Tetrahydrocannabinol and Two of its Metabolites in Whole Blood Using Liquid Chromatography-Tandem Mass Spectrometry

Journal of Analytical Toxicology, Vol. 32, October 2008 Tetrahydrocannabinol and Two of its Metabolites in Whole Blood Using Liquid Chromatography– Tandem Mass Spectrometry Cynthia Coulter, Elizabeth Miller, Katherine Crompton, and Christine Moore* Immunalysis Corporation, 829 Towne Center Drive, Pomona, California 91767 Abstract An analytical procedure for the determination of ∆9∆9tetrahydrocannabinol (THC), 11-nor-9-carboxy-∆ ∆9tetrahydrocannabinol (THCA), and 11-hydroxy-∆ tetrahydrocannabinol (11-OH-THC) in whole blood has been developed and validated using liquid chromatography with tandem mass spectral detection (MS). Cannabinoids present in the blood samples were quantified using solid-phase extraction followed by MS detection in positive electrospray ionization mode. For confirmation, two transitions were monitored and one ratio determined. Samples being reported as positive were required to have both transitions present, the ratio of quantifying transition to qualifying transition being within 20% of that determined from known calibration standards. The monitoring of the qualifying transition and requirement for its presence within a specific ratio to the primary ion has the potential of limiting the sensitivity of the assay, however, the additional confidence in the final result as well as forensic defensibility were considered to be of greater importance. The limit of quantitation was 0.5 ng/mL for THC, 5 ng/mL for THCA, and 2 ng/mL for 11-OH-THC. The limit of detection was 0.5 ng/mL for THC, 4 ng/mL for THCA, and 1 ng/mL for 11-OH-THC. The percentage recovery of the cannabinoids from whole blood at a concentration of 5 ng/mL was 71.5% for THC, 64.5% for 11-OH-THC, and 61.2% for THCA (n = 3). Introduction Marijuana is the most commonly used illicit drug in the U.S. and is frequently encountered in cases of driving under the influence of drugs. In a recent manuscript recommending cutoff concentrations for the analysis of drugs in blood and urine for driving cases, the majority of forensic laboratories reported cannabis as the most frequently encountered drug (1). There are various published procedures for the determination of THC and its metabolites in whole blood, which generally incorporate gas chromatography–mass spectrometry instrumentation (GC– MS) (2–4). However, the use of liquid chromatography with tandem mass spectrometry (LC–MS–MS) provides adequate sensitivity for the determination of these compounds in whole * Author to whom correspondence should be addressed. E-mail: . blood, without the need for derivatization, and usually a much shorter run-time. Many reported procedures monitor only one transition in the multiple-reaction monitoring (MRM) mode, which is inadequate for forensic defensibility of the result. Recently, several authors have focused on the need to monitor a second transition, allowing the ratio between the abundance of the primary and secondary transitions to be calculated, and establishing more confidence in the final result (5). Maralikova and Weinmann (6) noted that guidelines for confirmatory analysis using LC–MS–MS have not yet been established, and they suggested that the monitoring of at least two transitions is required to provide sufficient identification of drugs. They applied their recommendation to the detection of ∆9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THCA), and 11-hydroxy-∆9-tetrahydrocannabinol (11-OHTHC) in plasma as well as THCA-glucuronide using LC–MS– MS and monitoring two transitions. In this procedure, two transitions are monitored for the cannabinoids providing excellent sensitivity for their determination in whole blood. The extraction efficiency of the cannabinoids from blood, the degree of ion suppression caused by the blood matrix, and the stability of the extracted specimens over a 72-h period were also investigated. The method is useful in routine testing for the determination of cannabinoids in whole blood, providing forensic defensibility by the inclusion of two monitored transitions. Materials Standards and reagents Tri-deuterated internal standards of THC, THCA, and 11OH-THC as well as unlabelled drug standards were obtained from Cerilliant (Round Rock, TX). Solid-phase extraction columns (Cerex® Polychrom™ THC 682-0353) were purchased from SPEWare (Baldwin Park, CA). All solvents were HPLC grade or better and obtained from Spectrum Chemicals (Gardena, CA). All chemicals were ACS grade. Quality control (QC) Downloaded from https://academic.oup.com/jat/article-abstract/32/8/653/829920 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission. by guest on 27 June 2018 653 Journal of Analytical Toxicology, Vol. 32, October 2008 samples for whole blood at concentrations of 7.5 ng/mL (low QC) and 50 ng/mL (high QC) were purchased from UTAK Laboratories (Valencia, CA). Calibrators For the chromatographic calibration standards, a working solution for the deuterated internal standard was prepared in methanol at a concentration of 1000 ng/mL. Unlabelled drug standard was prepared in methanol at the same concentration. All the working solutions were stored at –20°C when not in use. For each batch, seven calibration standards were prepared in authentic human blood (1 mL), which had been previously analyzed and found to be negative for cannabinoids. Drug concentrations of 0.5, 1, 2.5, 5, 10, 20, and 100 ng/mL of whole blood were prepared (internal standard concentration: 10 ng/mL) for the calibration. For limit of quantitation and limit of detection experiments, standards at 0.125, 0.25, 2, 3, and 4 ng/mL were also included. Methods Sample preparation for chromatographic analysis column was a narrow-bore Zorbax Extend C18 threaded system (2.1 × 50 mm × 1.8 µm), designed for high-pressure operation. The double endcapping of the stationary phase protected the silica support from dissolution at high pH extending the allowable pH range of operation from 2 to 11.5. The column temperature was held at 40°C, and the injection volume was 5 µL. The mobile phase consisted of 20 mM ammonium formate (pH 8.6, Solvent A) and methanol (Solvent B). Initially, the assay was operated with the pH of the buffer at 6.4; however, the response of the THCA was significantly reduced. At the beginning of the run, the mobile phase composition was 30% A/70% B at a constant flow rate of 0.3 mL/min. After 5 min, the percentage of solvent B was 90%, remaining there for 3 min; finally at 10 min, the percentage of B returned to 70%. The equilibration time was 4 min. The gas temperature was 350°C, the gas flow was 8 L/min, and the nebulizer pressure was 35 psi. Nitrogen was used as the collision gas, and the capillary voltage was 3500 V. Two transitions were selected and optimized for each drug using flow injection analysis. The transitions and fragment voltages are shown in Table I. Each subsequent analysis required the ratio between the quantita (...truncated)