolled

Page 5 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.2001 Title: Acidic-Neutral Drug Screens Rev.: 16

5.4 Table 2: GC/MS Acquisition Parameters

OVEN INLET COLUMN INJECTOR Initial temp 50ºC Pulsed splitless DB-17MS or equivalent Injection volume 1-2 µL Helium Syringe size 10 µL Maximum temp: 250ºC 310ºC

MSD 280ºC ACQUISTION MS Source 230°C Mode: SCAN Run time up to 30 MS Quad 150°C Method: ACIDIC.M min Copy 5.5 Table 3: Target Ions

ANALYTE Target Ions Hexobarbital (IS) 221 Butalbital 168 Pentobarbital 156 Secobarbital 168 Phenobarbital 204 Carbamazepine 193 Phenytoin 180 Carisoprodol 158 Topiramate 324 Valproic acid 73 Ibuprofen 161 Meprobamate 83 Naproxen 185 Levetiracetam 126

6.0 Data Analysis / Interpretation/ Documentation

Uncontrolled6.1 Qualitative Results

6.1.1 All drugs identified by GCMS library match and retention time match are considered "Present” in a case sample if: a. The drug is seen in 2 different specimens or it is seen in 2 places (a parent and metabolite are both identified or if there is a prescription for the drug in question). b. The level of the drug is above the administrative cutoff.

6.1.2 If a control is not run with IFS cases, or the drug is only seen once, it may Page 6 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.2001 Title: Acidic-Neutral Drug Screens Rev.: 16

be reported only as “Presumptive Positive”.

6.1.3 Drug metabolites may be reported as “Present” if the parent drug is present with an acceptable library match and is accompanied by a positive control as specifically listed in TOXF.071: Drug Metabolites Reporting Criteria.

Note: Please refer to TOXF.071 for reporting instructions on drugs and their metabolites.

6.1.4 “Presumptive Positive” results must be reported with a qualified statement. For example, “Presumptive Positive- Quantity not sufficient for confirmation” or “Presumptive Positive- Confirmation not performed”. Copy 6.2 Semi-quantitative Results

6.2.1 Load the correct method

6.2.2 Select Calibrate and then Edit Compounds to update the internal standard and analyte retention times.

6.2.3 Select Calibrate and then Update and then Update One Level. When prompted “File has previously been Quantitated. Requant now?”, choose No.

6.2.4 Select Recalibrate, Calib Level ID 10, Replace Responses, and check the box to replace qualifier ion relative responses. Click Do Update.

6.2.5 Repeat steps 6.2.1 through 6.2.4 for the other control.

6.2.6 If the drug of interest is above the administrative cutoff level, but less than the control level run on the same day, the library match criteria are met, and it is seen in 2 different specimens or it is seen in 2 places (a parent and metabolite are both identified or if there is a prescription for the drug in Uncontrolledquestion), the case can be reported as "Less than …” the control level.

6.3 If the internal standard is not clearly visible due to the presence of interfering substances, the sample should be reported as “Specimen not satisfactory”.

6.4 If the concentration of a drug is above the control level and requires a quantitation, the data reviewer must enter any dilution factors in the notes field of the quantitation request in JusticeTrax, if necessary.

6.5 Cases with valproic acid concentrations above the control level will be sent to a

Page 7 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.2001 Title: Acidic-Neutral Drug Screens Rev.: 16

reference laboratory for quantitation.

7.0 Acceptance Criteria

7.1 Criteria for Accepting Results

7.1.1 Daily mass spectrometer tune criteria and test mix chromatographic criteria must be met.

7.1.2 All added compounds should be present in each extracted control and the following analytes must have the following responses:

QC# Representative Area Count Analyte Copy 1 Butalbital ≥10,000 2 Phenytoin ≥10,000 Internal Hexobarbital ≥ 20,000 standard

7.1.3 The administrative cutoff for carbamazepine is 1.0 mg/L. The administrative cut-off for all other acidic and neutral drugs is 2.0 mg/L. All drugs detected less than the administrative cut-offs are considered negative, except in instances where prescription information is available. When there is no semi-quantitation level for drugs detected in case specimens, consult a Toxicologist I or higher for instruction.

7.1.4 Corrective action should be taken to replace septum or glass insert, cut 0.3 meter from column, replace column, clean source, repeat extraction or remake standards or reagents if deviations occur in the mass spectrometer tune, test mix, or extracted controls.

7.1.5 The retention time of the analyte peak must be within 2% of the retention time of the same compound in a positive control sample run on the same day Uncontrolled 7.1.6 Refer to the “GC/MS LIBRARY SEARCH TECHNIQUES AND DATA RETENTION” SOP for the reviewing, and recording results

7.1.7 If individual acceptance criteria are not met consult a Toxicologist 1 or higher.

8.0 References

8.1 Baselt, R.C. and Cravey, R.H., Journal of Analytical Toxicology, 1: 2, 81-98 Page 8 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.2001 Title: Acidic-Neutral Drug Screens Rev.: 16

(1977).

9.0 Revision History

Revision Description of Change Reviewed By Date 0 Original document 1 2 3 0302 4 A. Beard 0806 Reformatted, added Table 1, included Positive QC 2 and Topiramate to Positive QC 1, added analyte and I. Bermudez 4 quant ions, GC/MS parameters CopyF. Guale 0907 Updated the drugs in the controls and added 5 commercial controls A. Beard 0208 2008 annual review-added acetaminophen and 6 naproxen in the commercial QC A. Scarafile 0708 Changed the extraction solvent-dichloromethane to 7 Toluene F. Guale 0808 Added naproxen to the controls and target ion table and updated the sample injection log, changed some stock standards to Cerilliant, added levetiracetam, and increased the volume of working internal standard 8 prepared. A. Beard 0409 9 Update to Current Practice A. Beard 0809 10 Name changed to Institute of Forensic Sciences T. Cao 0410 Changed some stock standards to Cerilliant, deleted methanol clean up steps because it’s not needed with the toluene extraction, and added sections 6.3, 6.4, 6.5 A. Beard 11 Changed storage temperature of commercial controls G. Thomas 0610 Changed the level of levetiracetam control, and 12 updated sections 5.2.1, 5.2.2 G. Thomas 0811 Uncontrolled2012 annual review; Header- changed Approved by to Dr. Roger Kahn and revision number to 13; changed amount of levetiracetam added to 10uL; Added section 13 6.1.2 Drug metabolite reporting criteria. D. Mike 1212

Page 9 of 10 Harris County Institute of Forensic Sciences Section: Toxicology Approved By: Toxicology Manager Document Type: GC & GC/MS Procedure No.: TOX07.2001 Title: Acidic-Neutral Drug Screens Rev.: 16

Added acceptance criteria for QC’s and administrative cutoff for drugs (7.1.2: a, b, c), updated CRM storage and expiration dates, added “or equivalent” to CRM, deleted non-critical instrument parameters, incorporated raise changes, added re-injection of positive controls, and instruction for set up of semi- F. Guale/ 14 quanitative results A. Beard 1013 2013 annual review- Changed revision number to 15 to 15 correlate with Q-pulse. D. Mike 1213 Added statement that presumptive positive results must 16 include a qualifying statement. D. Mike 0514

10.0 Attachment(s) or Appendices Copy 10.1 Not Applicable

Uncontrolled

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