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Simultaneous Quantification of 13 Cannabinoids and Metabolites in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry in Adult Epilepsy Patients

Roslawski, Michaela J., BA*; Remmel, Rory P., PhD; Karanam, Ashwin, PharmD*,†; Leppik, Ilo E., MD*,†,‡; Marino, Susan E., PhD*,†; Birnbaum, Angela K., PhD*,†

doi: 10.1097/FTD.0000000000000583
Original Article
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Background: A sensitive, robust method was developed and validated to quantitate 13 major natural cannabinoid parent and metabolite compounds in human plasma at or below 0.5 ng/mL.

Methods: A liquid chromatography tandem mass spectrometry method was developed and validated to measure 13 cannabinoid compounds: cannabidiol (CBD), cannabidiolic acid, cannabidivarin, cannabinol, cannabigerol, cannabigerolic acid, cannabichromene, Δ9-tetrahydocannabinol (THC), Δ9-tetrahydrocannabinolic acid A (THCA), Δ9-tetrahydrocannabivarin (THCV), 11-hydroxy-Δ9-tetrahydrocannbinol (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannbinol (THC-COOH), and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol glucuronide (THC-COOH-glu). Samples (200 µL) were extracted through protein precipitation and separated with a Kinetex EVO C18 column and a 65%–95% gradient of methanol and 0.2% ammonium hydroxide/H2O at a flow rate of 0.4 mL/min. Samples were obtained from patients with epilepsy receiving cannabis for the treatment of seizures.

Results: The extracted lower limit of quantification was 0.05 ng/mL for CBD, cannabidivarin, cannabinol, and 11-OH-THC; 0.10 ng/mL for cannabidiolic acid, cannabigerol, cannabichromene, cannabigerolic acid, THC, THCA, and THCV; and 0.50 ng/mL for THC-COOH and THC-COOH-glu. Mean quality control intraday accuracy and precision for all analytes ranged 96.5%–104% and 2.7%–4.9%, respectively, whereas interday accuracy and precision ranged 98%–103.3% and 0.2%–3.6%, respectively. An absolute matrix effect was observed for some analytes, however, with minimal relative matrix effect. Lack of nonspecific drug binding to extraction glass and plasticware was verified. Patient CBD levels ranged from 0.135 to 11.13 ng/mL.

Conclusions: The validated method met FDA guidelines for bioanalytical assays precision and accuracy criteria. The assay reliably confirmed the use of particular medical cannabis formulations in patient samples as well as reliably measured low CBD concentrations from single-dose CBD exposure.

*Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota;

Center for Clinical and Cognitive Neuropharmacology, College of Pharmacy, University of Minnesota; and

MINCEP Epilepsy Care, University of Minnesota Physicians, Minneapolis, Minnesota.

Correspondence: Angela K. Birnbaum, PhD, Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Room 463, 717, Delaware St, SE, Minneapolis, MN 55414 (e-mail: birnb002@umn.edu).

Supported by the Patricia L Nangle Fund through the Epilepsy Foundation, and MacMillan Innovative Epilepsy Research and Education Fund.

A. K. Birnbaum and M. J. Roslawski contributed to the conception and design of the study; M. J. Roslawski, R. P. Remmel, A. Karanam, and A. K. Birnbaum contributed to the conduct of the study and analysis of data; Data for proof of applicability were designed, acquired, and analyzed by A. K. Birnbaum, S. E. Marino, I. E. Leppik, M. J. Roslawski, and A. Karanam. M. J. Roslawski, A. K. Birnbaum, A. Karanam, S. E. Marino, R. P. Remmel, and I. E. Leppik contributed to drafting the text and preparing the figures.

A. K. Birnbaum reports grants from the Epilepsy Foundation, National Institutes of Health, Superunus Pharmaceuticals, and Veloxis Pharmaceutics during the conduct of the study. She also has a royalty agreement for intravenous carbamazepine with Lundbeck. S. E. Marino reports grants from the Epilepsy Foundation, National Institutes of Health, Superunus Pharmaceuticals, and Veloxis Pharmaceutics during the conduct of the study. She and R. P. Remmel have a royalty agreement for intravenous topiramate Ligand Pharmaceuticals. The remaining authors declare no conflict of interest.

Received June 09, 2018

Accepted October 11, 2018

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