Validation of a two-dimensional gas chromatography mass spectrometry method for the simultaneous quantification of cannabidiol, Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC, and 11-nor-9-carboxy-THC in plasma

A sensitive analytical method for simultaneous quantification of sub-nanogram concentrations of cannabidiol (CBD), Δ⁹-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) in plasma is presented for monitoring cannabinoid pharmacotherapy and illicit cannabis use. Analytes were extracted from 1 mL plasma by solid-phase extraction, derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane, and analyzed by two-dimensional gas chromatography mass spectrometry (2D-GCMS) with cryofocusing. The lower calibration curve was linear from 0.25–25 ng/mL for CBD and THC, 0.125-25 ng/mL for 11-OH-THC and 0.25-50 ng/mL for THCCOOH. A second higher linear range from 5–100 ng/mL, achieved through modification of injection parameters, was validated for THC, 11-OH-THC, and THCCOOH and was only implemented if concentrations exceeded the lower curve upper limit of linearity. This procedure prevented laborious re-extraction by allowing the same specimen to be re-injected for quantification on the high calibration curve. Intra- and inter-assay imprecision, determined at four quality control concentrations, were ≤7.8% CV. Analytical bias was within ±9.2% of target and extraction efficiencies were ≥72.9% for all analytes. Analytes were stable when stored at 22°C for 16 h, 4°C for 48 h, after three freeze–thaw cycles at −20°C and when stored on the autosampler for 48 h. This sensitive and specific 2D-GCMS assay provides a new means of simultaneously quantifying CBD, THC and metabolite biomarkers in clinical medicine, forensic toxicology, workplace drug testing, and driving under the influence of drugs programs.     

Cannabinoids and metabolites in expectorated oral fluid after 8 days of controlled around-the-clock oral THC administration

Oral fluid (OF) is an increasingly accepted matrix for drug testing programs, but questions remain about its usefulness for monitoring cannabinoids. Expectorated OF specimens (n = 360) were obtained from 10 adult daily cannabis smokers before, during, and after 37 20-mg oral Δ⁹-tetrahydrocannabinol (THC) doses over 9 days to characterize cannabinoid disposition in this matrix. Specimens were extracted and analyzed by gas chromatography–mass spectrometry with electron-impact ionization for THC, 11-hydroxy-THC, cannabidiol, and cannabinol, and negative chemical ionization for 11-nor-9-carboxy-THC (THCCOOH). Linear ranges for THC, 11-hydroxy-THC, and cannabidiol were 0.25–50 ng/mL; cannabinol 1–50 ng/mL; and THCCOOH 5–500 pg/mL. THCCOOH was the most prevalent analyte in 344 specimens (96.9%), with concentrations up to 1,390.3 pg/mL. 11-hydroxy-THC, cannabidiol, and cannabinol were detected in 1, 1, and 3 specimens, respectively. THC was detected in only 13.8% of specimens. The highest THC concentrations were obtained at admission (median 1.4 ng/mL, range 0.3–113.6) from previously self-administered smoked cannabis. A total of 2.5 and 3.7% of specimens were THC-positive at the recommended Substance Abuse and Mental Health Services Administration (2 ng/mL) and Driving Under the Influence of Drugs, Alcohol and Medicines (DRUID) (1 ng/mL) confirmation cutoffs, respectively. THC is currently the only analyte for monitoring cannabis exposure in OF; however, these data indicate chronic therapeutic oral THC administration and illicit oral THC use are unlikely to be identified with current guidelines. Measurement of THCCOOH may improve the detection and interpretation of OF cannabinoid tests and minimize the possibility of OF contamination from passive inhalation of cannabis smoke.     

Oral fluid cannabinoid concentrations following controlled smoked cannabis in chronic frequent and occasional smokers

Oral fluid (OF) is an alternative biological matrix for monitoring cannabis intake in drug testing, and drugged driving (DUID) programs, but OF cannabinoid test interpretation is challenging. Controlled cannabinoid administration studies provide a scientific database for interpreting cannabinoid OF tests. We compared differences in OF cannabinoid concentrations from 19 h before to 30 h after smoking a 6.8 % THC cigarette in chronic frequent and occasional cannabis smokers. OF was collected with the Statsure Saliva Sampler? OF device. 2D-GC-MS was used to quantify cannabinoids in 357 OF specimens; 65 had inadequate OF volume within 3 h after smoking. All OF specimens were THC-positive for up to 13.5 h after smoking, without significant differences between frequent and occasional smokers over 30 h. Cannabidiol (CBD) and cannabinol (CBN) had short median last detection times (2.5–4 h for CBD and 6–8 h for CBN) in both groups. THCCOOH was detected in 25 and 212 occasional and frequent smokers’ OF samples, respectively. THCCOOH provided longer detection windows than THC in all frequent smokers. As THCCOOH is not present in cannabis smoke, its presence in OF minimizes the potential for false positive results from passive environmental smoke exposure, and can identify oral THC ingestion, while OF THC cannot. THC?≥?1 μg/L, in addition to CBD?≥?1 μg/L or CBN?≥?1 μg/L suggested recent cannabis intake (≤13.5 h), important for DUID cases, whereas THC?≥?1 μg/L or THC?≥?2 μg/L cutoffs had longer detection windows (≥30 h), important for workplace testing. THCCOOH windows of detection for chronic, frequent cannabis smokers extended beyond 30 h, while they were shorter (0–24 h) for occasional cannabis smokers.     

Direct quantification of cannabinoids and cannabinoid glucuronides in whole blood by liquid chromatography–tandem mass spectrometry

The first method for quantifying cannabinoids and cannabinoid glucuronides in whole blood by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated. Solid-phase extraction followed protein precipitation with acetonitrile. High-performance liquid chromatography separation was achieved in 16 min via gradient elution. Electrospray ionization was utilized for cannabinoid detection; both positive (Δ⁹-tetrahydrocannabinol [THC] and cannabinol [CBN]) and negative (11-hydroxy-THC [11-OH-THC], 11-nor-9-carboxy-THC [THCCOOH], cannabidiol [CBD], THC-glucuronide, and THCCOOH-glucuronide) polarity were employed with multiple reaction monitoring. Calibration by linear regression analysis utilized deuterium-labeled internal standards and a 1/x ² weighting factor, yielding R ² values >0.997 for all analytes. Linearity ranged from 0.5 to 50 μg/L (THC-glucuronide), 1.0–100 μg/L (THC, 11-OH-THC, THCCOOH, CBD, and CBN), and 5.0–250 μg/L (THCCOOH-glucuronide). Imprecision was <10.5% CV, recovery was >50.5%, and bias within ±13.1% of target for all analytes at three concentrations across the linear range. No carryover and endogenous or exogenous interferences were observed. This new analytical method should be useful for quantifying cannabinoids in whole blood and further investigating cannabinoid glucuronides as markers of recent cannabis intake.     

11-Nor-9-carboxy-∆9-tetrahydrocannabinol quantification in human oral fluid by liquid chromatography–tandem mass spectrometry

Currently, ?9-tetrahydrocannabinol (THC) is the analyte quantified for oral fluid cannabinoid monitoring. The potential for false-positive oral fluid cannabinoid results from passive exposure to THC-laden cannabis smoke raises concerns for this promising new monitoring technology. Oral fluid 11-nor-9-carboxy-?9-tetrahydrocannabinol (THCCOOH) is proposed as a marker of cannabis intake since it is not present in cannabis smoke and was not measureable in oral fluid collected from subjects passively exposed to cannabis. THCCOOH concentrations are in the picogram per milliliter range in oral fluid and pose considerable analytical challenges. A liquid chromatography–tandem mass spectrometry (LCMSMS) method was developed and validated for quantifying THCCOOH in 1 mL Quantisal-collected oral fluid. After solid phase extraction, chromatography was performed on a Kinetex C18 column with a gradient of 0.01 % acetic acid in water and 0.01 % acetic acid in methanol with a 0.5-mL/min flow rate. THCCOOH was monitored in negative mode electrospray ionization and multiple reaction monitoring mass spectrometry. The THCCOOH linear range was 12–1,020 pg/mL (R ²?>?0.995). Mean extraction efficiencies and matrix effects evaluated at low and high quality control (QC) concentrations were 40.8–65.1 and ?2.4–11.5 %, respectively (n?=?10). Analytical recoveries (bias) and total imprecision at low, mid, and high QCs were 85.0–113.3 and 6.6–8.4 % coefficient of variation, respectively (n?=?20). This is the first oral fluid THCCOOH LCMSMS triple quadrupole method not requiring derivatization to achieve a <15 pg/mL limit of quantification. The assay is applicable for the workplace, driving under the influence of drugs, drug treatment, and pain management testing.

Simultaneous quantification of Delta9-tetrahydrocannabinol, 11-hydroxy-Delta9-tetrahydrocannabinol, and 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid in human plasma using two-dimensional gas chromatography, cryofocusing, and electron impact-mass spectrometry

A two-dimensional (2D) gas chromatography/electron impact-mass spectrometry (GC/EI-MS) method for simultaneous quantification of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC), and 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in human plasma was developed and validated. The method employs 2D capillary GC and cryofocusing for enhanced resolution and sensitivity. THC, 11-OH-THC, and THCCOOH were extracted by precipitation with acetonitrile followed by solid-phase extraction. GC separation of trimethylsilyl derivatives of analytes was accomplished with two capillary columns in series coupled via a pneumatic Deans switch system. Detection and quantification were accomplished with a bench-top single quadrupole mass spectrometer operated in electron impact-selected ion monitoring mode. Limits of quantification (LOQ) were 0.125, 0.25 and 0.125 ng/mL for THC, 11-OH-THC, and THCCOOH, respectively. Accuracy ranged from 86.0 to 113.0% for all analytes. Intra- and inter-assay precision, as percent relative standard deviation, was less than 14.1% for THC, 11-OH-THC, and THCCOOH. The method was successfully applied to quantification of THC and its 11-OH-THC and THCCOOH metabolites in plasma specimens following controlled administration of THC.     

Comparison of cannabinoid concentrations in oral fluid and whole blood between occasional and regular cannabis smokers prior to and after smoking a cannabis joint

A cross-over controlled administration study of smoked cannabis was carried out on occasional and heavy smokers. The participants smoked a joint (11 % Δ9-tetrahydrocannabinol (THC)) or a matching placebo on two different occasions. Whole blood (WB) and oral fluid (OF) samples were collected before and up to 3.5 h after smoking the joints. Pharmacokinetic analyses were obtained from these data. Questionnaires assessing the subjective effects were administered to the subjects during each session before and after the smoking time period. THC, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) were analyzed in the blood by gas chromatography or liquid chromatography (LC)-tandem mass spectrometry (MS/MS). The determination of THC, THCCOOH, cannabinol (CBN), and Δ9-tetrahydrocannabinolic acid A (THC-A) was carried out on OF only using LC-MS/MS. In line with the widely accepted assumption that cannabis smoking results in a strong contamination of the oral cavity, we found that THC, and also THC-A, shows a sharp, high concentration peak just after smoking, with a rapid decrease in these levels within 3 h. No obvious differences were found between both groups concerning THC median maximum concentrations measured either in blood or in OF; these levels were equal to 1,338 and 1,041 μg/L in OF and to 82 and 94 μg/L in WB for occasional and heavy smokers, respectively. The initial WB THCCOOH concentration was much higher in regular smokers than in occasional users. Compared with the occasional smokers, the sensation of confusion felt by the regular smokers was much less while the feeling of intoxication remained almost unchanged.

In Situ Decarboxylation-Pressurized Hot Water Extraction for Selective Extraction of Cannabinoids from Cannabis sativa. Chemometric Approach

Isolation of the therapeutic cannabinoid compounds from Cannabis Sativa L. (C. Sativa) is important for the development of cannabis-based pharmaceuticals for cancer treatment, among other ailments. The main pharmacological cannabinoids are THC and CBD. However, THC also induces undesirable psychoactive effects. The decarboxylation process converts the naturally occurring acidic forms of cannabinoids, such as cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA), to their more active neutral forms, known as cannabidiol (CBD) and tetrahydrocannabinol (THC). The purpose of this study was to selectively extract cannabinoids using a novel in situ decarboxylation pressurized hot water extraction (PHWE) system. The decarboxylation step was evaluated at different temperature (80–150 °C) and time (5–60 min) settings to obtain the optimal conditions for the decarboxylation-PHWE system using response surface methodology (RSM). The system was optimized to produce cannabis extracts with high CBD content, while suppressing the THC and CBN content. The identification and quantification of cannabinoid compounds were determined using UHPLC-MS/MS with external calibration. As a result, the RSM has shown good predictive capability with a p-value < 0.05, and the chosen parameters revealed to have a significant effect on the CBD, CBN and THC content. The optimal decarboxylation conditions for an extract richer in CBD than THC were set at 149.9 °C and 42 min as decarboxylation temperature and decarboxylation time, respectively. The extraction recoveries ranged between 96.56 and 103.42%, 95.22 and 99.95%, 99.62 and 99.81% for CBD, CBN and THC, respectively.     

Determination of cannabinoids in cannabis products using liquid chromatography-ion trap mass spectrometry

A method was developed and validated for the simultaneous determination of five cannabinoids, viz. cannabidiol (CBD), cannabidiol acid (CBD-COOH), cannabinol (CBN), delta9-tetrahydrocannabinol (THC), and 3'-carboxy-delta9-all-trans-tetrahydrocannabinol (THC-COOH) in cannabis products. The cannabinoids were extracted from the grinded cannabis samples with a mixture of methanol-chloroform and analysed using liquid chromatography with ion-trap-mass-spectrometry (LC-IT-MSn). For quantification the two most abundant diagnostic MS-MS ions of the analyte in the sample and external standard were monitored. For confirmation purposes the EU criteria as described in Commission Decision 2002/657/EC were followed. Fully satisfactory results were obtained, that is, unequivocal confirmation according to the most stringent EU criteria was possible. The limits of quantification were 0.1 g/kg for CBD, 0.04 g/kg for CBD-COOH, 0.03 g/kg for CBN, 0.28 g/kg for THC and 9.9 g/kg for THC-COOH. The repeatabilities, defined by R.S.D., were 2% for CBN, THC and THC-COOH at the concentration levels of respectively 0.023, 3.3 and 113 g/kg and 5% for CBD-COOH at the level of 0.34 g/kg (n = 6).     

Effect of hydrolysis on identifying prenatal cannabis exposure

Identification of prenatal cannabis exposure is important due to potential cognitive and behavioral consequences. A two-dimensional gas chromatography–mass spectrometry method for cannabinol, Δ⁹-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 8β,11-dihydroxy-THC, and 11-nor-9-carboxy-THC (THCCOOH) quantification in human meconium was developed and validated. Alkaline, enzymatic, and enzyme–alkaline tandem hydrolysis conditions were optimized with THC- and THCCOOH-glucuronide reference standards. Limits of quantification ranged from 10 to 15 ng/g, and calibration curves were linear to 500 ng/g. Bias and intra-day and inter-day imprecision were <12.3%. Hydrolysis efficiencies were analyte-dependent; THC-glucuronide was effectively cleaved by enzyme, but not base. Conversely, THCCOOH-glucuronide was most sensitive to alkaline hydrolysis. Enzyme–alkaline tandem hydrolysis maximized efficiency for both glucuronides. Identification of cannabinoid-positive meconium specimens nearly doubled following alkaline and enzyme–alkaline hydrolysis. Although no 11-OH-THC glucuronide standard is available, enzymatic hydrolysis improved 11-OH-THC detection in authentic specimens. Maximal identification of cannabis-exposed neonates and the widest range of cannabis biomarkers are achieved with enzyme–alkaline tandem hydrolysis.     

Simultaneous determination of Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol in oral fluid using isotope dilution liquid chromatography tandem mass spectrometry

The detection and confirmation of cannabinoids in oral fluid are important in forensic toxicology. Currently, the presence of Δ⁹-tetrahydrocannabinol (THC) is used for the detection of cannabis in oral fluid. A low concentration of 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH) is found in oral fluid, which suggested a convenient and low-sensitivity confirmation assay can be used in a routine forensic laboratory. In this study, a highly sensitive isotope dilution liquid chromatography–tandem mass spectrometry method following dansylation was successfully developed for simultaneous determination of THC and THC-COOH in oral fluid. The dansylated derivatives dramatically demonstrated and enhanced the sensitivity of THC and THC-COOH. To avoid signal influenced by the matrix, a 5-min liquid chromatography gradient program was evaluated and optimized, which reduced the sample diffusion and caused sharp peaks (less than 12 s) and thus helped to achieve detection at a low level. The sensitivity, accuracy, and precision were also evaluated, and high quantitative accuracy and precision were obtained. The limit of quantitation of this approach was 25 pg/mL for THC and 10 pg/mL for THC-COOH in oral fluid. Finally, the method was successfully applied to eight suspected cannabis users. Among them, in six oral fluid samples THC-COOH was determined at a concentration from 13.1 to 47.2 pg/mL.     

Development and validation of an LC‐MS/MS method for quantification of Δ9‐tetrahydrocannabinolic acid A (THCA‐A), THC,CBN and CBD in hair

For analysis of hair samples derived from a pilot study (‘in vivo’ contamination of hair by sidestream marijuana smoke), an LC‐MS/MS method was developed and validated for the simultaneous quantification of Δ9‐tetrahydrocannabinolic acid A (THCA‐A), Δ9‐tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD). Hair samples were extracted in methanol for 4 h under occasional shaking at room temperature, after adding THC‐D₃, CBN‐D₃, CBD‐D₃ and THCA‐A‐D₃ as an in‐house synthesized internal standard. The analytes were separated by gradient elution on a Luna C18 column using 0.1% HCOOH and ACN + 0.1% HCOOH. Data acquisition was performed on a QTrap 4000 in electrospray ionization‐multi reaction monitoring mode. Validation was carried out according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Limit of detection and lower limit of quantification were 2.5 pg/mg for THCA‐A and 20 pg/mg for THC, CBN and CBD. A linear calibration model was applicable for all analytes over a range of 2.5 pg/mg or 20 pg/mg to 1000 pg/mg, using a weighting factor 1/x. Selectivity was shown for 12 blank hair samples from different sources. Accuracy and precision data were within the required limits for all analytes (bias between ?0.2% and 6.4%, RSD between 3.7% and 11.5%). The dried hair extracts were stable over a time period of one to five days in the dark at room temperature. Processed sample stability (maximum decrease of analyte peak area below 25%) was considerably enhanced by adding 0.25% lecithin (w/v) in ACN + 0.1% HCOOH for reconstitution. Extraction efficiency for CBD was generally very low using methanol extraction. Hence, for effective extraction of CBD alkaline hydrolysis is recommended. Copyright © 2013 John Wiley & Sons, Ltd.     

Oral fluid/plasma cannabinoid ratios following controlled oral THC and smoked cannabis administration

Oral fluid (OF) is a valuable biological alternative for clinical and forensic drug testing. Evaluating OF to plasma (OF/P) cannabinoid ratios provides important pharmacokinetic data on the disposition of drug and factors influencing partition between matrices. Eleven chronic cannabis smokers resided on a closed research unit for 51 days. There were four 5-day sessions of 0, 30, 60, and 120 mg oral ?⁹-tetrahydrocannabinol (THC)/day followed by a five-puff smoked cannabis challenge on Day 5. Each session was separated by 9 days ad libitum cannabis smoking. OF and plasma specimens were analyzed for THC and metabolites. During ad libitum smoking, OF/P THC ratios were high (median, 6.1; range, 0.2–348.5) within 1 h after last smoking, decreasing to 0.1–20.7 (median, 2.1) by 13.0–17.1 h. OF/P THC ratios also decreased during 5-days oral THC dosing, and after the smoked cannabis challenge, median OF/P THC ratios decreased from 1.4 to 5.5 (0.04–245.6) at 0.25 h to 0.12 to 0.17 (0.04–5.1) at 10.5 h post-smoking. In other studies, longer exposure to more potent cannabis smoke and oromucosal cannabis spray was associated with increased OF/P THC peak ratios. Median OF/P 11-nor-9-carboxy-THC (THCCOOH) ratios were 0.3–2.5 (range, 0.1–14.7) ng/μg, much more consistent in various dosing conditions over time. OF/P THC, but not THCCOOH, ratios were significantly influenced by oral cavity contamination after smoking or oromucosal spray of cannabinoid products, followed by time-dependent decreases. Establishing relationships between OF and plasma cannabinoid concentrations is essential for making inferences of impairment or other clinical outcomes from OF concentrations.     

On-line SPE LC-MS/MS for the quantification of Δ9-tetrahydrocannabinol (THC) and its two major metabolites in human peripheral blood by liquid chromatography tandem mass spectrometry

A universal and robust analytical method for the determination of Δ9-tetrahydrocannabinol (THC) and two of its metabolites Δ9-(11-OH)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-carboxy-tetrahydrocannabinol (THC-COOH) in human whole blood was developed and validated for use in forensic toxicology. Protein precipitation, integrated solid phase extraction and on-line enrichment followed by high-performance liquid chromatography separation and detection with a triple quadrupole mass spectrometer were combined. The linear ranges used for the three cannabinoids were from 0.5 to 20 ng/mL for THC and 11-OH-THC and from 2.5 to 100 ng/mL for THC-COOH, therefore covering the requirements for forensic use. Correlation coefficients of 0.9980 or better were achieved for all three analytes. No relevant hydrolysis was observed for THC-COOH glucuronide with this procedure — in contrast to our previous GC-MS procedure, which obviously lead to an artificial increase of the THC-COOH concentration due to the hydrolysis of the glucuronide-conjugate occurring at high pH during the phase-transfer catalyzed methylation step.     

In vitro stability of free and glucuronidated cannabinoids in urine following controlled smoked cannabis

Analyte stability is an important factor in urine test interpretation, yet cannabinoid stability data are limited. A comprehensive study of Δ⁹-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol, cannabinol, THC-glucuronide, and THCCOOH-glucuronide stabilities in authentic urine was completed. Urine samples after ad libitum cannabis smoking were pooled to prepare low and high pools for each study participant; baseline concentrations were measured within 24 h at room temperature (RT), 4 °C and -20 °C. Stability at RT, 4 °C and ?20 °C was evaluated by Friedman tests for up to 1 year. THCCOOH, THC-glucuronide, and THCCOOH-glucuronide were quantified in baseline pools. RT THCCOOH baseline concentrations were significantly higher than ?20 °C, but not 4 °C baseline concentrations. After 1 week at RT, THCCOOH increased, THCCOOH-glucuronide decreased, but THC-glucuronide was unchanged. In RT low pool, total THCCOOH (THCCOOH?+?THCCOOH-glucuronide) was significantly lower after 1 week. At 4 °C, THCCOOH was stable 2 weeks, THCCOOH-glucuronide 1 month and THC-glucuronide for at least 6 months. THCCOOH was stable frozen for 1 year, but 6 months high pool results were significantly higher than baseline; THC-glucuronide and THCCOOH-glucuronide were stable for 6 months. Total THCCOOH was stable 6 months at 4 °C, and frozen 6 months (low) and 1 year (high). THC, cannabidiol and cannabinol were never detected in urine; although not detected initially, 11-OH-THC was detected in 2 low and 3 high pools after 1 week at RT. Substantial THCCOOH-glucuronide deconjugation was observed at RT and 4 °C. Analysis should be conducted within 3 months if non-hydrolyzed THCCOOH or THCCOOH-glucuronide quantification is required.

Direct Quantitation of Phytocannabinoids by One-Dimensional 1H qNMR and Two-Dimensional 1H-1H COSY qNMR in Complex Natural Mixtures

The widespread use of phytocannabinoids or cannabis extracts as ingredients in numerous types of products, in combination with the legal restrictions on THC content, has created a need for the development of new, rapid, and universal analytical methods for their quantitation that ideally could be applied without separation and standards. Based on previously described qNMR studies, we developed an expanded ¹H qNMR method and a novel 2D-COSY qNMR method for the rapid quantitation of ten major phytocannabinoids in cannabis plant extracts and cannabis-based products. The ¹H qNMR method was successfully developed for the quantitation of cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabielsoin (CBE), and cannabidivarin (CBDV). Moreover, cannabidivarinic acid (CBDVA) and Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA) can be distinguished from CBDA and Δ9-THCA respectively, while cannabigerovarin (CBGV) and Δ8-tetrahydrocannabivarin (Δ8-THCV) present the same ¹H-spectra as CBG and Δ8-THC, respectively. The COSY qNMR method was applied for the quantitation of CBD, CBDA, CBN, CBG/CBGA, and THC/THCA. The two methods were applied for the analysis of hemp plants; cannabis extracts; edible cannabis medium-chain triglycerides (MCT); and hemp seed oils and cosmetic products with cannabinoids. The ¹H-NMR method does not require the use of reference compounds, and it requires only a short time for analysis. However, complex extracts in ¹H-NMR may have a lot of signals, and quantitation with this method is often hampered by peak overlap, with 2D NMR providing a solution to this obstacle. The most important advantage of the COSY NMR quantitation method was the determination of the legality of cannabis plants, extracts, and edible oils based on their THC/THCA content, particularly in the cases of some samples for which the determination of THC/THCA content by ¹H qNMR was not feasible.     

Combining microwave-assisted extraction and liquid chromatography–ion-trap mass spectrometry for the analysis of hexabromocyclododecane diastereoisomers in marine sediments

An efficient microwave-assisted extraction (MAE) procedure coupled with high performance liquid chromatography–electrospray-ion-trap mass spectrometry (HPLC–ESI-ITMS) has been evaluated to determine hexabromocyclododecane diastereoisomers (α-, β- and γ-HBCD) in marine sediments. The composition of the LC mobile phase (consisting of water, methanol and acetonitrile) and the parameters of electrospray ionization (ESI) were evaluated to obtain chromatographic baseline separation and high sensitivity for the detection of these diastereoisomers. The effects of various operating parameters on the quantitative extraction of the HBCDs through MAE were systematically investigated. The three diastereoisomers were then quantitated by HPLC–ITMS employing ESI operated in the negative ionization mode. The HBCDs were extracted from the sediments through MAE using 40 mL of acetone/n-hexane (1/3, v/v) at 90 °C for 12 min. The limits of quantitation (LOQ) ranged from 25 to 40 pg/g (dry weight) in 5 g of the sediment samples. The recoveries of the HBCDs in spiked sediment samples ranged from 68 to 91% (relative standard derivation: 2–11%). The extraction efficiency of the MAE technique was also compared with Soxhlet extraction and pressurized liquid extraction.     

Quantitative analysis of delta9-tetrahydrocannabinol in preserved oral fluid by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method for the analysis of delta9-tetrahydrocannabinol (THC) in preserved oral fluid was developed and fully validated. Oral fluid was collected with the Intercept, a Food and Drug Administration (FDA) approved sampling device that is used on a large scale in the U.S. for workplace drug testing. The method comprised a simple liquid-liquid extraction with hexane, followed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis. Chromatographic separation was achieved using a XTerra MS C18 column, eluted isocratically with 1 mM ammonium formate-methanol (10:90, v/v). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. The use of the liquid-liquid extraction was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Validation of the method was performed using both 100 and 500 MicroL of oral fluid. The method was linear over the range investigated (0.5-100 ng/mL and 0. 1-10 ng/mL when 100 and 500 microL, respectively, of oral fluid were used) with an excellent intra-assay and inter-assay precision (relative standard deviations, RSD <6%) for quality control samples spiked at a concentration of 2.5 and 25 ng/mL and 0.5 and 2.5 ng/mL, respectively. Limits of quantification were 0.5 and 0.1 ng/mL when using 100 and 500 microL, respectively. In contrast to existing GC-MS methods, no extensive sample clean-up and time-consuming derivatisation steps were needed. The method was subsequently applied to Intercept samples collected at the roadside and collected during a controlled study with cannabis.     

Impact of enzymatic and alkaline hydrolysis on CBD concentration in urine

A sensitive and specific analytical method for cannabidiol (CBD) in urine was needed to define urinary CBD pharmacokinetics after controlled CBD administration, and to confirm compliance with CBD medications including Sativex—a cannabis plant extract containing 1:1 ?⁹-tetrahydrocannabinol (THC) and CBD. Non-psychoactive CBD has a wide range of therapeutic applications and may also influence psychotropic smoked cannabis effects. Few methods exist for the quantification of CBD excretion in urine, and no data are available for phase II metabolism of CBD to CBD-glucuronide or CBD-sulfate. We optimized the hydrolysis of CBD-glucuronide and/or -sulfate, and developed and validated a GC-MS method for urinary CBD quantification. Solid-phase extraction isolated and concentrated analytes prior to GC-MS. Method validation included overnight hydrolysis (16 h) at 37 °C with 2,500 units β-glucuronidase from Red Abalone. Calibration curves were fit by linear least squares regression with 1/x ² weighting with linear ranges (r ²?>?0.990) of 2.5–100 ng/mL for non-hydrolyzed CBD and 2.5–500 ng/mL for enzyme-hydrolyzed CBD. Bias was 88.7–105.3 %, imprecision 1.4–6.4 % CV and extraction efficiency 82.5–92.7 % (no hydrolysis) and 34.3–47.0 % (enzyme hydrolysis). Enzyme-hydrolyzed urine specimens exhibited more than a 250-fold CBD concentration increase compared to alkaline and non-hydrolyzed specimens. This method can be applied for urinary CBD quantification and further pharmacokinetics characterization following controlled CBD administration.     

超高效液相色谱-串联质谱法同时测定食用油中痕量的δ-9-四氢大麻酚、大麻酚和大麻二酚

建立了超高效液相色谱-串联质谱(UPLC-MS/MS)同时测定食用油中δ-9-四氢大麻酚(THC)、大麻酚(CBN)和大麻二酚(CBD)的方法。目标分析物经甲醇提取、中性氧化铝固相萃取柱净化后,采用UPLC-MS/MS分离和检测。实验以氘代四氢大麻酚(THC-D3)为内标物,采用同位素内标法定量。在3个添加水平下,目标物的平均回收率为68.0%～101.6%,相对标准偏差为7.0%～20.1%。方法检出限为0.06～0.17 μg/kg,定量限为0.20～0.52 μg/kg。该方法能够满足食用油中痕量四氢大麻酚、大麻酚和大麻二酚检测的需要。