Colorado Department of Public Health and Environment. Laboratory Services Division Toxicology Laboratory
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1 Colorado Department of Public Health and Environment Laboratory Services Division Toxicology Laboratory Carboxy-THC (11-nor-9-carboxy-delta-9- tetrahydrocannabinol), GC/MS, Urine Revision 1 SEPTEMBER 2004
2 TITLE 1 REFERENCES 1 METHOD 1 PRINCIPLE 1 SAMPLE 1 SAFETY 1 EQUIPMENT 1 REAGENTS 3 CHEMICALS 3 GASES 3 DERIVITIZING AGENTS 3 CONTROLS 3 STANDARDS/INTERNAL STANDARDS 3 REAGENT PREPARATION 4 PROCEDURE 4 STOCK STANDARD PREPARATION 4 WORKING STANDARD PREPARATION 5 CONTROL PREPARATION 5 SAMPLE PREPARATION 5 EXTRACTION 6 Column Preparation 6 Specimen Application 6 Specimen Elution 7 Eluant Concentration 7 Derivatization 7 GC/MS ANALYSIS 7 GC/MS Parameters 7 MSD SIM Program 8 Instrument Set Up 8 Injection Sequence 8 DATA INTERPRETATION AND QUALITY CONTROL 8 POLLUTION PREVENTION 9 WASTE MANAGEMENT 9
3 File: SOP_THC_R2 Section: Toxicology September 2004 TITLE Carboxy-THC(11-nor-9-carboxy-delta-9-tetrahydrocannabinol), GCMS, Urine. REFERENCES THC Metabolite in Urine for GC or GC/MS Confirmations Using 200 mg Clean Screen TM Extraction Column, Worldwide Monitoring United Chemical Technologies, Inc. Clean Screen TM Extraction Column Applications Manual, Worldwide Monitoring United Chemical Technologies, Inc. HP MS EnvironQuant Chemstation Software G1701BA Version B.01.00, Hewlett-Packard Company. HP 5971 Mass Selective Detector Hardware Manual, Hewlett-Packard Company. MS Chemstation User s Guide, HP G1034C Ms Chemstation Software, Hewlett-Packard Company. METHOD Gas chromatography/mass spectrometry. PRINCIPLE A solid phase extraction column is used to extract the major THC metabolite, 11-nor-9-carboxydelta-9-tetrahydrocannabinol from urine samples that have tested positive by an immunoassay screening procedure. The extracted drug(s) is derivatized and then analyzed by GC/MS. SAMPLE Urine, 3 ml, not preserved. SAFETY Use routine precautions found in the Chemical Hygiene Plan (Appendix I Safety Manual) when working in the laboratory. Follow the Bloodborne Pathogens Exposure Control Plan (Appendix G Safety Manual), when working with biological fluids or tissues. Read all Material Safety Data Sheets before handling unfamiliar reagents. EQUIPMENT mg Clean Screen TM extraction columns; Worldwide Monitoring United Chemical Technologies, Inc. Standard Operating Procedure Page 1 of 12
4 Section: Toxicology September Multiprep workstation: vacuum control assembly and gauge, column mounting plate, vacuum box, elution rack, and sample evaporator. 3. Adjustable volume pipettes. 4. Volumetric pipettes, (Class A required for standard preparation). 5. Microliter syringes (Hamilton or equivalent). 6. Graduated cylinders. 7. Volumetric flasks. 8. Beakers. 9. Heating block. 10. Vortex mixer. 11. Stir plate. 12. Heating plate with 100 o C capability x100 mm borosilicate glass disposable culture tubes. 14. ph paper. 15. Crimp top vials with 0.25 ml inserts, 11 mm. 16. Crimp caps 11 mm with teflon coated red silicone rubber septa. 17. Crimping tool. 18. Turbo-vap or N-vap evaporation and concentration apparatus. 19. GC/MSD system: a) GC with a fused silica column with a stationary phase of crosslinked methylsilicone - either 5% or 50%. b) Mass selective detector - (MSD). c) Automatic liquid sampler - (ALS). Standard Operating Procedure Page 2 of 11
5 Section: Toxicology September 2004 REAGENTS Reagent grade chemicals shall be used in all tests unless otherwise indicated. It is intended that all reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficient high purity to permit its use without lessening the accuracy of the determination. Chemicals 1. Acetonitrile - (ACN). 2. Ethyl Acetate - (EA). 3. Glacial Acetic Acid. 4. Hexane - (HEX). 5. Hydrochloric Acid, concentrated - (HCL). 6. Methanol - (MEOH). 7. Reagent water, ASTM Type II. Gases 1. Helium Gas Tank % purity. 2. Nitrogen Gas Tank - ultra high purity. Derivitizing Agents 1. Bis-(trimethylsilyl)trifluoroacetamide - (BSTFA) Controls 1. Biorad Liquicheck Urine Toxicology Controls, Biorad C1 and Biorad C4. 2. Negative urine UTAK Laboratories, Inc. Certified Drug Free (or donated by staff). Standards/Internal Standards 1. Carboxy-THC-D3 (THC-D3). Standard Operating Procedure Page 3 of 11
6 Section: Toxicology September Carboxy-THC (THC). REAGENT PREPARATION Note: All working chemical solutions, mixtures, or dilutions shall be labeled with the following information; chemical name, concentration, date prepared, analysts initials, special storage instructions, and expiration date. 1. Potassium Hydroxide (KOH), 10N. Into a 600 ml glass beaker, placed in a water bath on a stir plate, dissolve 140 g of KOH (Potassium Hydroxide) into 150 ml of reagent water. Cool. Transfer to a 250 ml plastic volumetric flask. Bring to volume with DI water. Stable at room temperature until used. CAUTION: CAUSTIC! Heat is generated in the making of this reagent. In the event of a spill, use a base spill kit to contain and clean up the spill. 2. Hydrochloric Acid (HCL) 100 mm. Into a 1000 ml glass volumetric flask add 800 ml reagent water. Add 8.4 ml concentrated HCl. Bring to volume with reagent water. Mix. Store at room temperature. Stable for 6 months. 3. Acetonitrile:Hydrochloric Acid (60:40): Using a graduated cylinder, add 120 ml of Acetonitrile. Transfer to glass storage container labeled 60:40 (Acetonitrile:HCL). Using the same graduated cylinder, add 80 ml of 100mM HCL. Add to Acetonitrile in glass storage container. Mix. Store at room temperature. Stable for 2 weeks. 4. Hexane:Ethyl Acetate (75:25): Using a graduated cylinder, add 150 ml of Hexane. Transfer to glass storage container labeled 75:25 (Hexane:EA). Using the same graduated cylinder, add 50 ml of Ethyl Acetate. Add to Hexane in glass storage container. Mix. Store at room temperature. Stable for 2 weeks. PROCEDURE Stock Standard Preparation 1. Carboxy-THC-D3 1.0 μg/ml internal standard. Current supplier: Cerilliant; received in ampoules at 100 μg/ml concentration. Transfer one ml of Carboxy-THC-D3 to a 100 ml volumetric flask; bring to volume with MEOH. Store in freezer (0 o C). Expiration date of 1 year. 2. Carboxy-THC 1.0 μg/ml standard. Current supplier: Cerilliant. Received in ampoules at 100 μg/ml concentration. Transfer one ml Carboxy-THC to a 100 ml volumetric flask; bring to volume with MEOH. Store in freezer (0 o C). Expiration date of 1 year. Standard Operating Procedure Page 4 of 11
7 Section: Toxicology September 2004 Working Standard Preparation 1. For the 5 ng/ml standard: Using a macropipette, pipette 3 ml of negative urine into the corresponding labeled tube. Use a micropipette to spike with 15 μl of 1.0 μg/ml Carboxy- THC standard. 2. For the 10 ng/ml standard: Using a macropipette, pipette 3 ml of negative urine into the corresponding labeled tube. Use a micropipette to spike with 30 μl of 1.0 μg/ml Carboxy- THC standard For the 50 ng/ml standard: Using a macropipette, pipette 3 ml of negative urine into the corresponding labeled tube. Use a micropipette to spike with 150 μl of 1.0 μg/ml Carboxy- THC standard. 5. For the 200 ng/ml standard: Using a macropipette, pipette 3 ml of negative urine into the corresponding labeled tube. Use a micropipette to spike with 600 μl of 1.0 μg/ml Carboxy- THC standard. Control Preparation 1. Negative control: Using a macropipette, pipette 3 ml of negative urine into the corresponding labeled tube. 2. Positive controls: Record controls used on worksheet. Use a macropipette to pipette 3 ml of C1 and C4 into the corresponding labeled tubes. Sample Preparation 1. For each sample (standards, controls, and patient specimens) to be analyzed label a 13x100 mm borosilicate tube in a supporting rack. 2. For all positive screened samples, use a macropipette to pipette 3 ml of urine into the corresponding labeled tube. A straight extraction in addition to the dilution is NOT necessary. These dilutions are generated from experience with the Carboxy-THC immunoassay screen: 3. Use a micropipette to add 100 μl of 1.0 μg/ml Carboxy-THC-D3 internal standard to each samples. 4. Using a repeat pipette, add 200 μl of 10N KOH, to each. 5. Cap tubes and vortex gently to mix. Standard Operating Procedure Page 5 of 11
8 Section: Toxicology September Place in heating blocks at 70 o C for 30 minutes. 7. Cool to room temperature. 8. Using a repeat pipette, add 2 ml of glacial acetic acid. 9. Vortex gently to mix. 10. Check ph of several samples (one or two per row). If not between 3.0 and 4.0, adjust with glacial acetic acid. Extraction NOTE: A repeat pipette is used for all additions during the extraction. NOTE: Elution rate must not exceed 1 ml/minute. Column Preparation 1. Place one 200 mg Clean Screen extraction column in the multiprep workstation vacuum mounting plate for each extraction to be performed. Label each, using a marker, with the standard name or position number (according to the worksheet) to be extracted. 2. Pull 3 ml of MEOH through each column. 3. Pull 3 ml of reagent water through each column. 4. Pull 1 ml of 100mM HCL through each column. NOTE: Columns should not go dry during this procedure Specimen Application 1. Pour each specimen onto the column, matching the labeled tube to each marked/numbered column. 2. Pull 2 ml of reagent water through each column. 3. Pull 2 ml of 60:40 (Acetonitrile:HCL) through each column. 4. Dry columns under full vacuum for 5 minutes. Note: If liquids do not elute freely by gravity flow, there is probably air trapped within the Standard Operating Procedure Page 6 of 11
9 Section: Toxicology September 2004 column bed. Tapping the column mounting plate onto the vacuum box should initiate flow. If this does not initiate flow, use a rubber bulb to gently push a few drops of eluation solvent and trapped air into the collection tube. Allow the remainder of solvent to flow by gravity or apply a gentle vacuum (<2 mm Hg). Specimen Elution 1. Using the elution rack, put a labeled 13x100mm culture tube in the appropriate spots for all samples. 2. Add 200 μl of Hexane to each column. 3. Add 3 ml of Hexane:EA (75:25) to each column. Let columns drip without vacuum then turn vacuum on and increase to collect the remaining eluant. Eluant Concentration 1. Evaporate the eluant under nitrogen at room temperature. Remove immediately after completion. Make sure all tubes are completely dry. Derivatization CAUTION - In the event of a spill, absorb the derivitizing agent with vermiculite. Place in a small autoclave bag, and seal with tape. This may then be disposed of in the normal trash. Ventilate area and wash spill site after material has been cleaned up. 1. Add 50 μl of BSTFA and 150 μl of EA to each tube. 2. Cap and vortex. 3. Incubate at 70 o C for 30 minutes. 4. Cool to room temperature. 5. Transfer, using a micropipette, to labeled crimp top GC/MS sample vials with inserts. 6. Cap using crimping tool. Make sure vials are tightly sealed. GC/MS Analysis GC/MS Parameters Current GC/MS method parameters are located in the GC/MS Methods Logbook and are automatically loaded into the instrument when the sequence runs. Standard Operating Procedure Page 7 of 11
10 Section: Toxicology September 2004 MSD SIM Program Drug BSTFA THC-D3 374, 476, 491 THC 371, 473, 488 Instrument Set Up 1. Perform routine maintenance and autotune as described in the Tuning and Troubleshooting an Agilent 597X MSD Standard Operating Procedure. Injection Sequence THC 5 ng neat (for setting retention times) THC 5 ng standard THC 10 ng standard THC 50 ng standard THC 200 ng standard THC Biorad C1 THC Biorad C4 WASH THC Negative Control Specimens END THC 5 ng standard END THC Biorad C1 END THC Negative Control DATA INTERPRETATION AND QUALITY CONTROL 1. Review all calibration data for compliance with Toxicology Laboratory requirements. Record any noncompliance(s) and corrective actions required. 2. Review all control sample data for compliance. Record any noncompliance(s) and corrective actions required. 3. Review all sample data to determine presence/absence of analytes and record appropriate data on the work sheet. 4. All comments/observations written on chromatograms are to be written in ink, initialed, and dated. 5. Record all quality control results in the Quality Control Log. 6. Assemble the data package containing initial calibration information, quantitation reports and chromatograms for all standards, controls and samples and submit to the Toxicology Quality Control Analyst in accordance with the Toxicology Data Review, Approval, and Reporting Instruction. 7. The Toxicology Quality Control Analyst will review each data package for accuracy, Standard Operating Procedure Page 8 of 11
11 Section: Toxicology September 2004 completeness, and appropriate corrective actions and initial the QC Review Checklist. 8. The data package will be given to the Toxicology Supervisor for final review and approval. 9. File original data in appropriate filing cabinets(s). POLLUTION PREVENTION 1. Pollution prevention encompasses any technique that reduces or eliminates the quantity or toxicity of waste at the point of generation. Numerous opportunities for pollution prevention exist in a laboratory operation. The United States Environmental Protection Agency (US EPA) has established a preferred hierarchy of environmental management techniques that places pollution prevention as the management option of first choice. Whenever feasible, laboratory personnel should use pollution prevention techniques to address waste generation. When wastes cannot feasibly be reduced at the source, the US EPA recommends recycling as the next best option. 2. The quantity of chemicals purchased should be based on expected usage during the shelf life and disposal cost of unused material. Actual reagent preparation volume should reflect anticipated usage and reagent stability. 3. For information about pollution prevention that may be applicable to laboratories, consult Less is Better: Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society s (ACS) Department of Governmental Regulations and Science Policy, th Street NW, Washington D.C , (202) WASTE MANAGEMENT 1. The US EPA requires that laboratory waste management practice be consistent with all applicable rules and regulations. Excess reagents, samples and method process wastes should be characterized and disposed of in an acceptable manner. The agency urges laboratories to protect the air, water, and land by minimizing and controlling all releases from hoods and bench operations, complying with the letter and spirit of any waste discharge permits and regulations, and by complying with all solid and hazardous waste regulations, particularly the hazardous waste identification rules and land disposal restrictions. For further information on waste management consult the Waste Management Manual for Laboratory Personnel, available from The American Chemical Society at the address listed above. 2. Dispose of all urine samples in the laboratory sink with a large volume of excess water after neutralization to ph 5-9 with sodium bicarbonate (NaHCO 3 ). 3. Contact the Chemical Hygiene Office for disposal recommendation of other chemicals or solutions. Standard Operating Procedure Page 9 of 11
12 Section: Toxicology September 2004 Written by: Laurel J. Farrell 5/30/94 Procedure Author Approved by: Laurel J. Farrell 5/30/94 Section Supervisor Alan Dunhill 5/31/94 Program Manager James D. McKinna 6/1/94 QA Officer Ronald L. Cada 6/2/94 LARS Director Effective 6/2/94 Deleted From Service Original File Name: TTHC Handwritten changes made by Laurel Farrell on 8/26/96 and approved by Alan Dunhill. Incorporated into Revision 1. Handwritten changes made by Harold Wells on 8/24/2000. Incorporated into Revision 1. Handwritten changes made and approved by Cynthia Burbach on 7/16/2001. Incorporated into Revision 1. Handwritten changes made by Cynthia Burbach on 3/3/04 and 6/17/2004. Incorporated into Revision 1. Annual review performed in Comments incorporated into Revision 1. Annual review performed in Comments incorporated into Revision 1. Standard Operating Procedure Page 10 of 11
13 Section: Toxicology September 2004 REVISIONS File Name: SOP_THC_R1 Revision: 1 09/13/04 Edited by: Vanessa N. Simmons 09/13/04 Approved by: Section Supervisor: Cynthia Burbach 09/13/04 Program Manager: Laurie Peterson-Wright Date 09/13/04 Quality Assurance Officer: David A. Butcher 09/13/04 Division Director: David A. Butcher 09/13/04 File Name: Revision: Approved by: Edited by: Section Supervisor: Program Manager: Quality Assurance Officer: Division Director: Date File Name: Revision: Approved by: Edited by: Section Supervisor: Program Manager: Quality Assurance Officer: Division Director: Date File Name: Revision: Approved by: Edited by: Section Supervisor: Program Manager: Quality Assurance Officer: Division Director: Date Standard Operating Procedure Page 11 of 11
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