An Atmospheric Pressure Chemical Ionization MS/MS Assay Using Online Extraction for the Analysis of 11 Cannabinoids and Metabolites in Human Plasma and Urine

Abstract

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, pharmaco- and toxicodynamic effects of cannabinoids such as Δ9-tetrahydrocannabinol (THC). Even less is known about other active cannabinoids. Methods To address this knowledge gap, an online extraction, high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of 11 cannabinoids and metabolites including THC, 11-hydroxy-Δ9-tetrahydrocannabinol (11OH-THC), 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide (THC-C-gluc), cannabinol (CBN), cannabidiol (CBD), cannabigerol (CBG), cannabidivarin (CBDV), Δ9-tetrahydrocannabivarin (THCV), and 11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (THCV-COOH) was developed and validated in human urine and plasma. Results In contrast to atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI) was associated with extensive ion suppression in plasma and urine samples. Thus, the APCI assay was validated showing a lower limit of quantification (LLOQ) ranging from 0.39 to 3.91 ng/mL depending on study compound and matrix. The upper limit of quantitation (ULOQ) was 400 ng/mL except for THC-C-gluc with a ULOQ of 2000 ng/mL. The linearity was r> 0.99 for all analyzed calibration curves. Acceptance criteria for intra- and inter-batch accuracy (85%-115%) and imprecision (<15%) were met for all compounds. In plasma, the only exceptions were THCV (75.3%-121.2% inter-batch accuracy) and CBDV (inter-batch imprecision, 15.7%-17.2%). In urine, THCV did not meet predefined acceptance criteria for intra-batch accuracy. Conclusions This assay allows not only for monitoring THC and its major metabolites, but also of major cannabinoids that are of interest for marijuana research and clinical practice.

A review on the recent advances in HPLC, UHPLC and UPLC analyses of naturally occurring cannabinoids (2010–2019)

“…Marijuana or hashish, a cocktail of at least 30 different major cannabinoids, generally prepared from crushing the leaves, flowers (inflorescenses) and even stems of C. sativa , is one of the oldest recreational and addictive natural products used by humans for centuries . However, the non‐medical use of Cannabis or marijuana is illegal in many countries, which prompts the use of analytical tools, like HPLC and UPLC, to analyse biological and forensic samples like blood, oral liquid, hair and urine, to confirm marijuana usage (Tables ) . After consumption of marijuana, THCA ( 13 ), which is present in the crude marijuana, but converted to THC ( 12 ) by heat during smoking, is excreted in the urine as its glucuronide conjugate, and can be analysed comfortably by LC methods.…”

“…In fact, blood, plasma and serum samples are quite extensively used in forensic analysis to detect the consumption of cannabinoids . HPLC and UPLC methods are commonly used for the analysis of cannabinoids in blood samples …”

“…Blood plasma samples are quite often used in forensic analysis for the detection of illegal cannabinoids consumption, and the use of HPLC and UPLC methods, both applying simple UV‐PDA or ESI‐MS/MS detection technologies is common . An HPLC method utilising APCI (atmospheric pressure chemical ionisation)‐MS/MS in positive ion mode associated with extensive ion suppression was employed to analyse naturally occurring cannabinoids in 352 human plasma samples, providing simultaneous quantification of several cannabinoids and their biotransformed products including CBC ( 1 ), CBD ( 3 ), CBDV ( 5 ), CBN ( 8 ), CBG ( 6 ), THC ( 12 ), 11‐OH‐THC ( 9 ), THCV ( 14 ), 11‐nor‐9‐carboxy‐Δ 9 ‐tetrahydrocannbinol (THC‐COOH, 10 ), Δ 9 ‐tetrahydrocannabivarin‐carboxylic acid (THCV‐COOH, 15 ), and THC‐COOH‐glucuronide . A Poroshell Eclipse C 18 column (40 mm × 4.6 mm; particle size: 2.7 μm) was used in this analysis and a gradient elution with the mobile phase was composed of 20% isopropanol, 20% MeOH and 60% ACN, and water containing 0.1% HCOOH was employed.…”

“…Human urine samples are popular in forensic toxicological analysis for the determination of various illegal drugs, including cannabinoids, and their metabolites . Table summarises the applications of HPLC and UPLC‐based analytical methods for naturally occurring cannabinoids and their biotransformation products in human urine samples , . Detection techniques used in these studies included simple UV, PDA and MS/MS methods.…”

“…An APCI‐MS/MS in positive ion mode was applied with an HPLC for the detection of several cannabinoids including CBD 1 , 3 , 5 , 6 , 8 – 10 , 12 , 14 , 15 and THC‐COOH‐glucuronide in human urine samples using rather a short column (40 mm) . A similar method, but using a 150 mm column, was also reported for the quantification of similar cannabinoids and their biotransformation products (Table ).…”