Division: Laboratory Services Unit: Chemistry Section: Toxicology Laboratory Toxicology Laboratory Delta 9 THC and Delta 9 THC COOH, LC MS/MS, Blood Revision 1 September 2011
Division: Laboratory Services Unit: Chemistry Section: Toxicology Table of Contents 1.0 Introduction 1 1.1 Principle 1.2 Flow Diagram 2.0 Reagents and Equipment 1 2.1 Reagents 2.1.1 Chemicals 2.1.2 Standards/Controls 2.1.3 Gases 2.2 Reagent Preparation 2.3 Equipment/Materials 3.0 LC MS/MS Conditions and Parameters. 3 4.0 Internal Standards and Standards.5 4.1 Internal Standards 4.1.1 Stock Internal Standard Preparation 4.1.2 Working Internal Standard Preparation 4.2 Standards 4.2.1 Stock Standard Preparation 4.2.2 Working Standard Preparation 4.3 Negative Blood Preparation 5.0 Quality Control 6 5.1 Controls 5.1.1 Stock Control Preparation 5.1.2 Working Control Preparation 6.0 Procedure.. 7 6.1 Blood Solid Phase Extraction 6.1.1 Specimen Preparation 6.1.2 Protein Precipitation 6.1.3 Calibrator Preparation/Extraction 6.1.4 Column Preparation and Conditioning 6.2 Sample Preparation for LC MS/MS 7.0 Data and Results. 9 7.1 Calculation of the Calibration Curve 7.2 Data 7.3 Interpretation of Specimen Data/Results 8.0 Criteria for Quality Control. 9 8.1 Results
Division: Laboratory Services Unit: Chemistry Section: Toxicology 9.0 Limitations/Procedure Notes. 10 10.0 Laboratory Safety Precautions..11 11.0 Waste Management..11 12.0 Pollution Prevention.12 13.0 References..13 14.0 Authorization..14
1 1.0 Introduction 1.1 Principle All samples that have screened presumptive positive for Delta 9 THC and/or Delta 9 THC COOH are confirmed by LC MS/MS. In summary, all of the samples are subject to protein precipitation, the addition of internal standards (Delta 9 THC d3 and Delta 9 THC COOH d3), the addition of buffer (ph 7), and then extracted via solid phase extraction (SPE). The organic phase of the sample is evaporated off and the sample is then reconstituted with methanol. The identification and quantification are then achieved by the use of the LC MS/MS in the Multiple Reaction Monitoring (MRM) mode. 1.2 Flow Diagram Blood Sample ACN Precipitation Clear Liquid ISTD, CTRLS, STDS, Buffer (ph7) Solid Phase Extraction (SPE) Evaporate 2.0 Reagents and Equipment Analyze by LC MS/MS 2.1 Reagents Reagent Grade Chemicals are used in all tests unless otherwise indicated. The reagents must 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.
2 2.1.1 Chemicals Formic Acid (FA); Acros Organics 147932500, or equivalent Hexane; J.T. Baker 9262 03, or equivalent Methanol; Fisher HPLC Grade A452 4, or equivalent Deionized Water (DI H2O) Ethyl Acetate; EMD EX0241 1, or equivalent Acetonitrile (ACN); Fisher HPLC Grade A998 4, or equivalent Buffer Solution ph 7; Negative Blood; Hemostat, or equivalent Sodium Fluoride; Acetic Acid; Fisher A490 212, or equivalent 2.1.2 Standards/Controls Delta 9 THC; Cerilliant T 005 (1 mg/ml), or equivalent Delta 9 THC COOH; Cerilliant T 018 (100 µg/ml), or equivalent Delta 9 THC d3; Cerilliant T 003(100 µg/ml), or equivalent Delta 9 THC COOH d3; Cerilliant T 004 (100 µg/ml), or equivalent Delta 9 THC; Lipomed THC 135 1LE (1 mg/ml), or equivalent Delta 9 THC COOH; Lipomed THC 318 0.1LM (1 mg/ml) or equivalent 2.1.3 Gases Nitrogen Gas 2.1 Reagent Preparation Mobile Phase A : 0.1% Formic Acid; add 1.0 ml FA to 1 liter DI H2O Mobile Phase B : 0.1% Formic Acid; add 1.0 ml FA to 1 liter ACN Elution Solvent: 25 ml of Ethyl Acetate: 25 ml Hexane: 1 ml of Glacial Acetic Acid 2.3 Equipment/Materials LC MS/MS, ABI 4000, equipped with Agilent 1100 series LC or equivalent LC Column Pipettor, variable sizes (5) ml, (100 1000) µl, (10 100) µl, (5 40) µl, (2 20) µl, and (0.5 10) µl Centrifuge Vortex TurboVap LV; Caliper or Equivalent
3 Test Tubes, 16mm X 100mm silanized borosilicate glass; Fisher Scientific or equivalent Amber Glass Wide Mouth Vials; Agilent, or equivalent Conical Glass Inserts; Agilent, or equivalent Screw Caps (Pre Slit); Agilent, or equivalent Solid Phase Extraction Columns; UCT Clean Screen Extraction THC Columns, or equivalent 3.0 LC MS/MS Conditions and Parameters LC Oven 30 C LC Binary Flow at 0.55 ml/min LC Pump Parameters: Table 3.1 MS/MS Parameters: Table 3.2 Target Monitoring Ions: Table 3.3 Table 3.1 Time (min) Module Event Parameter 1 0.00 pumps pump A/B concentration 90% / 10% 2 0.050 pumps pump A/B concentration 50% / 50% 3 1.50 pumps pump A/B concentration 10% / 90% 4 2.50 pumps pump A/B concentration 10% / 90% 5 5.00 controller pump A/B concentration 90% / 10% Table 3.2 Curtain Gas 15.0 psi Collision Gas 6 psi Ionspray Voltage 5000 volts Temperature 650 C Ion Source Gas 1 50 psi Ion source Gas 2 50 psi
4 Table 3.3 Internal Standards Name Q1/Q3 1 D3_THC 1 318.200/196.200 2 D3_THC 2 318.200/123.000 3 D3_COOH_THC 1 348.200/196.100 4 D3_COOH_THC 2 348.200/302.300 Analytes Name Internal Standards Q1/Q3 1 THC 1 D3_THC 1 315.300/193.200 2 THC 2 D3_THC 2 315.300/123.100 3 COOH_THC 1 D3_COOH_THC 1 345.200/299.200 4 COOH_THC 2 D3_COOH_THC 2 345.200/193.200 Injection Sequence: Blank THC 5 ng/ml Neat (for establishing retention times) Blank THC 1 ng/ml Calibrator THC 5 ng/ml Calibrator THC 10 ng/ml Calibrator THC 50 ng/ml Calibrator THC 100 ng/ml Calibrator Blank/Wash THC Negative Control Blank/Wash THC 5 ng/ml Control THC 10 ng/ml Control THC 40 ng/ml Control THC 80 ng/ml Control Blank/Wash Specimens (to avoid carry over perform blank/wash between each specimen and every 10 samples insert a control or standard for quality control) End THC 1 ng/ml Standard End THC 40 ng/ml Control End THC Negative Control
5 4.0 Internal Standards, Standards and Negative Blood *Note: ALL 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. 4.1 Internal Standards 4.1.1 Stock Internal Standards Preparation Delta 9 THC d3 (100µg/mL); Ceriliant Corporation, in sealed ampoules containing 100 µg/ml Delta 9 THC d3 in 1 ml methanol. Delta 9 THC COOH d3 (100µg/mL); Ceriliant Corporation, in sealed ampoules containing 100 µg/ml of Delta 9 THC COOH in 1 ml methanol. Stock Internal Standard Mix (Delta 9 THC d3 and Delta 9 THC COOH d3, 10 µg/ml) Using a syringe transfer exactly 1 ml of each Stock Internal Standard from Ceriliant Corporation into a 10 ml volumetric flask. Dilute to mark with methanol. Expiration date of 1 year and stored at less than 4 C. 4.1.2 Working Internal Standard Preparation Working Internal Standard Mix (Delta 9 THC d3 and Delta 9 THC COOH d3, 1 µg/ml) Using a syringe transfer exactly 1 ml of the stock internal standard mix into a 10 ml volumetric flask. Dilute to the mark with methanol. Expiration date of 1 year and stored at less than 4 C. 4.2 Standards 4.2.1 Stock Standards Preparation Delta 9 THC (1 mg/ml); Ceriliant Corporation, in sealed ampoules containing 1 mg/ml of Delta 9 THC in 1 ml methanol. THC (100 µg/ml): Using a syringe, transfer exactly 1 ml of Delta 9 THC into a 10 ml volumetric flask. Dilute to mark with methanol. Expiration date of 1 year and stored in freezer at less than 4 C. Delta 9 THC COOH (100 µg/ml); Ceriliant Corporation, in sealed ampoules containing 100 µg/ml of Delta 9 THC COOH in 1 ml methanol. Stock Standard Mix (Delta 9 THC and Delta 9 THC COOH 10 µg/ml) Using a syringe transfer exactly 1 ml of each stock standard mix into a 10 ml volumetric flask. Dilute to mark with methanol. Expiration date of 1 year and stored in freezer at less than 4 C.
6 4.2.2 Working Standard Preparation Working Standard Mix (Delta 9 THC and Delta 9 THC COOH 0.5 µg/ml) Place Exactly 0.5 ml of the stock Standard Mix (10 µg/ml) into a 10 ml volumetric flask. Dilute to mark with methanol. Stable for 1 year at less than 4 C. 4.3 Negative Blood Preparation 1% NaFl Place 10.0 grams of sodium fluoride into a 1000 ml bottle containing defibrinated sheep s blood (Note ensure blood is at room temperature or the volume may be inaccurate). Mix thoroughly. Transfer to 10 ml serum vials, label and cap tightly. Store refrigerated. Expiration date of 3 months. Blood must reach room temperature before use, to ensure proper percentage of sodium fluoride. 5.0 Quality Control 5.1 Controls Commercially available Delta 9 THC and Delta 9 THC COOH purchased from a lab independent from which the calibration standards were purchased. 5.1.1 Stock Controls Preparation Delta 9 THC (1 mg/ml); Lipomed, in sealed ampoules containing 1 mg/ml of Delta 9 THC in 1 ml methanol. Delta 9 THC COOH (1 mg/ml); Lipomed, in sealed ampoules containing 1 mg/ml of Delta 9 THC COOH in 1 ml methanol. Stock Control Mix (Delta 9 THC and Delta 9 THC COOH 10 µg/ml) Using a syringe transfer exactly 1 ml of each stock control mix into a 10 ml volumetric flask. Dilute to mark with methanol. Expiration date of 1 year and stored in freezer at less than 4 C. 5.1.2 Working Control Preparation Working Control Mix (Delta 9 THC and Delta 9 THC COOH 0.5 µg/ml) Place Exactly 0.5 ml of the stock control mix (10 µg/ml) into a 10 ml volumetric flask. Dilute to mark with methanol. Stable for 1 year at less than 4 C.
7 6.0 Procedure 6.1 Solid Phase Extraction 6.1.1 Specimen Preparation Obtain the test specimens, working standards/controls and negative sheep s blood from refrigerated storage. Place on rocker and allow them to reach room temperature for at least 10 minutes prior to use. Label (3 sets) of tubes for standards, controls, samples and extraction columns. Prepare samples by adding 1 ml of Negative blood to all standard and control tubes and 1 ml of sample blood into each of the appropriately labeled tubes. o 1 ng/ml Std, 5 ng/ml Std, 10 ng/ml Std, 50 ng/ml Std, 100 ng/ml Std, Negative Ctrl, 5 ng/ml Ctrl, 10 ng/ml Ctrl, 40 ng/ml Ctrl, 80 ng/ml Ctrl 6.1.2 Protein Precipitation To all blood specimens add 2.5 ml of cold ACN with a 5 ml pipette drop wise while mixing on a vortex. Allow mixture to settle before centrifuging at 5000 RPM for 10 mins. Carefully decant the clear liquid to the second set of appropriately labeled tubes. Evaporate decant for 10 mins at 40 C under a gentle stream of nitrogen (~12psi). 6.1.3 Calibrator Preparation/Extraction In the appropriately labeled tubes, add 5 ml of buffer ph 7. Transfer the following amounts of internal standard working mix, standard working mix or control working mix into each. Where possible, calibrators should be matrixmixed to specimens. For controls each batch should include a minimum of one positive and one negative control. Table 6.1.3.1 Standard Preparation Final Concentration Amount of Working Standard (0.5 µg/ml) Amount of Internal Standard (1.0 µg/ml) 1 ng/ml 2 µg 10 µg 5 ng/ml 10 µg 10 µg 10 ng/ml 20 µg 10 µg 50 ng/ml 100 µg 10 µg 100 ng/ml 200 µg 10 µg
8 Table 6.1.3.2 Control Preparation Final Concentration Amount of Working Control (0.5 µg/ml) Amount of Internal Standard (1.0 µg/ml) 5 ng/ml 10 µg 10 µg 10 ng/ml 20 µg 10 µg 40 ng/ml 80 µg 10 µg 80 ng/ml 160 µg 10 µg Negative 0 10 µg Once completed, cap and place on rocker for 10 mins. 6.1.4 Column Preparation and Conditioning Place labeled columns in appropriate position on vacuum manifold To each of the labeled columns add in order 3 ml of methanol 2 ml of DI Water 1 ml of buffer ph7 Decant specimen/buffer solution into the appropriately labeled column. Be careful not to transfer any cellular debris. Allow to gravity feed through the column. Wash all columns with 1 ml of buffer ph 7. Dry columns for a minimum of 15 minutes at 10 15 inches of Hg. Place another set of appropriately labeled tubes in an elution rack. Place the rack inside the vacuum tank with the appropriate test tubes located beneath the corresponding column. Elute with 3 ml of 25:25:1 Hexane/Ethyl Acetate/Acetic Acid at gravity (no vacuum). Once eluted, pull through with vacuum to collect any remaining solvent. 6.2 Sample Preparation for LC MS/MS Evaporate to dryness in TurboVap for 12 minutes at (~12 psi). Reconstitute with 100 µl of Methanol (HPLC grade). Thoroughly mix tubes on vortex. Transfer specimens to appropriately labeled vials with inserts and cap. Inject on LC MS/MS at a volume of 20 µl.
9 7.0 Data/Results 7.1 Calculation of the Calibration Curve The calibration curve is generated by the software, using all 5 calibrators (standards). Response of peak area ratios of analyte vs. deuterated internal standard are calculated for each concentration. The calibration curves are then generated for each drug, and all the results are calculated using these new curves. The calculations use a linear fit curve. The correlation coefficient for the calibration curve must be 0.98 for the acceptance. The qualifier ion ratio for all calibrators must be within ± 25% of the 1.0 ng/ml calibrator that the software uses to calculate the qualifiers. Make all the necessary adjustments to the calculations (e.g. multiply using the appropriate dilution factor) if less than 1 ml of sample is tested. 7.2 Data The retention time must be within ± 25% or ± 0.4 minute of the 1.0 ng/ml calibrator. If the concentration of the specimen exceeds the concentration of the highest calibrator, the value will be reported as greater than that calibrator value. The results must have the quantitative and qualitative ion ratios within ± 25% of the values for the 1.0 ng/ml calibrator. 7.3 Interpretation of Specimen Data/Results Positive samples will only be reported when: o Chromatography peak shape is acceptable. o Quantitative and qualitative ion ratio is within the acceptable range. o Retention times are within the acceptable range. o Quality control specimen results are within ±20% of the expected quantitative value. o Any deviation from this method must be noted in the case file and should be approved by the lead supervisor or his/her section designee. o Assemble a data packet containing all initial calibration information, quantitation reports, chromatograms for all standards, controls and samples and corrective action logs and submit to the Quality Control Analyst for review and the Toxicology Supervisor or designee for approval in accordance with the Toxicology Data Review, Approval, and Reporting Instruction. 8.0 Criteria for Quality Control 8.1 Results Positive quantitative control results must fall within ±20 % of the targeted value.
10 Negative control (s) must not quantitate above the LOD. All results are recorded. 9.0 Limitations/Procedure Notes Linearity: All levels of controls within linear range were analyzed. These controls included 1 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml, and 100 ng/ml. Samples were run each 10 times on Applied Biosystems API 4000 LC MS/MS. The software used was Analyst Software. Average recovery, relative standard deviation and correlation coefficient was calculated at each concentration level. Precision: Three levels of controls within linear range analyzed. These controls included 5 ng/ml, 10 ng/ml, and 50 ng/ml. Samples were run each 10 times on Applied Biosystems API 4000 LC MS/MS. The software used was Analyst Software. Average recovery and relative standard deviation was calculated at each concentration level. Accuracy: Three levels of controls within linear range analyzed. These controls included 5.0 ng/ml, 10 ng/ml, and 50 ng/ml. Samples were run each 10 times on Applied Biosystems API 4000 LC MS/MS. The software used was Analyst Software. Average recovery and relative standard deviation was calculated at each concentration level. Carryover: High Control (100 ng/ml) was analyzed twice followed by negative control analyzed twice. Samples were run six consecutive times on Applied Biosystems API 4000 LC MS/MS. The software used was Analyst Software. Specificity: The retention time, ion ratios and deuterated internal standards define the specificity for this analysis. Also, the specificity can be calculated from the relationship: True Negatives/ (True Negatives + False Positives). This relationship provides a specificity of less than 100% only when a false positive is encountered with the negative control. If the negative control is always found to be negative, the specificity for the analysis is 100%. Relative Recovery: Absolute recovery is not a requisite due to the use of the isotopic dilution with deuterated internal standards. L.O.D. (Limit of Detection) and L.O.Q. (Limit of Quantitation): The limit of detection and quantitation were established by analyzing the low calibrator (1.0 ng/ml). Samples were run six times on Applied Biosystems API 4000 LC MS/MS. The software used was Analyst Software. o LOD = mean + 3 (Std. Dev.) Delta 9 THC = 1.28 Delta 9 THC COOH = 1.28 o LOQ= mean + 10 (Std. Dev.) Delta 9 THC = 1.84 Delta 9 THC COOH = 1.84
11 o Limit of quantification 1.0 ng/ml for both Delta 9 THC and Delta 9 THC COOH o Limit of detection 1.0 ng/ml for both Delta 9 THC and Delta 9 THC COOH Interfering Substances: A sample containing various benzodiazepines, amphetamines and GC Scan Mixture was ran and no known interferences were noted. Validation of the 1 ng/ml Cut Off Concentrations: The procedure was performed in 10 replicates of a spiked standard of 1 ng/ml. The concentration was calculated using the calibration curve. The standard deviation was calculated to be 0.08 ng/ml for Delta 9 THC and Delta 9 THC COOH. Carryover: This was completed by analyzing the high control (100 ng/ml) twice followed by the negative control twice. The prepared samples were run six consecutive times. Acceptable performance yielded no carryover that is significant enough to cause change in quantitation for a sample. Chemicals prepared fresh on the day of use are determined to be acceptable on the basis of an acceptable performance of the positive and negative controls. THC is extensively protein bound and poorly distributed to red cells, the concentration of THC in whole blood is approximately half that found in serum or plasma. However, blood containing sodium fluoride can be used and is preferred when serum or plasma are not available. 10.0 Laboratory Safety Precautions All biological specimens must be considered pathogenic. All biological specimens must be handled wearing gloves. Refer to the lab safety manual for additional procedures. 11.0 Waste Management 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 be4nch 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. Dispose if all consumable contaminated by blood in appropriate biohazard waste containers for autoclaving. Contact the Chemical Hygiene Office for disposal recommendation of other chemicals or solutions.
12 12.0 Pollution Prevention 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 of Environmental Protection Agency (USEPA) 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. 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. 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, 1155 16 th Street NW, Washington D.C. 20036, (202) 872 4477. 13.0 References 1. Sunshine, Irving. Monograph: "Marijuana (THC). Marion Laboratories, Inc., Kansas City, MO, 1985. 2. E.G. Zimmerman, E.P. Yeager, J.R. Soares, L.E. Hollister, V.C. Reeve, Measurement of Delta- 9-Tetrahydrocannabinol in Whole Blood Samples from Impaired Motorists, Journal of Forensic Sciences, Vol. 28, 1983. 3. M.A. Huestis, J.E. Henningfield, and E.J. Cone, Blood Cannabinoids. Adsorption of THC and Formation of 11-OH-THC and THCCOOH During and After Smoking Marijuana. Journal of Analytical Toxicology, Vol 16, 1992. 4. Baselt, Randall G. Disposition of Toxic Drugs and Chemicals in Man. Biomedical Publications, CA 1982. 5. NIDA Research Monograph #42. "The Analysis of Cannabinoids in Biological Fluids." U.S. Department of Health and Human Services, 1982. 6. Felgate, P.D.,and Dinan, A.C., The determination of delta-9-thc and THCCOOH in whole blood using solvent extraction combined with polar solid-phase extraction. J. Anal. Tox., Vol 24, (2000), 127-132. 7. Communication with Tennessee Bureau of Investigation, Forensic Services Division, Aug. 2006. 8. Less is Better: Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society s (ACS) Department of Governmental Regulations and Science Policy, 1155 16 th Street NW, Washington D.C. 20036, (202) 872-4477. 9. Reagent Chemicals American Chemical Society Committee on Analytical Reagents, Washington, DC.
13 10. Waste Management Manual for Laboratory Personnel. American Chemical Society Department of Government Regulations and Science Policy, Washington, DC. 11. C.R. Goodall, and B.J. Basteyns, A Reliable Method for the Detection, Confirmation and Quantitation of Cannabinoids in Blood, JAT, 19: 419-426 (1995)
14 14.0 Authorization Written by: Kimberly Stephens Date: 10-12-2011 Procedure Author Approved by: Section Supervisor Date: Program Manager Date: QA Officer Date: LSD Director Date: Effective Date: Deleted From Service Date: