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Analysis of Cannabinoids and Their Metabolites in Human Urine.

ABSTRACT: Biologically monitoring marijuana exposure from active and passive use requires both a wide linear range and sensitive detection. We have developed and validated a multifunctional method using ultrahigh performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) for analysis of urinary ?9-tetrahydrocannabinol (THC), cannabidiol and cannabinol, and two major metabolites of THC, 11-nor-9-carboxy-THC and 11-hydroxy-THC, in active users and particularly in people exposed to secondhand marijuana smoke (SHMS). The method used positive electrospray ionization (ESI) mode to reach the sensitivity needed to detect trace SHMS exposure with limits of detection (LOD) ranging from 0.002 to 0.008 nanograms per milliliter (ng/mL) and 0.005 to 0.017 ng/mL for "free" (unconjugated forms) and "total" (unconjugated plus conjugated forms) measurements, respectively. These LODs were approximately 10-100 times more sensitive than those reported in the literature. To reduce or avoid time-consuming repetitive sample preparation and analysis, the method simultaneously monitored multiple reaction monitoring transitions in negative ESI mode to quantify high analyte levels typically found in the urine of active marijuana users (linear dynamic range of 12.5-800 ng/mL). The validation results indicated this method was accurate (average inter/intra-day bias, <10%), precise (inter/intra-day imprecision, <10%), and fast (6 min run time). In addition, sample preparation throughput was greatly improved using an automation liquid-handling system, meeting the needs for potential large-scale population studies.

SUBMITTER: Wei B

PROVIDER: S-EPMC5022557 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Analysis of Cannabinoids and Their Metabolites in Human Urine.

Wei Binnian B Wei Binnian B Wang Lanqing L Blount Benjamin C BC

Analytical chemistry 20151005 20

Biologically monitoring marijuana exposure from active and passive use requires both a wide linear range and sensitive detection. We have developed and validated a multifunctional method using ultrahigh performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) for analysis of urinary Δ9-tetrahydrocannabinol (THC), cannabidiol and cannabinol, and two major metabolites of THC, 11-nor-9-carboxy-THC and 11-hydroxy-THC, in active users and particularly in people exposed to ...[more]

PMID: 26411292

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Project description:BACKGROUND:Although, especially in the United States, there has been a recent surge of legalized cannabis for either recreational or medicinal purposes, surprisingly little is known about clinical dose-response relationships, pharmacodynamic and toxicodynamic effects of cannabinoids such as ?9-tetrahydrocannabinol (THC). Even less is known about other active cannabinoids. METHODS:To address this knowledge gap, an online extraction, high-performance liquid chromatography coupled with tandem mass spectrometry method for simultaneous quantification of 11 cannabinoids and metabolites including THC, 11-hydroxy-?9-tetrahydrocannabinol, 11-nor-?9-tetrahydrocannabinol-9-carboxylic acid, 11-nor-?9-tetrahydrocannabinol-9-carboxylic acid glucuronide (THC-C-gluc), cannabinol, cannabidiol, cannabigerol, cannabidivarin, ?9-tetrahydrocannabivarin (THCV), and 11-nor-9-carboxy-?9-tetrahydrocannabivarin (THCV-COOH) was developed and validated in human urine and plasma. RESULTS:In contrast to atmospheric pressure chemical ionization, electrospray ionization was associated with extensive ion suppression in plasma and urine samples. Thus, the atmospheric pressure chemical ionization assay was validated showing a lower limit of quantification ranging from 0.39 to 3.91 ng/mL depending on study compound and matrix. The upper limit of quantification was 400 ng/mL except for THC-C-gluc with an upper limit of quantification of 2000 ng/mL. The linearity was r > 0.99 for all analyzed calibration curves. Acceptance criteria for intrabatch and interbatch accuracy (85%-115%) and imprecision (<15%) were met for all compounds. In plasma, the only exceptions were THCV (75.3%-121.2% interbatch accuracy) and cannabidivarin (interbatch imprecision, 15.7%-17.2%). In urine, THCV did not meet predefined acceptance criteria for intrabatch accuracy. CONCLUSIONS:This assay allows for monitoring not only THC and its major metabolites but also major cannabinoids that are of interest for marijuana research and clinical practice.

Project description:Clandestine laboratories constantly produce new synthetic cannabinoids to circumvent legislative efforts, complicating toxicological analysis. No extensive synthetic cannabinoid quantitative urinary methods are reported in the literature. We developed and validated a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for simultaneously quantifying JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, JWH-398, RCS-4, AM-2201, MAM-2201, UR-144, CP 47,497-C7, CP 47,497-C8 and their metabolites, and JWH-203, AM-694, RCS-8, XLR-11 and HU-210 parent compounds in urine. Non-chromatographically resolved alkyl hydroxy metabolite isomers were considered semi-quantitative. ?-Glucuronidase hydrolyzed urine was extracted with 1ml Biotage SLE+ columns. Specimens were reconstituted in 150?L mobile phase consisting of 50% A (0.01% formic acid in water) and 50% B (0.01% formic acid in 50:50 methanol:acetonitrile). 4 and 25?L injections were performed to acquire data in positive and negative ionization modes, respectively. The LC-MS/MS instrument consisted of a Shimadzu UFLCxr system and an ABSciex 5500 Qtrap mass spectrometer with an electrospray source. Gradient chromatographic separation was achieved utilizing a Restek Ultra Biphenyl column with a 0.5ml/min flow rate and an overall run time of 19.5 and 11.4min for positive and negative mode methods, respectively. Quantification was by multiple reaction monitoring with CP 47,497 compounds and HU-210 ionized via negative polarity; all other analytes were acquired in positive mode. Lower and upper limits of linearity were 0.1-1.0 and 50-100?g/l (r(2)>0.994). Validation parameters were evaluated at three concentrations spanning linear dynamic ranges. Inter-day analytical recovery (bias) and imprecision (N=20) were 88.3-112.2% and 4.3-13.5% coefficient of variation, respectively. Extraction efficiencies and matrix effect (N=10) were 44-110 and -73 to 52%, respectively. We present a novel LC-MS/MS method for simultaneously quantifying 20 synthetic cannabinoids and 21 metabolites, and semi-quantifying 12 alkyl hydroxy metabolites in urine.

Dataset Information

Analysis of Cannabinoids and Their Metabolites in Human Urine.

Publications

Analysis of Cannabinoids and Their Metabolites in Human Urine.

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