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Anal Bioanal Chem. Author manuscript; available in PMC 2014 Jul 1.
Published in final edited form as:
Anal Bioanal Chem. 2013 Jul; 405(18): 6019–6027.
Published online 2013 May 17. doi:10.1007/s00216-013-7027-6
PMCID: PMC3773502
NIHMSID: NIHMS487497
PMID: 23681203
Karl B. Scheidweiler, Sarah K. Himes, Xiaohong Chen, Hua-Fen Liu, and Marilyn A. Huestis
Author information Copyright and License information PMC Disclaimer
The publisher's final edited version of this article is available at Anal Bioanal Chem
Abstract
Currently, Δ9-tetrahydrocannabinol (THC) is the analyte quantifiedfor oral fluid cannabinoid monitoring. The potential for false-positive oralfluid cannabinoid results from passive exposure to THC-laden cannabis smokeraises concerns for this promising new monitoring technology. Oral fluid11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) is proposed as amarker of cannabis intake since it is not present in cannabis smoke and was notmeasureable in oral fluid collected from subjects passively exposed to cannabis.THCCOOH concentrations are in the picogram per milliliter range in oral fluidand pose considerable analytical challenges. A liquidchromatography–tandem mass spectrometry (LCMSMS) method was developed andvalidated for quantifying THCCOOH in 1 mL Quantisal-collected oral fluid. Aftersolid phase extraction, chromatography was performed on a Kinetex C18 columnwith a gradient of 0.01 % acetic acid in water and 0.01 % acetic acid inmethanol with a 0.5-mL/min flow rate. THCCOOH was monitored in negative modeelectrospray ionization and multiple reaction monitoring mass spectrometry. TheTHCCOOH linear range was 12–1,020 pg/mL(R2>0.995). Mean extraction efficiencies andmatrix effects evaluated at low and high quality control (QC) concentrationswere 40.8–65.1 and −2.4–11.5 %, respectively(n=10). Analytical recoveries (bias) and total imprecisionat low, mid, and high QCs were 85.0–113.3 and 6.6–8.4 %coefficient of variation, respectively (n=20). This is thefirst oral fluid THCCOOH LCMSMS triple quadrupole method not requiringderivatization to achieve a <15 pg/mL limit of quantification. The assayis applicable for the workplace, driving under the influence of drugs, drugtreatment, and pain management testing.
Keywords: Cannabinoids, Carboxy THC, Oral fluid, Metabolites, Analytical method, LCMSMS
Introduction
Oral fluid testing is a useful monitoring tool for driving under theinfluence of cannabis and workplace, drug treatment, and parolee programs [1, 2].Currently, as a result of growing interest, the Substance Abuse and Mental HealthServices Administration (SAMHSA) is considering oral fluid testing guidelines forfederally mandated workplace drug testing. Although SAMHSA guidelines are not yetapproved, oral fluid testing in the US nonregulated sector is rapidly growing, andoral fluid testing programs are firmly in place in Australia and Europe [1, 3].
Oral fluid can be collected under direct observation without requiringsame-sex observers and reduces opportunities for sample adulteration compared tourine collection [2]. Risks to analysts frominfectious disease exposure is lower than for blood, and the presence of the parentdrug in oral fluid might provide better correlation with pharmacodynamic effectsthan urine testing [2]. Numerous commerciallyavailable oral fluid collection devices are capable of accurately collectingstandardized oral fluid volumes. Most devices include a collection pad andproprietary buffer for recovering and stabilizing drugs during storage prior toanalysis.
Oral fluid poses analytical challenges with small specimen volumes and drugconcentrations that are much lower than for urine [4]. This is confounded when there are multiple drug classes presentrequiring multiple drug confirmation analyses [4]. Furthermore, most collection device buffers include surfactants thatcould cause matrix effect challenges for liquid chromatography–massspectrometry analysis [2, 5].
Cannabis is the most commonly abused drug of abuse [6] and is often present in drug treatment, pain management, andforensic and workplace cases. Δ9-Tetrahydrocannabinol (THC) is theprimary psychoactive component in cannabis and is metabolized via cytochrome P450 toseveral metabolites, most prominently 11-hydroxy-THC (11-OH-THC) and11-nor-9-carboxy-THC (THCCOOH) [7, 8]. The conditions under which exposure tocannabis smoke can produce false-positive oral fluid THC results and for how longare still unclear. Parent THC predominates in oral fluid following controlled smokedcannabis administration [9-11] and was found in oral fluid during passiveexposure studies [12-14], but no THCCOOH was found in oral fluid collected fromnonsmokers 0.3–22 h after 3 h exposure to smoke from multiple cannabissmokers in a Groningen café [14].THCCOOH exceeded 7.5 pg/mL for up to 29 days in chronic, daily cannabis smokersduring sustained, monitored abstinence providing a longer detection window than THC[15]. Therefore, monitoring THCCOOH hasbeen proposed to minimize false-positive oral fluid results possibly caused bypassive cannabis exposure while providing effective detection of cannabis smoking[14]. THCCOOH also documents oral THCadministration with THCCOOH detected in oral fluid for at least 10.5 h after 15 mgoral THC, while THC was undetectable in most participants [16]; THC was only detected in 20.7 % of specimens and THCCOOHwas present in 98.2 % of specimens during 37 around-the-clock oral THCadministrations [17].
Monitoring picogram per milliliter THCCOOH concentrations in oral fluidpresents an analytical sensitivity challenge requiring two-dimensional gaschromatography negative chemical ionization mass spectrometry (2D-GCMS) [18, 19],gas chromatography—tandem mass spectrometry (GCMSMS) [20], or liquid chromatography–tandem mass spectrometryanalysis (LCMSMS) [21-23]. Lee et al. and Coulter et al. employed chemicalderivatization with dansyl chloride or triphenylphosphine and 2-picolylamine,respectively, prior to LCMSMS, achieving oral fluid limits of quantification (LOQ)of 5 and 10 pg/mL, respectively [21, 22]. He et al. employed drydown,reconstitution, and ultrafiltration (DRUF) prior to online trapping and microflowliquid chromatography with high-resolution Orbitrap MS achieving a THCCOOH LOQ of7.5 pg/mL [23]. Our aim was to develop andvalidate a simple, rapid, and robust method via traditional liquidchromatography–triple quadrupole mass spectrometry without derivatizationthat was capable of high-throughput picogram per milliliter THCCOOH quantificationin oral fluid. This assay is applicable for workplace, drug treatment, painmanagement, and forensic testing.
Methods
Reagents and supplies
THCCOOH and THCCOOH-d9 were purchased from Cerilliant (RoundRock, TX, USA). Acetonitrile, hexane, and ethyl acetate were obtained fromSigma-Aldrich (St. Louis, MO, USA). Methanol and acetic acid were acquired fromFisher Scientific (Fair Lawn, NJ). Water was purified in house with an ELGAPurelab Ultra Analytic purifier (Siemens Water Technologies, Lowell, MA, USA).All solvents were HPLC grade or better. Strata X-C columns (3 mL/30 mg,Phenomenex Inc, Torrance, CA, USA) were utilized for preparing samples.Specimens were extracted on a Cerex System 48 positive pressure manifold(SPEware Corp, Baldwin Park, CA, USA). Analytical chromatography was performedon a Kinetex C18 column (50×2.1 mm; 2.6 μm particle size) combinedwith a KrudKatcher Ultra frit purchased from Phenomenex. Quantisal™ oralfluid collection devices were from Immunalysis Corp. (Pomona, CA, USA). Oralsynthetic THC, Marinol®, was from Unimed Pharmaceuticals (Marietta, GA,USA).
Instrumentation
Tandem mass spectrometry was performed on an ABSciex 5500 QTrap®triple quadrupole/linear ion trap mass spectrometer with a TurboIonSpray source(ABSciex, Foster City, CA, USA). The high-performance liquid chromatography(HPLC) system consisted of a DGU-20A3 degasser, LC-20ADxr pumps, SIL-20ACxrautosampler, and a CTO-20 column oven (Shimadzu Corp, Columbia, MD, USA). Datawere acquired and analyzed with Analyst software version 1.6.1.
Calibrators, quality control, and internal standards
Blank oral fluid for preparation of calibrators and quality controls wascollected anonymously via expectoration from volunteers in our laboratory andwas evaluated with the methodology detailed in this manuscript to ensure absenceof detectable THCCOOH prior to fortification with working stock solutions toprepare calibrators and quality control samples.
Primary stock solution containing THCCOOH at 10 μg/mL wasprepared in methanol. Dilutions of the stock solution created calibrators at 12,30, 90, 360, 720, and 1,020 pg/mL when fortifying 25 μL standard solutioninto 250 μL of blank oral fluid. All sample concentrations are expressedas picogram per milliliter neat oral fluid throughout this manuscript.
Quality control (QC) samples were prepared with different lot numbers ofreference standard solutions than calibrators. 360, 2250 and 9000 pg/mL QCsolutions were prepared in methanol. QC samples were prepared by adding workingsolutions to 0.25 mL blank oral fluid to yield 36, 225, and 900 pg/mLTHCCOOH.
Primary stock solutions of THCCOOH-d9 were diluted inmethanol, producing a mixed internal standard working solution of 900 pg/mL.Fifty microliters internal standard working solution was added to 250 μLoral fluid yielding 180 pg/mL THCCOOH-d9. All primary and workingsolutions were stored at −20 °C in amber glass vials.
Solid phase extraction
Two hundred fifty microliters of blank oral fluid and 750 μLQuantisal buffer were aliquoted into a glass 13×100-mm test tube prior tofortification with native calibrator or control stock solution and internalstandard. Glacial acetic acid, 0.5 mL, was added to each specimen beforevortexing. Solid phase extraction (SPE) columns were conditioned with 3 mLmethanol, water, and 0.1 % hydrochloric acid in water prior to application ofprepared samples. Columns were washed with 2 mL water and 2 mL 0.1 %hydrochloric acid in water/acetonitrile (70:30v/v). Columns were dried at 207 kPA for 10min prior to eluting analytes into clean glass centrifuge tubes with 2 mL ofhexane/ethyl acetate/glacial acetic acid (78:20:2v/v/v). Eluates weredried completely under nitrogen at 40 °C in a Zymark TurboVap. Residueswere reconstituted in 150 μL mobile phase A/B (50:50v/v), vortexed briefly beforecentrifugation at 2,000×g, 4 °C for 5 min, andtransferred to autosampler vials containing 200 μL glass inserts. Fiftymicroliters was injected onto the LCMSMS instrument.
LCMSMS
Chromatographic separation was performed on a Kinetex C18 columnequipped with a KrudKatcher Ultra frit. Gradient elution was performed with (A)0.01 % acetic acid in water and (B) 0.01 % acetic acid in methanol at a flowrate of 0.5 mL/min. The initial gradient conditions were 20 % B, held for 1 min,then increased to 60 % B at 1.5 min and increased to 98 % B over 2 min.Ninety-eight percent of B was maintained for 3.5 min, at which time the columnwas reequilibrated to 20 % B over 0.1 min and held for 1.9 min (total runtime, 9min). Flow rate was increased to 1.0 mL/min at 3.7 to 7.2 min to increase columnrinsing efficiency. HPLC eluent was diverted to waste for the first 2.0 min andthe final 4 min of analysis. The column oven and autosampler were maintained at40 and 4 °C, respectively. Mass spectrometric data were collected vianegative mode electrospray ionization (ESI). MS/MS parameter settings (Table 1) were optimized via direct infusionof individual analytes (10 ng/mL in methanol) at 10 μL/min. Optimizedsource parameters were: gas-1, 55; gas-2, 55; curtain gas, 45; sourcetemperature, 750 °C. Nitrogen collision gas was set at medium for allexperiments. Quadrupoles one and three were set to unit resolution. Quantifierand qualifier ion transitions were monitored for THCCOOH andTHCCOOH-d9.
Table 1
Liquid chromatography–tandem mass spectrometry parameters for THCCOOH inoral fluid
| Analyte | Q1 mass (amu) | Q3 mass (amu) | Dwell time (ms) | Declustering potential (V) | Entrance potential (V) | Collision energy (V) | Cell exit potential (V) | Retention time (min) |
|---|---|---|---|---|---|---|---|---|
| THCCOOH | 342.9 | 245.2 | 20 | −140 | −10 | −38 | −21 | 3.53 |
| 342.9 | 191.0 | 20 | −140 | −10 | −42 | −21 | ||
| THCCOOH-d9 | 351.9 | 254.2 | 20 | −160 | −10 | −38 | −26 | 3.51 |
| 351.9 | 194.0 | 20 | −160 | −10 | −51 | −13 |
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Bold masses depict quantification transitions
Q1 quadrupole 1, Q3 quadrupole 3
Data analysis
Peak area ratios of THCCOOH to THCCOOH-d9 were calculated foreach concentration to construct daily calibration curves via linear leastsquares regression with a 1/x2 weighting factor.THCCOOH calibration curves were linear from 12 to 1,020 pg/mL.
Method validation
Specificity, sensitivity, linearity, imprecision, analytical recovery,extraction efficiency, matrix effect, stability, dilution integrity, andcarry-over were evaluated during method validation.
Specificity
Analyte peak identification criteria were relative retention time within±0.1 min of the lowest calibrator and qualifier/quantifier transitionpeak area ratios ±20 % of mean calibrator transition ratios. Potentialendogenous interferences were assessed by analyzing 12 oral fluid specimens fromdifferent individuals. In addition, potential interferences from commonly useddrugs were evaluated by fortifying drugs into low QC samples prepared along withcalibrators in neat solutions. Final interferent concentrations were 1,000 ng/mLcocaine, benzoylecgonine, norcocaine, norbenzoylecgonine, ecgonine ethyl ester,ecgonine methyl ester, ecgonine, anhydroecgonine methyl ester,m-hydroxycocaine, p-hydroxycocaine,m-hydroxybenzoylecgonine,p-hydroxybenzoylecgonine, morphine, normorphine,morphine-3-beta-D-glucuronide,morphine-6-beta-D-glucuronide, codeine, norcodeine, 6-acetylmorphine,6-acetylcodeine, buprenorphine, norbuprenorphine, methadone,2-ethyl-5-methyl-3,3-diphenylpyrroline,2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, hydrocodone, hydromorphone,oxycodone, diazepam, lorazepam, oxazepam, alprazolam, amphetamine,methamphetamine, 3,4-methylenedioxymethamphetamine,3,4-methylenedioxyamphetamine, clonidine, ibuprofen, pentazocine, caffeine,aspirin, acetaminophen, phencyclidine, nicotine, cotinine, and norcotinine. Nointerference was noted if all analytes in the low QC sample quantified within±20 % of target concentrations with acceptable qualifier/quantifiertransition ratios.
Sensitivity and linearity
Limit of detection (LOD) was evaluated in triplicate experiments withduplicates from three different oral fluid sources and defined as the lowestconcentration producing a peak eluting within ±0.1 min of analyteretention time for the lowest calibrator, Gaussian peak shape, andqualifier/quantifier transition peak area ratios ±20 % of mean calibratortransition ratios for all replicates. Limit of quantification (LOQ) also wasevaluated in triplicate experiments with duplicates from three different oralfluid sources and defined as the lowest concentration that met LOD criteria andmeasured concentration within ±20 % of target. Performance at the LOQ wasconfirmed in each batch of specimens.
Preliminary experiments with five sets of calibrators determined themost appropriate calibration model comparing goodness of fit for unweightedlinear least squares, linear least squares employing 1/x and1/x2 weighting. Calibration curves were fit bylinear least squares regression with six concentrations across the lineardynamic range for THCCOOH. Calibrators were required to quantify within±15 %, except ±20 % at LOQ, and correlation coefficients(R2) were required to exceed 0.995.
Analytical recovery and imprecision
Intra-day and inter-day analytical recovery (bias) and imprecision weredetermined from four replicates at three different QC concentrations across thelinear dynamic range of the assay. Analytical recovery was determined bycomparing the mean result for all analyses to the nominal concentration value(i.e., mean percent expected concentration). Inter-day imprecision andanalytical recovery were evaluated on five different runs with four replicatesin each run, analyzed on five separate days (n=20). Imprecisionwas expressed as percent coefficient of variation (% CV) of the calculatedconcentrations. The guidelines detailed by Krouwer and Rabinowitz [24] were employed to calculate pooledintra-day, inter-day, and total imprecision.
Extraction efficiency and matrix effect
Extraction efficiency and matrix effect were evaluated via three sets ofsamples as described by Matuszewski et al. (n=10 for each set)[25]. In the first set, oral fluidsamples from ten individuals were fortified with analytes and internal standardsprior to SPE. In set 2, oral fluid samples from ten individuals were fortifiedwith analytes and internal standards after SPE, and the third set containedanalytes and internal standards in mobile phase. Extraction efficiency,expressed as a percentage, was calculated by dividing analyte mean peak areas ofset 1 by set 2. Absolute matrix effect was calculated by dividing the mean peakarea of the analyte in set 2 by the mean analyte area in set 3. The value wasconverted to a percentage and subtracted from 100 to represent the amount ofsignal suppressed by the presence of matrix. As an additional evaluation ofmatrix effect, ten blank oral fluid lots were fortified with low QC solution andinternal standard and were processed along with calibrators prepared using aseparate lot of blank oral fluid to verify accurate quantification.
Stability
Stability was evaluated with blank oral fluid fortified with analytes ofinterest at low and high QC concentrations (n=3). Short-termtemperature stability was evaluated for fortified oral fluid stored in the darkin polypropylene cryovials for 16 h at room temperature, 96 h at 4 °C,and after three freeze–thaw cycles at −20 °C. On the day ofanalysis, internal standard was added to each specimen and analyzed asdescribed. Autosampler stability was assessed by re-injecting QC specimens after72 h on the autosampler (4 °C) and comparing calculated concentrations tovalues obtained against the original calibration curve.
Dilution integrity
Dilution integrity was evaluated by diluting a fortified oral fluidsample (n = 3) containing 900 pg/mL THCCOOH in blank oralfluid/Quantisal buffer to achieve 1:5 (v/v)dilution. Internal standards were added and samples extracted as described.Dilution integrity was maintained if specimens quantified within ±20 % ofexpected diluted concentration.
Carry-over
Carry-over was investigated in triplicate by injecting extracted blankoral fluid samples containing internal standards immediately after samplescontaining target analytes at twice the ULOQ. Blank oral fluid specimeninjections could not meet LOD criteria to document absence of carry-over.
Clinical study
Oral fluid was collected with Quantisal devices from a single healthycannabis smoker that provided written informed consent to participate in aNational Institute on Drug Abuse Institutional Review Board-approved protocolcomparing Sativex oromucosal spray versus oral Marinol pharmaco*kinetics andpharmacodynamics following controlled administration. The participant resided ona secure research unit during the study. Oral fluid was collected with Quantisaldevices from −0.5 to 10.5 h after 5 and 15 mg Marinol oraladministration. The device collects 1.0±0.1 mL oral fluid with anabsorptive cellulose pad. Pads were placed into a plastic tube containing 3 mLelution/stabilization buffer for at least 24 h to elute drugs. The oralfluid/buffer mixture was decanted into Nunc® cryotubes and was stored at−20 °C until analysis. Participants were required to rinse theirmouth with water after eating during the study, and collection devices werevisually inspected after collection for presence of blood or other materialprior to placement in storage buffer.
Results
Evaluation of potential SPE elution solvents
Initial experiments using the validated SPE columns and procedure but 2mL 5 % ammonium hydroxide in methanol instead of hexane/ethyl acetate/aceticacid (78:20:2, v/v) elution solvent yieldedbetter THCCOOH recoveries (77.6–83.1 %, n=6) but morematrix suppression (−99.4 to −99.5 %, n=6) thanwe found with the validated approach (see Table2).
Table 2
Mean THCCOOH extraction efficiency and matrix effect for Quantisal oral fluiddevices, with authentic oral fluid fortified at 36 (low) and 900 (high) pg/mLconcentrations
| Analyte | Extractionefficiency (%, N=10) | Matrix effect (% ofsignal suppressed, N=10) | ||
|---|---|---|---|---|
| Low | High | Low | High | |
| THCCOOH | 44.6 | 64.5 | −2.4 | 10.8 |
| THCCOOH-d9 | 40.8 | 65.1 | −1.2 | 11.5 |
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Specificity
There were no interfering peaks in oral fluid from 12 cannabis-abstinentindividuals when mixed with device buffer. None of the 50 potential exogenousinterferences fortified at 1,000 ng/mL into neat low QC samples producedtransition ratio or quantification criteria failure except for 11-OH-THC andTHCCOOH-glucuronide which produced higher than expected QC concentrations.11-OH-THC and THCCOOH-glucuronide did not significantly alter low QCconcentrations (within 80–120 % of expected concentration) when fortifiedat 10 and 1 ng/mL, respectively. Multiple reaction monitoring ion chromatogramsfrom Quantisal-collected blank oral fluid, Quantisal-collected blank oral fluidfortified at the LOQ, and an authentic specimen collected after 5 mg Marinoloral administration are shown in Fig.1.
Fig. 1
Multiple reaction monitoring ion chromatograms for THCCOOH quantifier andqualifier transitions: from a blank oral fluid sample (A andB), blank oral fluid fortified at the 12 pg/mL limit ofquantification (C and D), and an authenticspecimen containing 26 pg/mL THCCOOH collected 0.25 h after 5 mg oral Marinol(E and F)
Sensitivity and linearity
Initial experiments were conducted with five sets of calibration curvesfit via unweighted linear least squares and linear least squares with1/x and 1/x2 weighting factorto identify the most appropriate calibration model. Inspection of residualsindicated linear least squares with 1/x2 weightingfactor produced the best fit for the calibration data. All correlationcoefficients exceeded 0.995.
LOD and LOQ were 9 and 12 pg/mL, respectively; THCCOOH linear range was12 to 1,020 pg/mL. Mean calibration curve slopes were 1.24 (SD=0.08),y intercepts were 0.02 (SD=0.02), and all correlationcoefficients (R2) exceeded 0.996.
Analytical recovery and imprecision
Analytical recovery and imprecision were evaluated at threeconcentrations across the linear dynamic range. Analytical recovery ranged from85.0 to 113.3 % of expected concentrations for intra-day and inter-dayanalytical recoveries (Table 3). Pooledintra-day, inter-day, and total imprecision were 4.1–6.6, 0–7.3,and 6.6–8.4 % CV, respectively (Table3).
Table 3
Analytical recovery and imprecision data for THCCOOH in oral fluid by liquidchromatography-tandem mass spectrometry
| Concentration (pg/mL) | Analytical recovery (% ofexpected concentration) | Imprecision (% coefficient ofvariation, N=20) | ||||||
|---|---|---|---|---|---|---|---|---|
| Intra-day, N=4 | Inter-day, N=20 | Pooled Intra-day | Inter-day | Total | ||||
| Mean | Range | Mean | Range | |||||
| THCCOOH | 36 | 96.3 | 87.8–110.8 | 97.1 | 85.0–110.8 | 6.6 | 0 | 6.6 |
| 225 | 97.2 | 91.1–104.9 | 100.4 | 89.3–112.4 | 4.1 | 5.7 | 7.0 | |
| 900 | 90.9 | 88.6–96.9 | 98.2 | 87.2–113.3 | 4.2 | 7.3 | 8.4 | |
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Extraction efficiency and matrix effect
Extraction efficiencies and matrix effects for THCCOOH in oral fluid arepresented in Table 2. Mean extractionefficiencies were 44.6–64.5 %, and mean matrix effects (percentsuppressed signal) were −2.4 to 10.8 % (n=10, Table 2).
Stability, dilution integrity, and carry-over
Analytes at low and high QC concentrations were stable for 72 h at 4°C in the autosampler (Table 4).THCCOOH was stable for 16 h at room temperature, 96 h at 4 °C, and afterthree freeze/thaw cycles (Table 4).
Table 4
THCCOOH stability in Quantisal-collected oral fluid, fortified at 36 (low) and900 (high)pg/mL concentrations
| Analyte | 72 h autosampler (%difference, n=4) | 16 h room temperature (%difference, n=4) | 96 h, 4 °C (%difference, n=4) | 3 Freeze/thaw cycles (%difference, n=4) | ||||
|---|---|---|---|---|---|---|---|---|
| Low | High | Low | High | Low | High | Low | High | |
| THCCOOH | 6.1 | −8.7 | 4.7 | −0.5 | −18.9 | −14.5 | −16.0 | −12.3 |
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Dilution integrity was acceptable (within ±20 % of expecteddiluted concentration) after diluting 1:5 with blank oral fluid/device buffer.There was no evidence of carry-over; negative specimens injected after samplescontaining twice the ULOQ did not contain analyte peaks satisfying assay LODcriteria (n=3).
Proof of method
The method was applied to measurement of THCCOOH specimens collectedwith Quantisal devices after administering 5 (Fig.1) and 15 mg (Table 5) oralsynthetic THC (Marinol) to a single participant.
Table 5
THCCOOH concentrations in oral fluid collected from a single participant withQuantisal device after 15 mg oral synthetic THC (Marinol) administration. TheLOQ was 12 pg/mL
| Time since administration (h) | THCCOOH (pg/mL) |
|---|---|
| −0.5 | <LOQ |
| 0.3 | 29.1 |
| 4.5 | <LOQ |
| 7.5 | 55.5 |
| 10.5 | 27.2 |
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Discussion
A validated and sensitive LCMSMS method for quantifying THCCOOH in oralfluid is necessary for workplace, pain management, drug treatment, and forensic drugtesting programs. Monitoring THCCOOH is critical for distinguishing between passiveenvironmental exposure and active cannabis smoking. THC was identified in oral fluidafter passive cannabis exposure with concentrations from 0.3 to 1.2 [13] and 0.7 to 17 ng/mL [14]. THCCOOH was not present following passive cannabisexposure at an LOQ of 2 pg/mL [14]. Anotheradvantage of THCCOOH monitoring is that this analyte exceeded 7.5 pg/mL in oralfluid from abstinent chronic cannabis smokers for up to 29 days of sustainedmonitored abstinence [15]. Therefore, THCCOOHprovides adequate windows of detection for workplace, pain management, drugtreatment, and forensic applications, while distinguishing passive exposure fromactive cannabis intake. THCCOOH also provides more effective monitoring of oral THCadministration than THC since THCCOOH was detected in oral fluid for at least 10.5 hafter 15 mg oral THC, while THC was undetectable in most participants [16]. THCCOOH was present in 98.2 % of oralfluid samples collected during and after 37 oral 20 mg THC doses, and profiles weresimilar to THCCOOH plasma profiles; THC was only present in 20.7 % of oral fluidsamples, and concentrations actually decreased during oral THC dosing [17, 26].THCCOOH analytical sensitivity is a significant challenge for oral fluid testingsince median oral fluid concentrations were less than 100 pg/mL 1 h after smoking asingle 6.8 % THC cigarette [10, 11]. We present a validated method measuringTHCCOOH in oral fluid collected with Quantisal device achieving a linear range of12–1,020 pg/mL of neat oral fluid after solid phase extraction and directinjection onto an LCMSMS triple quadrupole instrument that will be useful forhigh-throughput drug treatment, pain management, and forensic and workplace testinglaboratories.
We demonstrated the utility of the method’s sensitivity and linearityby analyzing oral fluid specimens collected via Quantisal after administration of 15mg Marinol to a single participant (Table 5).It is interesting that THCCOOH was less than the method LOQ at 4.5 h but was 29.1and 55.1 pg/mL at 0.3 and 7.5 h, respectively, after Marinol administration. It isimportant to note that the data are from a single session for a single participant.These specimens from a single participant illustrate the usefulness of our method.Analysis of additional specimens from more participants should reveal whether adecreasing THCCOOH trend at 4.5 h after Marinol is significant.THCCOOH-d9 peak areas were consistent for all specimens, indicatingvariable SPE recovery is not confounding the participant’s THCCOOHconcentration profile. Variable analyte recovery from collection devices may alsoexplain variable THCCOOH concentrations. However, Moore et al. previously reportedconsistent 80 % THCCOOH recovery from Quantisal devices with an 8.2 % coefficient ofvariation (n=6) [18].
Most oral fluid collection devices employ elution/stabilization bufferscontaining detergents that can cause problematic matrix effects during LCMSMSanalysis [2, 5]. During method development, we achieved 77.6–83.1 % THCCOOHrecoveries but observed greater than 95 % matrix suppression, unacceptable accuracy,and imprecision with a 5 % ammonium hydroxide in methanol elution solvent. Selectinga hexane/ethyl acetate/acetic acid (78:20:2;v/v/v) elution solventproduced lower THCCOOH recoveries but yielded less matrix effect (−2.4 to10.8 %), accuracies within ±15 % of target concentration, and <10 %imprecision. We achieved our desired assay sensitivity despite lower recovery, andthe reduced matrix effect enabled acceptable THCCOOH accuracy and imprecision.
Two reports describe 2D-GCMS methods for THCCOOH in Quantisal-collected oralfluid with a specimen volume equivalent to 250 μL oral fluid, similar to ourmethod, achieving LOQs of 2 and 7.5 pg/mL [18, 19]. Day et al. presented aTHCCOOH GCMSMS method for 100 μL oral fluid collected via Intercept deviceachieving an LOQ of 10 pg/mL [20]. All threeGCMS methods employed solid phase extraction and derivatization with trifluoraceticacid [18, 19] or pentafluoroacetic acid anhydride [20] and hexafluoropropanol. Our THCCOOH LCMSMS assay LOQ is similar orslightly less sensitive than these GCMS methods using similar oral fluid specimenvolumes while affording time and cost savings by avoiding derivatization.
Lee et al. reported a LCMSMS method for THCCOOH in 250 μLexpectorated oral fluid that employed acetonitrile precipitation, derivatizationwith dansyl chloride, and liquid-liquid extraction prior to LC triple quadrupole MSachieving an LOQ of 5 pg/mL [22]. Coulter etal. recently reported a LCMSMS method for THCCOOH in a Quantisal-collected oralfluid with a specimen volume equivalent to 250 μL oral fluid, similar to ourcurrent method, employing solid phase extraction prior to derivatization withtriphenylphosphine and 2-picolylamine prior to LC triple quadrupole MS achieving a10 pg/mL LOQ [21]. Our current LCMSMS methodachieves similar LOQs with identical oral fluid specimen volumes while avoidingcostly derivatization required for these other two LCMSMS methods [21, 22].
He et al. describe a microflow LC high-resolution MS method for THCCOOH inOralEze-collected samples with a specimen volume including 133 μL oral fluidachieving a 7.5 pg/mL LOQ [23]. This methodemployed an elaborate DRUF sample pretreatment and online trapping prior to LCMSMSanalysis. Samples were dried down and reconstituted in 30 % methanol, centrifuged at17,000×g for 5 min, and filtered through hydrophilicPTFE filters before injection onto an aQ trapping column and ultimatelychromatographed on an aQ LC column with a total runtime of 12.5 min with dataacquired at 40,000 resolution during MSMS analysis [23]. Although this method achieves sensitivity appropriate formonitoring THCCOOH in oral fluid, it requires extensive sample pretreatment stepsthat are not amenable to automation and requires high-resolution instrumentationthat is cost prohibitive in many drug testing laboratories. Our current methodemploys solid phase extraction that could be automated and employs standard triplequadrupole MS instrumentation that is available in most oral fluid testinglaboratories.
Quintela et al. reported a THCCOOH high-resolution LCquadrupole-time-of-flight MS method using 167 μL Intercept device-collectedoral fluid [27]. This method achieved a 500pg/mL THCCOOH LOQ that is inadequate for cannabis oral fluid testing.
This is the first validated oral fluid THCCOOH LCMSMS method employingLCMSMS triple quadrupole instrumentation, not requiring derivatization to achieve anLOQ below 15 pg/mL. This method provides an approach appropriate for high-throughputoral fluid drug testing in routine workplace, pain management, drug treatment, andforensic testing laboratories. THCCOOH recoveries were greater than 41 % and matrixeffect less than 12 %. Intra- and inter-day accuracy were within ±15 %, withpooled intra-day, inter-day, and total imprecision better than 9.4 % CV. THCCOOHlinear range was 12-1,020 pg/mL, which is appropriate for monitoring oral fluidTHCCOOH since reports indicate THCCOOH oral fluid concentrations below 763 pg/mL[10, 11, 15, 18, 20, 22, 23,28]. This LCMSMS method provides a rapidand reliable means of differentiating passive environmental cannabis exposure fromactive cannabis intake.
Acknowledgments
This research was supported by the Intramural Research Program of the NationalInstitute on Drug Abuse, National Institutes of Health.
Contributor Information
Karl B. Scheidweiler, Chemistry and Drug Metabolism, Intramural Research Program,National Institute on Drug Abuse, National Institutes of Health, BiomedicalResearch Center, 251 Bayview Boulevard Suite 200 Room 05A-721, Baltimore, MD21224, USA.
Sarah K. Himes, Chemistry and Drug Metabolism, Intramural Research Program,National Institute on Drug Abuse, National Institutes of Health, BiomedicalResearch Center, 251 Bayview Boulevard Suite 200 Room 05A-721, Baltimore, MD21224, USA.
Xiaohong Chen, ABSciex, 353 Hatch Drive, Foster City, CA 94404, USA.
Hua-Fen Liu, ABSciex, 353 Hatch Drive, Foster City, CA 94404, USA.
Marilyn A. Huestis, Chemistry and Drug Metabolism, Intramural Research Program,National Institute on Drug Abuse, National Institutes of Health, BiomedicalResearch Center, 251 Bayview Boulevard Suite 200 Room 05A-721, Baltimore, MD21224, USA.
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