Drug Testing and Analysis
Research article Received: 23 August 2014
Revised: 7 October 2014
Accepted: 24 October 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1753
Quantification of six cannabinoids and metabolites in oral fluid by liquid chromatography-tandem mass spectrometry Nathalie A. Desrosiers,a,b† Karl B. Scheidweilera† and Marilyn A. Huestisa* Δ9-Tetrahydrocannabinol (THC) is the most commonly analyzed cannabinoid in oral fluid (OF); however, its metabolite 11-nor-9carboxy-THC (THCCOOH) offers the advantage of documenting active consumption, as it is not detected in cannabis smoke. Analytical challenges such as low (ng/L) THCCOOH OF concentrations hampered routine OF THCCOOH monitoring. Presence of minor cannabinoids like cannabidiol and cannabinol offer the advantage of identifying recent cannabis intake. Published OF cannabinoids methods have limitations, including few analytes and lengthy derivatization. We developed and validated a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for THC, its metabolites, 11-hydroxy-THC and THCCOOH quantification, and other natural cannabinoids including tetrahydrocannabivarin (THCV), cannabidiol (CBD), and cannabigerol (CBG) in 1 mL OF collected with the Quantisal device. After solid-phase extraction, chromatography was performed on a Selectra PFPP column with a 0.15% formic acid in water and acetonitrile gradient with a 0.5 mL/min flow rate. All analytes were monitored in positive mode atmospheric pressure chemical ionization (APCI) with multiple reaction monitoring. Limits of quantification were 15 ng/L THCCOOH and 0.2 μg/L for all other analytes. Linear ranges extended to 3750 ng/L THCCOOH, 100 μg/L THC, and 50 μg/L for all other analytes. Inter-day analytical recoveries (bias) and imprecision at low, mid, and high quality control (QC) concentrations were 88.7-107.3% and 2.3-6.7%, respectively (n = 20). Mean extraction efficiencies and matrix effects evaluated at low and high QC were 75.9–86.1% and 8.4–99.4%, respectively. This method will be highly useful for workplace, criminal justice, drug treatment and driving under the influence of cannabis OF testing. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Keywords: cannabinoids; oral fluid; THC; THCCOOH; LC-MS/MS
Introduction Oral fluid (OF) testing offers advantages of non-invasiveness, observed and gender-neutral specimen collection, ease of multiple collections, and lower potential for adulteration compared to urine. In addition, OF may better correlate to blood concentrations than urine, although inter-subject variability suggests that predicting blood concentrations from OF concentrations is inaccurate and not recommended.[1] OF can be collected by passive drool, expectoration, or OF collection devices. OF collection devices contain storage buffers that reduce OF viscosity and adsorption to the collection pad or device containers, and increase analyte stability. Furthermore, OF collection with a collection device is preferable for donors and collectors over passive drool and expectoration. Analytical methods for multiple cannabinoids quantification in OF are needed for drug testing programs. Δ9-Tetrahydrocannabinol (THC) is the primary and sole analyte included in most published OF cannabinoid methods.[2–6] THC in cannabis smoke contaminates the oromucosal cavity, producing high THC OF concentrations. There is rapid initial drug clearance from the oral cavity, as >1000 μg/L median THC OF concentrations immediately after cannabis smoking fall to approximately 100-200 μg/L by 1 h[7,8] and approximately 15 μg/L by 6 h.[9] Hence, large linear dynamic ranges are required for OF THC. However, THC also can be detected in OF following passive exposure to environmental cannabis smoke confounding interpretation with legal implications.[10] THC’s inactive metabolite, 11-nor-9-carboxy-THC (THCCOOH), is not present in
Drug Test. Analysis (2014)
cannabis smoke and is found at low ng/L concentrations in OF. Therefore, monitoring THCCOOH is important, as it helps differentiate active cannabis intake from passive environmental cannabis smoke exposure.[10] Interestingly, THCCOOH-glucuronide was observed in OF, with approximately equal concentrations of free and glucuronidated THCCOOH.[11] OF hydrolysis may double THCCOOH concentrations, improving detectability and perhaps, windows of OF THCCOOH detection. However, due to its low concentrations and associated analytical challenges, few OF cannabinoid methods include THCCOOH. Currently published OF cannabinoids methods with THCCOOH at biologically relevant sub 80 ng/L concentrations are limited.[12–21] No existing methods achieve the required sensitivity without
* Correspondence to: Marilyn A. Huestis, PhD, Chief, Chemistry and Drug Metabolism IRP, National Institute on Drug Abuse, National Institutes of Health, Biomedical Research Center, 251 Bayview Boulevard Suite 200 Room 05A-721, Baltimore, MD 21224, SA. E-mail: [email protected] †
These authors contributed equally to this work.
a Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Boulevard, Baltimore, MD 21224, USA b Program in Toxicology, University of Maryland Baltimore, 620 W. Lexington St, Baltimore, MD 21201, USA
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Drug Testing and Analysis
N. A. Desrosiers, K. B. Scheidweiler and M. A. Huestis
employing complicated two-dimensional gas chromatographymass spectrometry (GM-MS), derivatization prior to triple quadrupole liquid chromatography-tandem mass spectrometry (LC-MS/MS) or LC-high resolution tandem mass spectrometry (LC-HRMS(/MS). Published methods include two-dimensional gas chromatography negative chemical ionization mass spectrometry (2D-GC-MS),[12,13] LC-MS/MS,[14–19] and gas chromatography-tandem mass spectrometry (GC-MS/MS)[20,21] assays. Some of these methods include only THCCOOH,[18,21] THC and THCCOOH,[15–17] and/or require derivatization.[12,16,17] Other methods include THCCOOH, but with limits of quantification (LOQs) >80 ng/L that are too high for identifying most smoked cannabis intake.[22–25] Furthermore, only one published method hydrolyzed THCCOOH-glucuronide, which approximately doubled THCCOOH concentrations, thereby enabling wider windows for detecting cannabis intake.[16] Minor cannabinoids, cannabidiol (CBD) and cannabinol (CBN), were previously suggested for documenting recent cannabis consumption, helping differentiate recent intake from residual cannabinoid excretion in frequent cannabis smokers.[7,9,26] However, the inclusion of these analytes adds further analytical challenges and few methods also included CBD and CBN.[12,14,19] To the best of our knowledge, the minor cannabinoids tetrahydrocannabivarin (THCV, the propyl analogue of THC) and cannabigerol (CBG, a biosynthetic precursor of THC and CBD) have not been evaluated in OF. Therefore, inclusion of these analytes in analytical methods for OF cannabinoids could assist interpretation. The analytical challenges of achieving a low 15 ng/L LOQ for THCCOOH, the lack of time-efficient methods for quantification of THCCOOH at biologically relevant concentrations, and the advantages of including minor cannabinoids to improve interpretation of cannabinoid OF results, led us to develop and validate a simple and rapid quantitative method for OF THC, 11-OH-THC, THCCOOH, THCV, CBD, and CBG analysis. Sample preparation included hydrolysis and solid-phase extraction (SPE) before LC-MS/MS. This assay will be useful for OF cannabinoid analysis for workplace, drug treatment, pain management, and criminal justice and driving under the influence of cannabis testing.
Material and methods Reagents and supplies THC, 11-OH-THC, THCCOOH, CBD, d3-THC, d3-11-OH-THC, d9THCCOOH, and d3-CBD standards were purchased from Cerilliant (Round Rock, TX, USA). CBG was acquired from Restek (Bellefonte, PA, USA) and THCV from RTI International (Research Triangle Park, NC, USA). Ammonium acetate, formic acid, and acetonitrile (LCMS grade) were obtained from Sigma-Aldrich (St Louis, MO, USA). Methanol, LC-MS grade water (for mobile phase A), isopropanol, methylene chloride, ammonium hydroxide, hydrochloric acid, and glacial acetic acid were purchased from Fisher Scientific (Fair Lawn, NY, USA). All solvents were high performance liquid chromatography (HPLC) grade unless otherwise stated. Water (for all purposes except mobile phase A) was purified in house with an ELGA Purelab Ultra Analytical purifier (Siemens Water Technologies, Lowell, MA, USA). Red abalone beta-glucuronidase solution containing 100 000 units/mL beta-glucuronidase was diluted with distilled water to contain 15, 625 units/mL beta-glucuronidase activity for enzymatic hydrolysis (Kura Biotech, Inglewood, CA, USA). Strata X-C columns (3 mL/30 mg, Phenomenex Inc., Torrance, CA, USA) were utilized for SPE. Specimens were extracted on a Cerex System 48 positive pressure manifold (SPEware Corp., Baldwin Park, CA,
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USA). Chromatography was performed with a United Chemical Technologies Selectra PFPP column (100 × 2.1 mm, 3 μm particle size) combined with a matched guard column (10 × 2.1 mm, 3 μm particle size) (Bristol, PA, USA). Quantisal buffer and OF collection devices were from Immunalysis Corp (Pomona, CA, USA). Instrumentation Tandem mass spectrometry was performed on an ABSciex 6500 QTRAPW triple quadrupole/linear ion trap mass spectrometer with an IonDrive™ Turbo V source (ABSciex, Foster City, CA, USA). The HPLC system consisted of a DGU-20A3r degasser, LC-20ADxr pumps, SIL-20ACxr autosampler, and a CTO-20A column oven (Shimadzu Corp, Columbia, MD, USA). Data were acquired and analyzed with Analyst (version 1.6.2) and Multiquant (version 2.1), respectively. Calibrators, quality control, and internal standards Blank OF for calibrators and quality controls (QC) were obtained (via expectoration) anonymously from volunteers in our laboratory and was evaluated for absence of cannabinoids following our described method. Primary stock solutions containing individual cannabinoids were prepared at 10 μg/L in methanol. Mixed analyte calibrator solutions were prepared via dilution in methanol creating calibrators at 0.2, 0.5, 2, 5, 20, 50 and 100 μg/L for THC, at 0.2, 0.5, 2, 5, 20, and 50 μg/L for 11-OH-THC, THCV, CBD, and CBG, and 15, 37.5, 150, 375, 1500, and 3750 ng/L for THCCOOH when fortifying 25 μL standard solution into 0.25 mL blank OF. QC samples were prepared with reference standard solutions from different lot numbers than calibrators or with separate ampules than used for preparing calibrators. Mixed analyte QC solutions were prepared via dilution in methanol creating QC solutions at 0.6, 6, and 90 μg/L for THC, 0.6, 6, and 45 μg/L for 11-OHTHC, THCV, CBD, and CBG, and 45, 450, and 3375 ng/L for THCCOOH when fortifying 25 μL QC solution into 0.25 mL blank OF. Primary stock solutions of THC-d3, 11-OH-THC-d3, THCCOOH-d9, and CBD-d3 were diluted in methanol, producing a mixed internal standard solution of 5 μg/L for THC-d3, 11-OH-THC-d3, and CBD-d3 and 37.5 ng/L for THCCOOH-d9, when fortifying 25 μL internal standard solution into 0.25 mL blank OF. No commercially available deuterated internal standards were available for THCV and CBG; CBD-d3 was the internal standard for these analytes. Hydrolysis Blank OF (250 μL) and 750 μL Quantisal buffer were aliquoted into 10-mL threaded glass centrifuge tubes prior to fortification with calibrator or QC solution and internal standard. Ammonium acetate buffer (1 M, pH 4, 0.3 mL) and 40 μL 15,625 U/mL beta glucuronidase solution (625 units) were added to each tube. Samples were capped, vortexed and incubated at 55 °C for 60 min. One mL authentic Quantisal specimen (containing 250 μL OF with 750 μL device buffer) was fortified with internal standard and 25 μL methanol before being processed identically along with calibrators and QCs. SPE Following hydrolysis, samples were acidified with 0.5 mL glacial acetic acid and vortexed. SPE columns were conditioned with 3 mL methanol, water, and 0.1% hydrochloric acid (HCl) before sample loading. Columns were washed with 2 mL water and 2 mL 0.1% HCl:acetonitrile (70:30, v/v). Columns were dried at 207 kPa for
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Drug Test. Analysis (2014)
Drug Testing and Analysis
Quantification of cannabinoids in oral fluid 15 min. Analytes were eluted into clean 10 mL glass centrifuge tubes with 2 mL dichloromethane:isopropanol: ammonium hydroxide (78:20:2, v/v/v). Eluates were completely dried under nitrogen at 40 °C in a Zymark TurboVap. Residues were reconstituted in 150 μL mobile phase A/B (70:30), vortexed briefly before centrifugation at 1800 × g, 4 °C for 5 min. Samples were transferred to 1.5 mL polypropylene microcentrifuge tubes, centrifuged at 15,000 × g, 4 °C for 5 min and 125 μL was transferred to autosampler vials containing 350 μL glass inserts. Fifty μL was injected onto the LC-MS/ MS instrument.
LC-MS/MS Chromatographic separation was performed on a Selectra PFPP column via gradient elution with 0.15% formic acid in water (A) and 0.15% formic acid in acetonitrile (B). Flow rate was set at 0.5 mL/min, with an initial gradient of 30% A. Mobile phase B increased to 78.5% over 8.5 min, increased to 98% over 0.2 min and then held at 98% for 3 min. The column was re-equilibrated to 30% B over 0.2 min and held for 2.1 min (total run time 14 min). HPLC eluent was diverted to waste for the first 4 min and for the final 5 min of the analysis. The autosampler and column oven temperatures were 4 °C and 40 °C, respectively. All data were acquired via positive mode atmospheric pressure chemical ionization (APCI). MS/MS parameter settings were optimized via direct infusion of individual analytes (10 ng/mL in methanol: 10 mM ammonium acetate in water; 50:50 v/v) at 10 μL/min (Table 1). Optimized source settings were: gas-1 45, curtain gas 45, and source temperature 450 °C. Nebulizer current was set at 4.0 V for periods 1 (11-OH-THC and THCCOOH) and 3 (THC) and at 3.0 V for period 2 (THCV, CBD, and CBG). Nitrogen collision gas was set at medium for all periods. Quadrupoles one and three were set to unit resolution. Quantifier and qualifier ion transitions were monitored for each analyte and internal standard.
Data analysis Peak area ratios of analytes corresponding to internal standards were calculated for each concentration to construct daily calibration curves via linear least-squares regression with a 1/x2 weighting factor. Calibration curves were from 0.2–100 μg/L for THC, 0.2–50 μg/L for 11-OH-THC, THCV, CBD, and CBG, and 15–3750 ng/L for THCCOOH.
Method validation Specificity, sensitivity, linearity, imprecision, analytical recovery (bias), extraction efficiency, matrix effect, stability, dilution integrity, and carry-over were evaluated during method validation according to SWGTOX guidelines. Specificity Analyte peak identification criteria included relative retention time within ±0.1 min of the lowest calibrator and qualifier/quantifier transition peak area ratios within ±20% of mean calibrator ratios. Endogenous interferences were evaluated by analyzing 10 different OF blanks from 10 different individuals. Potential interference from commonly ingested drugs and medications were evaluated by fortifying drugs into low QC samples at a final interferent concentration of 200 μg/L (Table 2). No interference was noted if all analytes in the low QC quantified within ±20% of target concentrations with acceptable qualifier/quantifier transition peak area ratios. Individual cannabinoids also were fortified into low QC samples at final concentrations of 50 μg/L (THC, CBD, cannabinol, CBG, THCV, Δ9-tetrahydrocannabinolic acid A [THCAA]) or 1 μg/L (11-OH-THC, THCCOOH, carboxy-THCV) and into negative OF at THC concentrations of 1000, 2500, and 5000 μg/L. No interference was noted if all analytes in the low QC quantified within ±20% of target low QC concentrations with acceptable qualifier/quantifier transition peak area ratios. The potential degradation of CBD and
Table 1. Liquid chromatography-tandem mass spectrometry parameters for cannabinoids in human oral fluid Analyte THC THC-d3 11-OH-THC 11-OH-THC-d3 THCCOOH THCCOOH-d9 THCV CBD CBD-d3 CBG
Drug Test. Analysis (2014)
Q1 mass (amu)
Q3 mass (amu)
Dwell (ms)
Declustering potential (V)
Entrance potential (V)
Collision energy (V)
Cell exit potential (V)
Retention Time (min)
315.0 315.0 318.1 318.1 331.0 331.0 334.1 334.1 345.0 345.0 354.0 354.0 287.0 287.0 315.0 315.0 318.0 318.0 317.1 317.1
193.0 123.1 196.1 123.0 193.0 201.0 196.1 201.1 299.2 193.1 308.2 196.0 165.1 123.0 193.0 123.0 196.1 123.0 193.1 122.9
25 25 25 25 20 20 20 20 20 20 20 20 35 35 35 35 35 35 35 35
66 66 31 31 36 36 51 51 56 56 66 66 46 46 51 51 26 26 36 36
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
31 43 33 45 33 33 35 33 27 37 29 37 31 41 29 41 29 43 23 45
10 8 14 8 10 14 12 10 14 12 14 10 14 6 10 6 10 10 6 14
7.92
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7.91 5.89 5.89 6.05 6.04 7.05 7.22 7.22 7.30
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morphine normorphine morphine-3-beta-D-glucuronide morphine-6-beta-D-glucuronide codeine norcodeine 6-acetylmorphine 6-acetylecodeine buprenorphine norbuprenorphine hydrocodone hydromorphone
oxycodone noroxycodone oxymorphone nor-oxymorphone methadone 2-ethyl-5-methyl-3,3-diphenyl1-pyrroline 2-ethylidene-1,5-dimethyl-3,3diphenylpyrrolidine propoxyphene pentazocine
m-hydroxybenzoylecgonine p-hydroxybenzoylecgonine
Opioids
cocaine benzoylecgonine norcocaine norbenzoylecgonine ecgonine ethyl ester ecgonine methyl ester anhydroecgonine methyl ester ecgonine cocaethylene norcocatheylene m-hydroxycocaine p-hydroxycocaine
Cocaine
clonazepam flurazepam
diazepam lorazepam oxazepam alprazolam 7-aminoclonazepam 7-aminoflunitrazepam 7-aminonitrazepam nitrazepam flunitrazepam temazepam nordiazepam bromazepam
Benzodiazepines
Table 2. List of exogenous interferences evaluated at 200 μg/L in oral fluid during method validation
imipramine clomipramine fluoxetine norfluoxetine paroxetine
Antidepressants
Published 2014. This article is a U.S. Government work and is in the public domain in the USA. phentermine
phenylpropanolamine
para-methoxyamphetamine para-methoxymethylamphetamine methamphetamine amphetamine hydroxyl-methamphetamine hydroxyl-amphetamine 4-hydroxy-3-methoxymethamphetamine 4-hydroxy-3-methoxyamphetamine 3,4-methylenedioxyamphetamine 3,4-methylenedioxymethamphetamine 3,4-methylenedioxyphenyl-2-butanamine methyl-1-(3,4-methylenedioxyphenyl)2-butanamine R-cathinone ethylamphetamine 4-bromo-2,5-dimethoxyphenethylamine fenfluramine ephedrine pseudoephedrine
Amphetamines and other amines
norcotinine hydroxycotinine
clonidine ibuprofen caffeine diphenhydramine chlorpheniramine bromopheniramine Aspirin Tylenol Ketamine dextromethorphan Nicotine Cotinine
Others
Drug Testing and Analysis N. A. Desrosiers, K. B. Scheidweiler and M. A. Huestis
Drug Test. Analysis (2014)
Drug Testing and Analysis
Quantification of cannabinoids in oral fluid THCAA to THC under acidic conditions was evaluated by fortifying 50 μg/L CBD or THCAA and internal standard into blank OF. Sensitivity and linearity Limits of detection (LODs) and LOQs were evaluated over three days with duplicates from three different OF sources. LOD was defined as the lowest concentration producing a peak eluting within ±0.1 min of analyte retention time for the lowest calibrator, Gaussian peak shape, qualifier/quantifier transition peak area ratios ±20% of mean calibrator transitions for all replicates, and software-determined signal-to-noise ratio ≥3. LOQ was defined as the lowest concentration to meet the LOD criteria, measured within ±20% of target concentrations and software-determined signal-tonoise ratio ≥10. With these criteria, LOD could equal LOQ. Linearity was preliminarily assessed with five sets of calibrators to determine the best calibration model by comparing goodness of fit for unweighted linear least squares and linear least squares employing 1/x or 1/x2. Calibration curves were fit by linear least squares regression with six concentrations across the linear dynamic range for each analyte (seven THC calibrators). Calibrators were required to quantify within ±15%, except at the LOQ, which was required to quantify within ±20%. Correlation coefficients (R2) were required to exceed 0.995. Analytical recovery and imprecision Intra-day analytical recovery (bias) and imprecision were evaluated with 4 replicates at 3 QC concentrations. Analytical recovery was determined by comparing the mean result for all analyses to the theoretical concentration (e.g. mean percent expected concentration). Imprecision was expressed as the percent coefficient of variation (%CV) of the calculated concentrations. Inter-day analytical recovery and imprecision were evaluated with 4 replicates at 3 QC concentrations over 5 days (n = 20). One-way analysis of variation (ANOVA) was conducted on low, medium, and high QCs to evaluate inter- and intra-day differences in analyte concentrations. Extraction efficiency and matrix effect Extraction efficiency and matrix effects were evaluated using three sets of samples for low and high QC, as described by Matuszewski et al. (n = 10 for each set).[27] In set A, OF from 10 individuals were fortified with analytes and internal standard prior to SPE. In set B, OF from 10 individuals were fortified with analytes and internal standard after SPE. In set C, elution solvent was fortified with analytes and internal standard. Extraction efficiency was calculated by dividing analyte mean peak areas of set A by set B and expressed as a percentage. Matrix effect was determined by dividing the mean analyte peak area of set B by set C; this was converted to a percentage and subtracted from 100 to represent the amount of signal suppressed or enhanced by the presence of matrix.
Dilution integrity Dilution integrity was evaluated by fortifying blank OF:buffer mixture (n = 3) to a final concentration of 7500 ng/L THCCOOH and 100 μg/L THC, 11-OH-THC, THCV, CBD, and CBG and diluting it with blank OF:buffer mixture to achieve a 1:20 (v/v) dilution. Internal standards were added and samples were extracted as described. Dilution integrity was maintained if specimens quantified within ±20% of expected diluted concentration. Carry-over Carry-over was evaluated by fortifying blank OF:buffer mixture to a final concentration of 1000 μg/L for THC or twice the upper LOQ (ULOQ) for all other analytes (n = 3). Negative OF samples followed each replicate. Samples could not meet LOD criteria to document absence of carry-over. Hydrolysis THC-glucuronide and THCCOOH-glucuronide were initially evaluated during method development with base hydrolysis and enzymatic hydrolysis with different pH buffers, times, temperatures, and amounts of enzyme. Hydrolysis was validated by fortifying THC-glucuronide and THCCOOH-glucuronide to final concentrations of 50 μg/L and 2000 ng/L, respectively, and processing samples as described herein. Samples fortified at the same concentrations but not undergoing hydrolysis also were extracted and analyzed to serve as controls. Proof of applicability OF was collected from one individual who participated in an Institutional Review Board-approved study evaluating the effects of inhaled cannabis containing 6.7% THC. Vapors of 500 mg cannabis were inhaled ad libitum by vaporization over 10 min via the VolcanoW Medic. OF was collected at baseline, 0.17, 1.4, 2.3, 3.3, 4.3, 5.3, 6.3, 7.3, and 8.3 h after inhalation with the Quantisal device. The samples were stored at 4 °C for 12 h in buffer to elute analytes from the pad, and transferred to polypropylene tubes stored at 4 °C until analysis.
Stability Stability was evaluated in blank OF:buffer mixture fortified with analytes of interest at low and high QC (n = 4 for each). Short-term stability was assessed in polypropylene cryotubes under various conditions: 16 h at room temperature, 72 h at 4 °C and after 3 freeze-thaw cycles. Internal standards were added immediately prior to extraction and concentrations were compared to freshly prepared QCs that were extracted along with stability samples. Analyte autosampler stability was evaluated by injecting low and high QC specimens 84 h after extraction and comparing concentrations to freshly prepared QCs.
Drug Test. Analysis (2014)
Figure 1. Extracted ion chromatograms of target analyte and deuterated internal standard quantifier MRMs, highlighting chromatographic resolution between our analytes.
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
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Drug Testing and Analysis
N. A. Desrosiers, K. B. Scheidweiler and M. A. Huestis Negative
LOQ
THC m/z 315 - 193
2.0e4
11-OH-THC m/z 331 - 193
8.00
8.40
3.0e3
THCCOOH m/z 345 - 299
8.00
8.40
5.90
6.4
0 5.55
0 7.60
8.00
8.40
6.0e3
5.90
5.91
3.0e3
6.4
5.90
1.0e4
0 5.55 3.0e4
5.90
6.4 6.05
6.05
5.0e3
5.0e3
0 5.55 4.0e4
THCV m/z 287 - 165
0 7.60 6.0e3
3.0e3
1.0e4
6.05
6.55
0 5.55 4.0e4
6.05
1.5e4
6.55
0 5.55 4.0e4
6.05
6.55 7.04
7.05 2.0e4
2.0e4
2.0e4
0 6.55 7.0e4
CBD m/z 315 - 193
1.0e6
1.0e4
1.0e4
0 5.55
7.05
7.55
0 6.55 7.0e4
7.05
7.55
3.5e4
3.5e4
0 6.55 7.0e4
7.05
7.55 7.22
3.5e4 7.22
0 6.60 1.4e5
CBG m/z 317 - 193
7.91
7.91
0 7.60 6.0e3
CPS
Authentic sample 2.0e6
2.0e4
7.15
7.60
0 6.60 1.4e5
7.15
7.60
0 6.60 1.4e5
7.15
7.60 7.28
7.0e4
0 6.75
7.29
7.0e4
7.18
7.60
0 6.75
7.18
7.60
7.0e4
0 6.75
7.18
7.60
Minutes Figure 2. Multiple reaction monitoring ion chromatograms for quantifier transitions from THC, 11-OH-THC, THCCOOH, THCV, CBD, and CBG from negative oral fluid, limit of quantification (LOQ), and authentic sample collected 5.3 h after vaporizing 500 mg of cannabis containing 6.7% THC. Concentrations were 19.2 μg/L for THC, 0.996. Analytical recovery and imprecision Analytical recovery and imprecision were evaluated at low, mid, and high concentrations across the linear dynamic range (Table 4). Analytical recovery ranged from 83.9 to 113.5% and 88.7 to 107.3% of expected concentrations for intra-day and inter-day recoveries, respectively. Imprecision ranged from 0.5 to 9.6% and 2.3 to 6.7% for intra-day and inter-day imprecision, respectively (Table 4). There were few significant effects of day on most analyte QC concentrations (F4,5 = 0.347-2.906, P > 0.05); however, there were 5 exceptions (F4,5 = 4.498-7.703, P < 0.05), including high THCCOOH, low 11-OH-THC, mid THCV, high THCV, and high CBG. Extraction efficiency and matrix effect
Sensitivity and linearity Table 3 details LOD, LOQ, linearity, and mean calibration results. LODs were 0.1 μg/L for THC, 11-OH-THC, THCV, CBD, and CBG,
Extraction efficiencies and matrix effects are presented in Table 5. Mean extraction efficiencies were 75.9–86.1% (n = 10) and mean matrix effects (% effect) were 8.4–99.4% (n = 10).
Table 3. Cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry limits of detection (LODs), limits of quantification (LOQs), linear ranges, and calibration results (n = 5) Analyte
Internal Standard
THC 11-OH-THC THCCOOH THCV CBD CBG
THC-d3 11-OH-THC-d3 THCCOOH-d9 CBD-d3 CBD-d3 CBD-d3
LOD
LOQ
Linear range
Y-intercept Mean ± SD
0.1 μg/L 0.1 μg/L 15 ng/L 0.1 μg/L 0.1 μg/L 0.1 μg/L
0.2 μg/L 0.2 μg/L 15 ng/L 0.2 μg/L 0.2 μg/L 0.2 μg/L
0.2-100 μg/L 0.2-50 μg/L 15-3750 ng/L 0.2-50 μg/L 0.2-50 μg/L 0.2-50 μg/L
4.4 × 10 ± 2.4 × 10 -2 -3 1.4 × 10 ± 4.4 × 10 -2 -3 2.7 × 10 ± 8.1 × 10 -3 -3 2.4 × 10 ± 2.6 × 10 -4 -3 4.1 × 10 ± 1.5 × 10 -3 -3 1.7 × 10 ± 8.2 × 10
-3
-3
2
Slope Mean ± SD -4
R (range) -6
2.0 × 10 ± 3.4 × 10 -4 -6 2.0 × 10 ± 4.2 × 10 -3 -4 4.5 × 10 ± 1.9 × 10 -4 -6 2.4 × 10 ± 6.6 × 10 -4 -6 2.1 × 10 ± 3.8 × 10 -4 -5 6.7 × 10 ± 1.6 × 10
0.997-1.000 0.999-1.000 0.996-0.999 0.997-1.000 0.997-1.000 0.997-1.000
Table 4. Analytical recovery (% target concentration) and imprecision (% coefficient of variation) data for cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry Analyte
THC
11-OH-THC
THCCOOH
THCV
CBD
CBG
Drug Test. Analysis (2014)
Concentration
Analytical Recovery (%)
Imprecision (%)
Intra-day
Inter-day
Intra-day
Inter-day
μg/L
N=4
N = 20
N=4
N = 20
0.6 6 90 0.6 6 45 45 ng/L 450 ng/L 3375 ng/L 0.6 6 45 0.6 6 45 0.6 6 45
103.5 101.7 102.1 102.7 104.8 98.8 90.4 92.5 89.9 106.7 104.4 100.3 108.6 105.9 102.4 106.6 102.5 99.4
100.9 100.8 102.4 103.1 104.4 101.4 88.7 91.6 90.8 106.6 105.8 104.2 107.3 106.6 104.6 106.0 105.4 103.7
1.9 2.4 0.5 1.3 2.6 2.3 2.3 2.9 2.9 4.6 1.4 0.9 4.2 1.0 0.6 4.4 1.5 1.7
4.5 3.8 2.3 5.1 3.8 3.2 5.4 6.7 3.5 4.8 4.8 5.3 3.2 3.7 2.7 3.7 4.4 3.9
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
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N. A. Desrosiers, K. B. Scheidweiler and M. A. Huestis
Table 5. Mean extraction efficiency and matrix effect for cannabinoids extracted from human oral fluid by solid-phase extraction Analyte
Extraction efficiency (n = 10)
THC THC-d3 11-OH-THC 11-OH-THC-d3 THCCOOH THCCOOH-d9 THCV CBD CBD-d3 CBG
Matrix effects (% difference, n = 10)
Low
High
Low
High
80.9% 79.7% 81.0% 78.9% 86.1% 81.1% 77.6% 79.9% 78.6% 78.2%
78.3% 80.9% 78.8% 79.0% 80.8% 79.2% 77.2% 78.1% 79.4% 75.9%
20.9% 20.0% 31.6% 25.3% 67.4% 64.0% 30.2% 40.0% 41.4% 99.4%
11.6% 11.0% 8.4% 10.4% 64.3% 70.7% 25.2% 33.3% 28.0% 75.3%
Stability, dilution integrity, and carry-over
Discussion
Stability was evaluated in oral fluid after 16 h at RT, 72 h at 4 °C and after three freeze-thaw cycles; autosampler stability was evaluated after extraction and storage for 84 h on the 4 °C autosampler. All concentrations were within ±12% of freshly prepared QC concentrations (N = 4). Dilution integrity was acceptable (within ±20% of target concentrations) for all analytes after diluting 1:20 with blank OF:buffer mixture. Analytes quantified within 82.1–89.9% of expected concentrations. There was no evidence of carry-over for any cannabinoid. Negative specimens injected after samples containing 1000 μg/L for THC or twice the ULOQ of other cannabinoids did not contain analyte peaks satisfying assay LOD criteria (n = 3).
We present an LC-MS/MS method for the simultaneous quantification of six cannabinoids and metabolites in OF. This novel LC-MS/ MS triple quadrupole method employs simple sample preparation without requiring derivatization, monitors THCCOOH at clinically relevant concentrations for documenting active cannabis consumption and includes minor cannabinoids that can improve interpretation of OF cannabinoid results. This method can be applied for cannabinoid analysis in workplace, pain management, drug treatment, criminal justice, and driving under the influence of cannabis testing. We quantified the minor cannabinoids THCV and CBG for the first time in OF, enabling future investigation of these new OF cannabinoid markers. Table 6 lists cannabinoid OF concentrations from a single participant following cannabis inhalation, highlighting the applicability of our new method. This method will be utilized in our upcoming clinical study examining cannabinoid disposition in OF following smoked, vaporized, and oral cannabis administration. During method development, we evaluated various sample preparation approaches and chromatography conditions for OF cannabinoid quantification. Evaluated SPE columns included BondElut Plexa (Aglient), Reprep C18 (Restek), Polychrom-THC (Cerex), Oasis Max (Waters), SLE + (Biotage), Strata-X (Phenomenex) and Strata-XC (Phenomenex), with different washing and elution conditions. While the Polychrom-THC successfully extracted all cannabinoids, LC backpressure and quadrupole charging became problematic, possibly due to the detergents in the OF collection buffer. Electrospray ionization in positive and negative mode and
Hydrolysis Hydrolysis efficiency was 67.7% for THC-glucuronide and 97.7% for THCCOOH-glucuronide.
Proof of applicability Ten OF samples collected after vaporized cannabis inhalation containing 6.7% (w/w) THC were analyzed for cannabinoids with the new method. Concentrations are presented in Table 6 and representative chromatograms are illustrated in Fig. 2.
Table 6. Cannabinoid concentrations in oral fluid following inhalation of 500 mg of vaporized cannabis containing 6.7% THC cannabis Sample 1 2 3 4 5 6 7 8 9 10
Time of collection (h since vaporization)
THC μg/L
11-OH-THC ng/L
THCCOOH ng/L
THCVμg/L
CBD μg/L
CBG μg/L
Baseline 0.15 1.4 2.3 3.3 4.3 5.3 6.3 7.3 8.2
13.4 3,126 317 96.3 67.3 8.5 19.2 11.3 3.4 7.6
200 0 0 346 351 0 0 226 0 0
175 381 203 243 180 61.6 136 126 41 135
0 32.8 4.7 1.4 1.2 0 0.3 0 0 0
0 120 16.8 8.6 3.9 0.4 0.8 0.5 0.3 0.3
0.31 115 14.3 6.6 3.1 0.3 0.5 0.3 0 0.2
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Drug Testing and Analysis
Quantification of cannabinoids in oral fluid APCI in negative mode were evaluated along with various mobile phases; however, APCI in positive mode with an acidic mobile phase provided optimal THCCOOH sensitivity with reduced matrix interferences. LC columns evaluated included Kinetex C18 (Phenomenex), Raptor Biphenyl (Restek), and the PFPP (UCT) with different mobile phases and gradients. While we achieved the desired LOQ with the Kinetex C18 column with fortified OF-buffer samples, authentic samples had THCCOOH interferences not present in calibrators or QCs, likely from pyrolysis products from cannabis smoke or other cannabinoids. The PFPP LC column from UCT was selected for optimal chromatography, as it minimized chromatographic interferences observed in authentic samples. The authentic specimen chromatogram (Fig. 2) depicts the worst resolution obtained between THCCOOH and the preceding interfering peak observed in all calibrators, QCs and authentic specimens. The co-eluting peak was more resolved in all other chromatograms compared to the included chromatogram. It should be noted that calibrators, QCs and authentic specimens were computer integrated with identical parameters. Therefore, while an interference eluted just before THCCOOH, all calibrators and QCs fulfilled MRM ratio, accuracy and imprecision criteria. We observed >88.7% inter-day analytical recovery (bias) and 75.9%. Matrix effects ranged from 8.4 to 99.4%, but deuterated internal standards compensated for matrix effects enabling QC quantification within ±20% for all analytes. We employed CBD-d3 as internal standard for CBG and THCV for which matched deuterated internal standards were unavailable. CBD-d3 was selected as the internal standard for these analytes because they had similar SPE efficiencies and eluted closest to these analytes. Matrix effects were similar between CBD-d3 and THCV, but CBG had higher % signal suppressed. Nevertheless, all THCV and CBG QCs fulfilled analytical recovery (bias) and imprecision criteria throughout validation. Dilution integrity and analyte stability were acceptable for all analytes. No exogenous interferences were noted; however, THCV at 50 μg/L produced 0.6 and 0.7 μg/L CBD and CBG. Given that CBD and CBG also were present in neat standard solutions, these are thought to be an impurity in the standard. Certificate of analysis for THCV indicated that purity was at 94.8 ± 0.12%, possibly explaining the presence of CBG and CBG in these standards. THC, CBD and CBG concentrations were 0.1, 1.5, and 1.3% of fortified THCV concentrations, respectively. Finally, THCCOOH-glucuronide hydrolysis to THCCOOH via Red Abalone glucuronidase neared completion (97.7%). CBD conversion to THC is negligible, as OF THC concentrations are generally >10x the CBD concentrations following cannabis inhalation (see table 6) or smoking,[7,9] mirroring the relative concentrations of these drugs in the cannabis plant. Therefore, any additional THC from CBD conversion would not be clinically significant, as this would represent
Research article Received: 23 August 2014
Revised: 7 October 2014
Accepted: 24 October 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1753
Quantification of six cannabinoids and metabolites in oral fluid by liquid chromatography-tandem mass spectrometry Nathalie A. Desrosiers,a,b† Karl B. Scheidweilera† and Marilyn A. Huestisa* Δ9-Tetrahydrocannabinol (THC) is the most commonly analyzed cannabinoid in oral fluid (OF); however, its metabolite 11-nor-9carboxy-THC (THCCOOH) offers the advantage of documenting active consumption, as it is not detected in cannabis smoke. Analytical challenges such as low (ng/L) THCCOOH OF concentrations hampered routine OF THCCOOH monitoring. Presence of minor cannabinoids like cannabidiol and cannabinol offer the advantage of identifying recent cannabis intake. Published OF cannabinoids methods have limitations, including few analytes and lengthy derivatization. We developed and validated a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for THC, its metabolites, 11-hydroxy-THC and THCCOOH quantification, and other natural cannabinoids including tetrahydrocannabivarin (THCV), cannabidiol (CBD), and cannabigerol (CBG) in 1 mL OF collected with the Quantisal device. After solid-phase extraction, chromatography was performed on a Selectra PFPP column with a 0.15% formic acid in water and acetonitrile gradient with a 0.5 mL/min flow rate. All analytes were monitored in positive mode atmospheric pressure chemical ionization (APCI) with multiple reaction monitoring. Limits of quantification were 15 ng/L THCCOOH and 0.2 μg/L for all other analytes. Linear ranges extended to 3750 ng/L THCCOOH, 100 μg/L THC, and 50 μg/L for all other analytes. Inter-day analytical recoveries (bias) and imprecision at low, mid, and high quality control (QC) concentrations were 88.7-107.3% and 2.3-6.7%, respectively (n = 20). Mean extraction efficiencies and matrix effects evaluated at low and high QC were 75.9–86.1% and 8.4–99.4%, respectively. This method will be highly useful for workplace, criminal justice, drug treatment and driving under the influence of cannabis OF testing. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Keywords: cannabinoids; oral fluid; THC; THCCOOH; LC-MS/MS
Introduction Oral fluid (OF) testing offers advantages of non-invasiveness, observed and gender-neutral specimen collection, ease of multiple collections, and lower potential for adulteration compared to urine. In addition, OF may better correlate to blood concentrations than urine, although inter-subject variability suggests that predicting blood concentrations from OF concentrations is inaccurate and not recommended.[1] OF can be collected by passive drool, expectoration, or OF collection devices. OF collection devices contain storage buffers that reduce OF viscosity and adsorption to the collection pad or device containers, and increase analyte stability. Furthermore, OF collection with a collection device is preferable for donors and collectors over passive drool and expectoration. Analytical methods for multiple cannabinoids quantification in OF are needed for drug testing programs. Δ9-Tetrahydrocannabinol (THC) is the primary and sole analyte included in most published OF cannabinoid methods.[2–6] THC in cannabis smoke contaminates the oromucosal cavity, producing high THC OF concentrations. There is rapid initial drug clearance from the oral cavity, as >1000 μg/L median THC OF concentrations immediately after cannabis smoking fall to approximately 100-200 μg/L by 1 h[7,8] and approximately 15 μg/L by 6 h.[9] Hence, large linear dynamic ranges are required for OF THC. However, THC also can be detected in OF following passive exposure to environmental cannabis smoke confounding interpretation with legal implications.[10] THC’s inactive metabolite, 11-nor-9-carboxy-THC (THCCOOH), is not present in
Drug Test. Analysis (2014)
cannabis smoke and is found at low ng/L concentrations in OF. Therefore, monitoring THCCOOH is important, as it helps differentiate active cannabis intake from passive environmental cannabis smoke exposure.[10] Interestingly, THCCOOH-glucuronide was observed in OF, with approximately equal concentrations of free and glucuronidated THCCOOH.[11] OF hydrolysis may double THCCOOH concentrations, improving detectability and perhaps, windows of OF THCCOOH detection. However, due to its low concentrations and associated analytical challenges, few OF cannabinoid methods include THCCOOH. Currently published OF cannabinoids methods with THCCOOH at biologically relevant sub 80 ng/L concentrations are limited.[12–21] No existing methods achieve the required sensitivity without
* Correspondence to: Marilyn A. Huestis, PhD, Chief, Chemistry and Drug Metabolism IRP, National Institute on Drug Abuse, National Institutes of Health, Biomedical Research Center, 251 Bayview Boulevard Suite 200 Room 05A-721, Baltimore, MD 21224, SA. E-mail: [email protected] †
These authors contributed equally to this work.
a Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Boulevard, Baltimore, MD 21224, USA b Program in Toxicology, University of Maryland Baltimore, 620 W. Lexington St, Baltimore, MD 21201, USA
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Drug Testing and Analysis
N. A. Desrosiers, K. B. Scheidweiler and M. A. Huestis
employing complicated two-dimensional gas chromatographymass spectrometry (GM-MS), derivatization prior to triple quadrupole liquid chromatography-tandem mass spectrometry (LC-MS/MS) or LC-high resolution tandem mass spectrometry (LC-HRMS(/MS). Published methods include two-dimensional gas chromatography negative chemical ionization mass spectrometry (2D-GC-MS),[12,13] LC-MS/MS,[14–19] and gas chromatography-tandem mass spectrometry (GC-MS/MS)[20,21] assays. Some of these methods include only THCCOOH,[18,21] THC and THCCOOH,[15–17] and/or require derivatization.[12,16,17] Other methods include THCCOOH, but with limits of quantification (LOQs) >80 ng/L that are too high for identifying most smoked cannabis intake.[22–25] Furthermore, only one published method hydrolyzed THCCOOH-glucuronide, which approximately doubled THCCOOH concentrations, thereby enabling wider windows for detecting cannabis intake.[16] Minor cannabinoids, cannabidiol (CBD) and cannabinol (CBN), were previously suggested for documenting recent cannabis consumption, helping differentiate recent intake from residual cannabinoid excretion in frequent cannabis smokers.[7,9,26] However, the inclusion of these analytes adds further analytical challenges and few methods also included CBD and CBN.[12,14,19] To the best of our knowledge, the minor cannabinoids tetrahydrocannabivarin (THCV, the propyl analogue of THC) and cannabigerol (CBG, a biosynthetic precursor of THC and CBD) have not been evaluated in OF. Therefore, inclusion of these analytes in analytical methods for OF cannabinoids could assist interpretation. The analytical challenges of achieving a low 15 ng/L LOQ for THCCOOH, the lack of time-efficient methods for quantification of THCCOOH at biologically relevant concentrations, and the advantages of including minor cannabinoids to improve interpretation of cannabinoid OF results, led us to develop and validate a simple and rapid quantitative method for OF THC, 11-OH-THC, THCCOOH, THCV, CBD, and CBG analysis. Sample preparation included hydrolysis and solid-phase extraction (SPE) before LC-MS/MS. This assay will be useful for OF cannabinoid analysis for workplace, drug treatment, pain management, and criminal justice and driving under the influence of cannabis testing.
Material and methods Reagents and supplies THC, 11-OH-THC, THCCOOH, CBD, d3-THC, d3-11-OH-THC, d9THCCOOH, and d3-CBD standards were purchased from Cerilliant (Round Rock, TX, USA). CBG was acquired from Restek (Bellefonte, PA, USA) and THCV from RTI International (Research Triangle Park, NC, USA). Ammonium acetate, formic acid, and acetonitrile (LCMS grade) were obtained from Sigma-Aldrich (St Louis, MO, USA). Methanol, LC-MS grade water (for mobile phase A), isopropanol, methylene chloride, ammonium hydroxide, hydrochloric acid, and glacial acetic acid were purchased from Fisher Scientific (Fair Lawn, NY, USA). All solvents were high performance liquid chromatography (HPLC) grade unless otherwise stated. Water (for all purposes except mobile phase A) was purified in house with an ELGA Purelab Ultra Analytical purifier (Siemens Water Technologies, Lowell, MA, USA). Red abalone beta-glucuronidase solution containing 100 000 units/mL beta-glucuronidase was diluted with distilled water to contain 15, 625 units/mL beta-glucuronidase activity for enzymatic hydrolysis (Kura Biotech, Inglewood, CA, USA). Strata X-C columns (3 mL/30 mg, Phenomenex Inc., Torrance, CA, USA) were utilized for SPE. Specimens were extracted on a Cerex System 48 positive pressure manifold (SPEware Corp., Baldwin Park, CA,
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USA). Chromatography was performed with a United Chemical Technologies Selectra PFPP column (100 × 2.1 mm, 3 μm particle size) combined with a matched guard column (10 × 2.1 mm, 3 μm particle size) (Bristol, PA, USA). Quantisal buffer and OF collection devices were from Immunalysis Corp (Pomona, CA, USA). Instrumentation Tandem mass spectrometry was performed on an ABSciex 6500 QTRAPW triple quadrupole/linear ion trap mass spectrometer with an IonDrive™ Turbo V source (ABSciex, Foster City, CA, USA). The HPLC system consisted of a DGU-20A3r degasser, LC-20ADxr pumps, SIL-20ACxr autosampler, and a CTO-20A column oven (Shimadzu Corp, Columbia, MD, USA). Data were acquired and analyzed with Analyst (version 1.6.2) and Multiquant (version 2.1), respectively. Calibrators, quality control, and internal standards Blank OF for calibrators and quality controls (QC) were obtained (via expectoration) anonymously from volunteers in our laboratory and was evaluated for absence of cannabinoids following our described method. Primary stock solutions containing individual cannabinoids were prepared at 10 μg/L in methanol. Mixed analyte calibrator solutions were prepared via dilution in methanol creating calibrators at 0.2, 0.5, 2, 5, 20, 50 and 100 μg/L for THC, at 0.2, 0.5, 2, 5, 20, and 50 μg/L for 11-OH-THC, THCV, CBD, and CBG, and 15, 37.5, 150, 375, 1500, and 3750 ng/L for THCCOOH when fortifying 25 μL standard solution into 0.25 mL blank OF. QC samples were prepared with reference standard solutions from different lot numbers than calibrators or with separate ampules than used for preparing calibrators. Mixed analyte QC solutions were prepared via dilution in methanol creating QC solutions at 0.6, 6, and 90 μg/L for THC, 0.6, 6, and 45 μg/L for 11-OHTHC, THCV, CBD, and CBG, and 45, 450, and 3375 ng/L for THCCOOH when fortifying 25 μL QC solution into 0.25 mL blank OF. Primary stock solutions of THC-d3, 11-OH-THC-d3, THCCOOH-d9, and CBD-d3 were diluted in methanol, producing a mixed internal standard solution of 5 μg/L for THC-d3, 11-OH-THC-d3, and CBD-d3 and 37.5 ng/L for THCCOOH-d9, when fortifying 25 μL internal standard solution into 0.25 mL blank OF. No commercially available deuterated internal standards were available for THCV and CBG; CBD-d3 was the internal standard for these analytes. Hydrolysis Blank OF (250 μL) and 750 μL Quantisal buffer were aliquoted into 10-mL threaded glass centrifuge tubes prior to fortification with calibrator or QC solution and internal standard. Ammonium acetate buffer (1 M, pH 4, 0.3 mL) and 40 μL 15,625 U/mL beta glucuronidase solution (625 units) were added to each tube. Samples were capped, vortexed and incubated at 55 °C for 60 min. One mL authentic Quantisal specimen (containing 250 μL OF with 750 μL device buffer) was fortified with internal standard and 25 μL methanol before being processed identically along with calibrators and QCs. SPE Following hydrolysis, samples were acidified with 0.5 mL glacial acetic acid and vortexed. SPE columns were conditioned with 3 mL methanol, water, and 0.1% hydrochloric acid (HCl) before sample loading. Columns were washed with 2 mL water and 2 mL 0.1% HCl:acetonitrile (70:30, v/v). Columns were dried at 207 kPa for
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Quantification of cannabinoids in oral fluid 15 min. Analytes were eluted into clean 10 mL glass centrifuge tubes with 2 mL dichloromethane:isopropanol: ammonium hydroxide (78:20:2, v/v/v). Eluates were completely dried under nitrogen at 40 °C in a Zymark TurboVap. Residues were reconstituted in 150 μL mobile phase A/B (70:30), vortexed briefly before centrifugation at 1800 × g, 4 °C for 5 min. Samples were transferred to 1.5 mL polypropylene microcentrifuge tubes, centrifuged at 15,000 × g, 4 °C for 5 min and 125 μL was transferred to autosampler vials containing 350 μL glass inserts. Fifty μL was injected onto the LC-MS/ MS instrument.
LC-MS/MS Chromatographic separation was performed on a Selectra PFPP column via gradient elution with 0.15% formic acid in water (A) and 0.15% formic acid in acetonitrile (B). Flow rate was set at 0.5 mL/min, with an initial gradient of 30% A. Mobile phase B increased to 78.5% over 8.5 min, increased to 98% over 0.2 min and then held at 98% for 3 min. The column was re-equilibrated to 30% B over 0.2 min and held for 2.1 min (total run time 14 min). HPLC eluent was diverted to waste for the first 4 min and for the final 5 min of the analysis. The autosampler and column oven temperatures were 4 °C and 40 °C, respectively. All data were acquired via positive mode atmospheric pressure chemical ionization (APCI). MS/MS parameter settings were optimized via direct infusion of individual analytes (10 ng/mL in methanol: 10 mM ammonium acetate in water; 50:50 v/v) at 10 μL/min (Table 1). Optimized source settings were: gas-1 45, curtain gas 45, and source temperature 450 °C. Nebulizer current was set at 4.0 V for periods 1 (11-OH-THC and THCCOOH) and 3 (THC) and at 3.0 V for period 2 (THCV, CBD, and CBG). Nitrogen collision gas was set at medium for all periods. Quadrupoles one and three were set to unit resolution. Quantifier and qualifier ion transitions were monitored for each analyte and internal standard.
Data analysis Peak area ratios of analytes corresponding to internal standards were calculated for each concentration to construct daily calibration curves via linear least-squares regression with a 1/x2 weighting factor. Calibration curves were from 0.2–100 μg/L for THC, 0.2–50 μg/L for 11-OH-THC, THCV, CBD, and CBG, and 15–3750 ng/L for THCCOOH.
Method validation Specificity, sensitivity, linearity, imprecision, analytical recovery (bias), extraction efficiency, matrix effect, stability, dilution integrity, and carry-over were evaluated during method validation according to SWGTOX guidelines. Specificity Analyte peak identification criteria included relative retention time within ±0.1 min of the lowest calibrator and qualifier/quantifier transition peak area ratios within ±20% of mean calibrator ratios. Endogenous interferences were evaluated by analyzing 10 different OF blanks from 10 different individuals. Potential interference from commonly ingested drugs and medications were evaluated by fortifying drugs into low QC samples at a final interferent concentration of 200 μg/L (Table 2). No interference was noted if all analytes in the low QC quantified within ±20% of target concentrations with acceptable qualifier/quantifier transition peak area ratios. Individual cannabinoids also were fortified into low QC samples at final concentrations of 50 μg/L (THC, CBD, cannabinol, CBG, THCV, Δ9-tetrahydrocannabinolic acid A [THCAA]) or 1 μg/L (11-OH-THC, THCCOOH, carboxy-THCV) and into negative OF at THC concentrations of 1000, 2500, and 5000 μg/L. No interference was noted if all analytes in the low QC quantified within ±20% of target low QC concentrations with acceptable qualifier/quantifier transition peak area ratios. The potential degradation of CBD and
Table 1. Liquid chromatography-tandem mass spectrometry parameters for cannabinoids in human oral fluid Analyte THC THC-d3 11-OH-THC 11-OH-THC-d3 THCCOOH THCCOOH-d9 THCV CBD CBD-d3 CBG
Drug Test. Analysis (2014)
Q1 mass (amu)
Q3 mass (amu)
Dwell (ms)
Declustering potential (V)
Entrance potential (V)
Collision energy (V)
Cell exit potential (V)
Retention Time (min)
315.0 315.0 318.1 318.1 331.0 331.0 334.1 334.1 345.0 345.0 354.0 354.0 287.0 287.0 315.0 315.0 318.0 318.0 317.1 317.1
193.0 123.1 196.1 123.0 193.0 201.0 196.1 201.1 299.2 193.1 308.2 196.0 165.1 123.0 193.0 123.0 196.1 123.0 193.1 122.9
25 25 25 25 20 20 20 20 20 20 20 20 35 35 35 35 35 35 35 35
66 66 31 31 36 36 51 51 56 56 66 66 46 46 51 51 26 26 36 36
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
31 43 33 45 33 33 35 33 27 37 29 37 31 41 29 41 29 43 23 45
10 8 14 8 10 14 12 10 14 12 14 10 14 6 10 6 10 10 6 14
7.92
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7.91 5.89 5.89 6.05 6.04 7.05 7.22 7.22 7.30
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morphine normorphine morphine-3-beta-D-glucuronide morphine-6-beta-D-glucuronide codeine norcodeine 6-acetylmorphine 6-acetylecodeine buprenorphine norbuprenorphine hydrocodone hydromorphone
oxycodone noroxycodone oxymorphone nor-oxymorphone methadone 2-ethyl-5-methyl-3,3-diphenyl1-pyrroline 2-ethylidene-1,5-dimethyl-3,3diphenylpyrrolidine propoxyphene pentazocine
m-hydroxybenzoylecgonine p-hydroxybenzoylecgonine
Opioids
cocaine benzoylecgonine norcocaine norbenzoylecgonine ecgonine ethyl ester ecgonine methyl ester anhydroecgonine methyl ester ecgonine cocaethylene norcocatheylene m-hydroxycocaine p-hydroxycocaine
Cocaine
clonazepam flurazepam
diazepam lorazepam oxazepam alprazolam 7-aminoclonazepam 7-aminoflunitrazepam 7-aminonitrazepam nitrazepam flunitrazepam temazepam nordiazepam bromazepam
Benzodiazepines
Table 2. List of exogenous interferences evaluated at 200 μg/L in oral fluid during method validation
imipramine clomipramine fluoxetine norfluoxetine paroxetine
Antidepressants
Published 2014. This article is a U.S. Government work and is in the public domain in the USA. phentermine
phenylpropanolamine
para-methoxyamphetamine para-methoxymethylamphetamine methamphetamine amphetamine hydroxyl-methamphetamine hydroxyl-amphetamine 4-hydroxy-3-methoxymethamphetamine 4-hydroxy-3-methoxyamphetamine 3,4-methylenedioxyamphetamine 3,4-methylenedioxymethamphetamine 3,4-methylenedioxyphenyl-2-butanamine methyl-1-(3,4-methylenedioxyphenyl)2-butanamine R-cathinone ethylamphetamine 4-bromo-2,5-dimethoxyphenethylamine fenfluramine ephedrine pseudoephedrine
Amphetamines and other amines
norcotinine hydroxycotinine
clonidine ibuprofen caffeine diphenhydramine chlorpheniramine bromopheniramine Aspirin Tylenol Ketamine dextromethorphan Nicotine Cotinine
Others
Drug Testing and Analysis N. A. Desrosiers, K. B. Scheidweiler and M. A. Huestis
Drug Test. Analysis (2014)
Drug Testing and Analysis
Quantification of cannabinoids in oral fluid THCAA to THC under acidic conditions was evaluated by fortifying 50 μg/L CBD or THCAA and internal standard into blank OF. Sensitivity and linearity Limits of detection (LODs) and LOQs were evaluated over three days with duplicates from three different OF sources. LOD was defined as the lowest concentration producing a peak eluting within ±0.1 min of analyte retention time for the lowest calibrator, Gaussian peak shape, qualifier/quantifier transition peak area ratios ±20% of mean calibrator transitions for all replicates, and software-determined signal-to-noise ratio ≥3. LOQ was defined as the lowest concentration to meet the LOD criteria, measured within ±20% of target concentrations and software-determined signal-tonoise ratio ≥10. With these criteria, LOD could equal LOQ. Linearity was preliminarily assessed with five sets of calibrators to determine the best calibration model by comparing goodness of fit for unweighted linear least squares and linear least squares employing 1/x or 1/x2. Calibration curves were fit by linear least squares regression with six concentrations across the linear dynamic range for each analyte (seven THC calibrators). Calibrators were required to quantify within ±15%, except at the LOQ, which was required to quantify within ±20%. Correlation coefficients (R2) were required to exceed 0.995. Analytical recovery and imprecision Intra-day analytical recovery (bias) and imprecision were evaluated with 4 replicates at 3 QC concentrations. Analytical recovery was determined by comparing the mean result for all analyses to the theoretical concentration (e.g. mean percent expected concentration). Imprecision was expressed as the percent coefficient of variation (%CV) of the calculated concentrations. Inter-day analytical recovery and imprecision were evaluated with 4 replicates at 3 QC concentrations over 5 days (n = 20). One-way analysis of variation (ANOVA) was conducted on low, medium, and high QCs to evaluate inter- and intra-day differences in analyte concentrations. Extraction efficiency and matrix effect Extraction efficiency and matrix effects were evaluated using three sets of samples for low and high QC, as described by Matuszewski et al. (n = 10 for each set).[27] In set A, OF from 10 individuals were fortified with analytes and internal standard prior to SPE. In set B, OF from 10 individuals were fortified with analytes and internal standard after SPE. In set C, elution solvent was fortified with analytes and internal standard. Extraction efficiency was calculated by dividing analyte mean peak areas of set A by set B and expressed as a percentage. Matrix effect was determined by dividing the mean analyte peak area of set B by set C; this was converted to a percentage and subtracted from 100 to represent the amount of signal suppressed or enhanced by the presence of matrix.
Dilution integrity Dilution integrity was evaluated by fortifying blank OF:buffer mixture (n = 3) to a final concentration of 7500 ng/L THCCOOH and 100 μg/L THC, 11-OH-THC, THCV, CBD, and CBG and diluting it with blank OF:buffer mixture to achieve a 1:20 (v/v) dilution. Internal standards were added and samples were extracted as described. Dilution integrity was maintained if specimens quantified within ±20% of expected diluted concentration. Carry-over Carry-over was evaluated by fortifying blank OF:buffer mixture to a final concentration of 1000 μg/L for THC or twice the upper LOQ (ULOQ) for all other analytes (n = 3). Negative OF samples followed each replicate. Samples could not meet LOD criteria to document absence of carry-over. Hydrolysis THC-glucuronide and THCCOOH-glucuronide were initially evaluated during method development with base hydrolysis and enzymatic hydrolysis with different pH buffers, times, temperatures, and amounts of enzyme. Hydrolysis was validated by fortifying THC-glucuronide and THCCOOH-glucuronide to final concentrations of 50 μg/L and 2000 ng/L, respectively, and processing samples as described herein. Samples fortified at the same concentrations but not undergoing hydrolysis also were extracted and analyzed to serve as controls. Proof of applicability OF was collected from one individual who participated in an Institutional Review Board-approved study evaluating the effects of inhaled cannabis containing 6.7% THC. Vapors of 500 mg cannabis were inhaled ad libitum by vaporization over 10 min via the VolcanoW Medic. OF was collected at baseline, 0.17, 1.4, 2.3, 3.3, 4.3, 5.3, 6.3, 7.3, and 8.3 h after inhalation with the Quantisal device. The samples were stored at 4 °C for 12 h in buffer to elute analytes from the pad, and transferred to polypropylene tubes stored at 4 °C until analysis.
Stability Stability was evaluated in blank OF:buffer mixture fortified with analytes of interest at low and high QC (n = 4 for each). Short-term stability was assessed in polypropylene cryotubes under various conditions: 16 h at room temperature, 72 h at 4 °C and after 3 freeze-thaw cycles. Internal standards were added immediately prior to extraction and concentrations were compared to freshly prepared QCs that were extracted along with stability samples. Analyte autosampler stability was evaluated by injecting low and high QC specimens 84 h after extraction and comparing concentrations to freshly prepared QCs.
Drug Test. Analysis (2014)
Figure 1. Extracted ion chromatograms of target analyte and deuterated internal standard quantifier MRMs, highlighting chromatographic resolution between our analytes.
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
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Drug Testing and Analysis
N. A. Desrosiers, K. B. Scheidweiler and M. A. Huestis Negative
LOQ
THC m/z 315 - 193
2.0e4
11-OH-THC m/z 331 - 193
8.00
8.40
3.0e3
THCCOOH m/z 345 - 299
8.00
8.40
5.90
6.4
0 5.55
0 7.60
8.00
8.40
6.0e3
5.90
5.91
3.0e3
6.4
5.90
1.0e4
0 5.55 3.0e4
5.90
6.4 6.05
6.05
5.0e3
5.0e3
0 5.55 4.0e4
THCV m/z 287 - 165
0 7.60 6.0e3
3.0e3
1.0e4
6.05
6.55
0 5.55 4.0e4
6.05
1.5e4
6.55
0 5.55 4.0e4
6.05
6.55 7.04
7.05 2.0e4
2.0e4
2.0e4
0 6.55 7.0e4
CBD m/z 315 - 193
1.0e6
1.0e4
1.0e4
0 5.55
7.05
7.55
0 6.55 7.0e4
7.05
7.55
3.5e4
3.5e4
0 6.55 7.0e4
7.05
7.55 7.22
3.5e4 7.22
0 6.60 1.4e5
CBG m/z 317 - 193
7.91
7.91
0 7.60 6.0e3
CPS
Authentic sample 2.0e6
2.0e4
7.15
7.60
0 6.60 1.4e5
7.15
7.60
0 6.60 1.4e5
7.15
7.60 7.28
7.0e4
0 6.75
7.29
7.0e4
7.18
7.60
0 6.75
7.18
7.60
7.0e4
0 6.75
7.18
7.60
Minutes Figure 2. Multiple reaction monitoring ion chromatograms for quantifier transitions from THC, 11-OH-THC, THCCOOH, THCV, CBD, and CBG from negative oral fluid, limit of quantification (LOQ), and authentic sample collected 5.3 h after vaporizing 500 mg of cannabis containing 6.7% THC. Concentrations were 19.2 μg/L for THC, 0.996. Analytical recovery and imprecision Analytical recovery and imprecision were evaluated at low, mid, and high concentrations across the linear dynamic range (Table 4). Analytical recovery ranged from 83.9 to 113.5% and 88.7 to 107.3% of expected concentrations for intra-day and inter-day recoveries, respectively. Imprecision ranged from 0.5 to 9.6% and 2.3 to 6.7% for intra-day and inter-day imprecision, respectively (Table 4). There were few significant effects of day on most analyte QC concentrations (F4,5 = 0.347-2.906, P > 0.05); however, there were 5 exceptions (F4,5 = 4.498-7.703, P < 0.05), including high THCCOOH, low 11-OH-THC, mid THCV, high THCV, and high CBG. Extraction efficiency and matrix effect
Sensitivity and linearity Table 3 details LOD, LOQ, linearity, and mean calibration results. LODs were 0.1 μg/L for THC, 11-OH-THC, THCV, CBD, and CBG,
Extraction efficiencies and matrix effects are presented in Table 5. Mean extraction efficiencies were 75.9–86.1% (n = 10) and mean matrix effects (% effect) were 8.4–99.4% (n = 10).
Table 3. Cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry limits of detection (LODs), limits of quantification (LOQs), linear ranges, and calibration results (n = 5) Analyte
Internal Standard
THC 11-OH-THC THCCOOH THCV CBD CBG
THC-d3 11-OH-THC-d3 THCCOOH-d9 CBD-d3 CBD-d3 CBD-d3
LOD
LOQ
Linear range
Y-intercept Mean ± SD
0.1 μg/L 0.1 μg/L 15 ng/L 0.1 μg/L 0.1 μg/L 0.1 μg/L
0.2 μg/L 0.2 μg/L 15 ng/L 0.2 μg/L 0.2 μg/L 0.2 μg/L
0.2-100 μg/L 0.2-50 μg/L 15-3750 ng/L 0.2-50 μg/L 0.2-50 μg/L 0.2-50 μg/L
4.4 × 10 ± 2.4 × 10 -2 -3 1.4 × 10 ± 4.4 × 10 -2 -3 2.7 × 10 ± 8.1 × 10 -3 -3 2.4 × 10 ± 2.6 × 10 -4 -3 4.1 × 10 ± 1.5 × 10 -3 -3 1.7 × 10 ± 8.2 × 10
-3
-3
2
Slope Mean ± SD -4
R (range) -6
2.0 × 10 ± 3.4 × 10 -4 -6 2.0 × 10 ± 4.2 × 10 -3 -4 4.5 × 10 ± 1.9 × 10 -4 -6 2.4 × 10 ± 6.6 × 10 -4 -6 2.1 × 10 ± 3.8 × 10 -4 -5 6.7 × 10 ± 1.6 × 10
0.997-1.000 0.999-1.000 0.996-0.999 0.997-1.000 0.997-1.000 0.997-1.000
Table 4. Analytical recovery (% target concentration) and imprecision (% coefficient of variation) data for cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry Analyte
THC
11-OH-THC
THCCOOH
THCV
CBD
CBG
Drug Test. Analysis (2014)
Concentration
Analytical Recovery (%)
Imprecision (%)
Intra-day
Inter-day
Intra-day
Inter-day
μg/L
N=4
N = 20
N=4
N = 20
0.6 6 90 0.6 6 45 45 ng/L 450 ng/L 3375 ng/L 0.6 6 45 0.6 6 45 0.6 6 45
103.5 101.7 102.1 102.7 104.8 98.8 90.4 92.5 89.9 106.7 104.4 100.3 108.6 105.9 102.4 106.6 102.5 99.4
100.9 100.8 102.4 103.1 104.4 101.4 88.7 91.6 90.8 106.6 105.8 104.2 107.3 106.6 104.6 106.0 105.4 103.7
1.9 2.4 0.5 1.3 2.6 2.3 2.3 2.9 2.9 4.6 1.4 0.9 4.2 1.0 0.6 4.4 1.5 1.7
4.5 3.8 2.3 5.1 3.8 3.2 5.4 6.7 3.5 4.8 4.8 5.3 3.2 3.7 2.7 3.7 4.4 3.9
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
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Drug Testing and Analysis
N. A. Desrosiers, K. B. Scheidweiler and M. A. Huestis
Table 5. Mean extraction efficiency and matrix effect for cannabinoids extracted from human oral fluid by solid-phase extraction Analyte
Extraction efficiency (n = 10)
THC THC-d3 11-OH-THC 11-OH-THC-d3 THCCOOH THCCOOH-d9 THCV CBD CBD-d3 CBG
Matrix effects (% difference, n = 10)
Low
High
Low
High
80.9% 79.7% 81.0% 78.9% 86.1% 81.1% 77.6% 79.9% 78.6% 78.2%
78.3% 80.9% 78.8% 79.0% 80.8% 79.2% 77.2% 78.1% 79.4% 75.9%
20.9% 20.0% 31.6% 25.3% 67.4% 64.0% 30.2% 40.0% 41.4% 99.4%
11.6% 11.0% 8.4% 10.4% 64.3% 70.7% 25.2% 33.3% 28.0% 75.3%
Stability, dilution integrity, and carry-over
Discussion
Stability was evaluated in oral fluid after 16 h at RT, 72 h at 4 °C and after three freeze-thaw cycles; autosampler stability was evaluated after extraction and storage for 84 h on the 4 °C autosampler. All concentrations were within ±12% of freshly prepared QC concentrations (N = 4). Dilution integrity was acceptable (within ±20% of target concentrations) for all analytes after diluting 1:20 with blank OF:buffer mixture. Analytes quantified within 82.1–89.9% of expected concentrations. There was no evidence of carry-over for any cannabinoid. Negative specimens injected after samples containing 1000 μg/L for THC or twice the ULOQ of other cannabinoids did not contain analyte peaks satisfying assay LOD criteria (n = 3).
We present an LC-MS/MS method for the simultaneous quantification of six cannabinoids and metabolites in OF. This novel LC-MS/ MS triple quadrupole method employs simple sample preparation without requiring derivatization, monitors THCCOOH at clinically relevant concentrations for documenting active cannabis consumption and includes minor cannabinoids that can improve interpretation of OF cannabinoid results. This method can be applied for cannabinoid analysis in workplace, pain management, drug treatment, criminal justice, and driving under the influence of cannabis testing. We quantified the minor cannabinoids THCV and CBG for the first time in OF, enabling future investigation of these new OF cannabinoid markers. Table 6 lists cannabinoid OF concentrations from a single participant following cannabis inhalation, highlighting the applicability of our new method. This method will be utilized in our upcoming clinical study examining cannabinoid disposition in OF following smoked, vaporized, and oral cannabis administration. During method development, we evaluated various sample preparation approaches and chromatography conditions for OF cannabinoid quantification. Evaluated SPE columns included BondElut Plexa (Aglient), Reprep C18 (Restek), Polychrom-THC (Cerex), Oasis Max (Waters), SLE + (Biotage), Strata-X (Phenomenex) and Strata-XC (Phenomenex), with different washing and elution conditions. While the Polychrom-THC successfully extracted all cannabinoids, LC backpressure and quadrupole charging became problematic, possibly due to the detergents in the OF collection buffer. Electrospray ionization in positive and negative mode and
Hydrolysis Hydrolysis efficiency was 67.7% for THC-glucuronide and 97.7% for THCCOOH-glucuronide.
Proof of applicability Ten OF samples collected after vaporized cannabis inhalation containing 6.7% (w/w) THC were analyzed for cannabinoids with the new method. Concentrations are presented in Table 6 and representative chromatograms are illustrated in Fig. 2.
Table 6. Cannabinoid concentrations in oral fluid following inhalation of 500 mg of vaporized cannabis containing 6.7% THC cannabis Sample 1 2 3 4 5 6 7 8 9 10
Time of collection (h since vaporization)
THC μg/L
11-OH-THC ng/L
THCCOOH ng/L
THCVμg/L
CBD μg/L
CBG μg/L
Baseline 0.15 1.4 2.3 3.3 4.3 5.3 6.3 7.3 8.2
13.4 3,126 317 96.3 67.3 8.5 19.2 11.3 3.4 7.6
200 0 0 346 351 0 0 226 0 0
175 381 203 243 180 61.6 136 126 41 135
0 32.8 4.7 1.4 1.2 0 0.3 0 0 0
0 120 16.8 8.6 3.9 0.4 0.8 0.5 0.3 0.3
0.31 115 14.3 6.6 3.1 0.3 0.5 0.3 0 0.2
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Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Drug Test. Analysis (2014)
Drug Testing and Analysis
Quantification of cannabinoids in oral fluid APCI in negative mode were evaluated along with various mobile phases; however, APCI in positive mode with an acidic mobile phase provided optimal THCCOOH sensitivity with reduced matrix interferences. LC columns evaluated included Kinetex C18 (Phenomenex), Raptor Biphenyl (Restek), and the PFPP (UCT) with different mobile phases and gradients. While we achieved the desired LOQ with the Kinetex C18 column with fortified OF-buffer samples, authentic samples had THCCOOH interferences not present in calibrators or QCs, likely from pyrolysis products from cannabis smoke or other cannabinoids. The PFPP LC column from UCT was selected for optimal chromatography, as it minimized chromatographic interferences observed in authentic samples. The authentic specimen chromatogram (Fig. 2) depicts the worst resolution obtained between THCCOOH and the preceding interfering peak observed in all calibrators, QCs and authentic specimens. The co-eluting peak was more resolved in all other chromatograms compared to the included chromatogram. It should be noted that calibrators, QCs and authentic specimens were computer integrated with identical parameters. Therefore, while an interference eluted just before THCCOOH, all calibrators and QCs fulfilled MRM ratio, accuracy and imprecision criteria. We observed >88.7% inter-day analytical recovery (bias) and 75.9%. Matrix effects ranged from 8.4 to 99.4%, but deuterated internal standards compensated for matrix effects enabling QC quantification within ±20% for all analytes. We employed CBD-d3 as internal standard for CBG and THCV for which matched deuterated internal standards were unavailable. CBD-d3 was selected as the internal standard for these analytes because they had similar SPE efficiencies and eluted closest to these analytes. Matrix effects were similar between CBD-d3 and THCV, but CBG had higher % signal suppressed. Nevertheless, all THCV and CBG QCs fulfilled analytical recovery (bias) and imprecision criteria throughout validation. Dilution integrity and analyte stability were acceptable for all analytes. No exogenous interferences were noted; however, THCV at 50 μg/L produced 0.6 and 0.7 μg/L CBD and CBG. Given that CBD and CBG also were present in neat standard solutions, these are thought to be an impurity in the standard. Certificate of analysis for THCV indicated that purity was at 94.8 ± 0.12%, possibly explaining the presence of CBG and CBG in these standards. THC, CBD and CBG concentrations were 0.1, 1.5, and 1.3% of fortified THCV concentrations, respectively. Finally, THCCOOH-glucuronide hydrolysis to THCCOOH via Red Abalone glucuronidase neared completion (97.7%). CBD conversion to THC is negligible, as OF THC concentrations are generally >10x the CBD concentrations following cannabis inhalation (see table 6) or smoking,[7,9] mirroring the relative concentrations of these drugs in the cannabis plant. Therefore, any additional THC from CBD conversion would not be clinically significant, as this would represent
