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Oral fluid (OF) is increasingly used for clinical, forensic and workplace drug testing as an alternative to urine. Uncertainties surrounding OF collection device performance, drug stability and testing reproducibility may be partially responsible for delays in the implementation of OF testing in regulated drug testing programs. Stability of Δ(9)-tetrahydrocannabinol (THC) fortified and authentic specimens was examined after routine collection, transport and laboratory testing. Acceptable recovery and stability were observed when THC-fortified OF (1.5 and 4.5 ng/mL) was applied to Oral-Eze devices. Neat OF samples collected with Oral-Eze, processed per the package insert, and fortified with THC (3 and 6 ng/mL) were stable (±20%) at room temperature (21-25°C), refrigerated (2-8°C) and frozen (-25 to -15°C) conditions up to 1 month, while samples collected with Intercept devices showed decreases at refrigerated and room temperatures. After long-term refrigerated or frozen storage, maximum reductions in THC concentrations were 42% for Oral-Eze and 69% for Intercept. After ≥1 year frozen storage, 80.7% of laboratory specimens positive for THC (3 ng/mL cut-off) by GC-MS were reconfirmed positive (within 25%), with an average THC decrease of 4.2%. Specimens (n = 47) processed with Oral-Eze (diluted) and tested via enzyme immunoassay were concordant with LC-MS-MS results and showed 100% sensitivity and 95% specificity. Paired specimens collected with Oral-Eze and Intercept exhibited 98% overall agreement between the immunoassay test systems. Collectively, these data demonstrate consistent and reproducible recovery and stability of THC in OF after collection, transport and laboratory testing using the Oral-Eze OF Collection System., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Δ⁹-Tetrahydrocannabinol (THC) is the illicit drug most frequently observed in accident and driving under the influence of drugs investigations. Whole blood is often the only available specimen collected during such investigations, yet few studies have examined relationships between cannabis effects and whole blood concentrations following cannabis smoking. Nine male and one female heavy, chronic cannabis smokers resided on a closed research unit and smoked ad libitum one 6.8% THC cannabis cigarette. THC, 11-hydroxy-THC and 11-nor-9-carboxy-THC were quantified in whole blood and plasma. Assessments were performed before and up to 6 h after smoking, including subjective [visual analog scales (VAS) and Likert scales], physiological (heart rate, blood pressure and respirations) and psychomotor (critical-tracking and divided-attention tasks) measures. THC significantly increased VAS responses and heart rate, with concentration-effect curves demonstrating counter-clockwise hysteresis. No significant differences were observed for critical-tracking or divided-attention task performance in this cohort of heavy, chronic cannabis smokers. The cannabis influence factor was not suitable for quantifying psychomotor impairment following cannabis consumption and was not precise enough to determine recent cannabis use with accuracy. These data inform our understanding of impairment and subjective effects following acute smoked cannabis and interpretation of whole blood cannabinoid concentrations in forensic investigations.

This paper describes the determination of tetrahydrocannabinol (THC) and its metabolite, 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid using solid-phase extraction and liquid chromatography with tandem mass spectral detection (LC-MS-MS) and its application to proficiency specimens. The method employs collection of oral fluid with the Quantisal™ device, base hydrolysis, solid-phase extraction and LC-MS-MS in positive ion electrospray mode. Because the concentration of the metabolite in oral fluid is quite low, extremely sensitive analytical methods are necessary. The requisite sensitivity was achieved by a simple, rapid derivatization of the compound after extraction. The derivatization conditions did not affect parent THC. The method was fully validated using standard parameters including linearity, sensitivity, accuracy, intra-day and inter-day imprecision, drug recovery from the collection pad, limit of quantitation, limit of detection and matrix effects. The procedure was applied to oral fluid proficiency specimens previously analyzed to assess the stability of THC-COOH.

Federal workplace drug testing was initiated during the late 1980s. Since then, numerous methods have been employed to subvert these drug tests, adulteration of urine samples being the most common. A wide variety of adulterants has been reported to date along with suitable methods of their detection. Recently, websites have claimed that zinc sulfate can be an effective adulterant to bypass drug testing. Herein, these claims are investigated using standard drug detection kits and urine samples adulterated with zinc. Drug-free urine samples were fortified with different amounts methamphetamines and benzoylecgonine, to which zinc sulfate was added to study its effect. Urine samples from acute marijuana smokers were also obtained in order to study the effects of zinc supplements on THC drug testing. All urine drug testing was performed using ELISA detection kits manufactured by Immunalysis. Both zinc sulfate and zinc supplements are effective in interfering with the detection of all three drugs by Immunalysis drug detection kits. Also, no suitable method could be established to detect zinc in urine samples. Zinc can be an effective adulterant in urine for some illicit drugs that are commonly screened under routine drug testing.

The present work describes the validation of a novel aqueous in situ derivatization procedure with trimethyloxonium tetrafluoroborate (TMO) as methylating agent for the simultaneous, quantitative analysis of Δ(9)-tetrahydrocannabinol (THC) and 11-nor-Δ(9)-tetrahydrocannabinol carboxylic acid (THC-COOH) in human urine. The derivatizing agent is directly added to the urine sample and the methyl-derivatives are then recovered by liquid-liquid extraction procedure. Gas chromatography-mass spectrometry was used to detect the derivatives in selected ion monitoring mode. The limits of detection were 0.7 ng/mL for THC and 0.5 ng/mL for THC-COOH, whereas the limits of quantification were 1.9 and 0.9 ng/mL, respectively. The method has been applied to 60 real samples both positive and negative to immunochemical screening test resulting to be very useful and reliable in routine analysis of THC-COOH in human urine for toxicological and forensic purposes.

In this study, solid-phase extraction (SPE) is described using a novel fluorinated [heptadecafluorotetrahydrodecyl (C(10)H(4)F(17))] phase to isolate THC and its primary metabolite carboxy-THC from whole blood samples. SPE was performed in hydrophobic mode after samples of whole blood were precipitated with acetonitrile. After applying the sample to the SPE column in aqueous phosphate buffer (pH 7), the sorbent was washed with deionized water and phosphate buffer (pH 7) and dried. The SPE column was eluted with a solvent consisting of ethyl acetate/hexanes (50:50) containing 2% acetic acid. The eluate was collected, evaporated to dryness, and dissolved in mobile phase (50 microL) for analysis by fast liquid chromatography-tandem mass spectrometry in positive/negative multiple reaction monitoring mode. Chromatography was performed in gradient mode employing a C(18) column and a mobile phase consisting of acetontitrile (containing 0.1% formic acid) and 0.1% aqueous formic acid. The total run time for each analysis was less than 5 min. The limits of detection/quantification for this method were determined to be 0.1 and 0.25 ng/mL, respectively. The method was found to be linear from 0.25 to 50 ng/mL (r(2) >or= 0.995). Recoveries of the individual cannabinoids were found to be greater than 85%. In this report, results of authentic samples analyzed for THC and carboxy-THC are reported using this new methodology.

To stabilize urinary solutions against adsorptive loss of metabolites of Delta9-tetrahydrocannabinol (THC), a non-ionic surfactant, Tergitol, was investigated to reduce the need for special handling and storage of such solutions. Addition of surfactant up to 20 times the critical micelle concentration (CMC) did not adversely affect the analytical process. Yet, at only two times CMC, surfactant was found to mitigate adsorptive loss of THC analytes under a variety of storage and handling conditions including exposure to glass and plastic surfaces, after storage in a refrigerator or freezer, and at reduced pH, where adsorptive losses were expected to be significant. On average, micellar solubilization of analyte increased the assayed concentration by 10% with a range of 3 to 20%, depending on condition, relative to solutions without surfactant. Solutions with surfactant did not fail (i.e., deviate in concentration by +/-20%) over a 49-week period, whereas those without surfactant failed by 21 weeks. These results indicate that addition of small amounts of non-ionic surfactant to solutions of urinary THC metabolites is a simple method to improve both the accuracy and precision of analyte concentrations, as determined by gas chromatography-mass spectrometry, in such solutions by mitigating adsorptive losses during storage and handling events.

A sensitive and specific method for extraction and quantification of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THCCOOH) in human urine was developed and fully validated. To ensure complete hydrolysis of conjugates and capture of total analyte content, urine samples were hydrolyzed by two methods in series. Initial hydrolysis was with Escherichia coli beta-glucuronidase (Type IX-A) followed by a second hydrolysis utilizing 10N NaOH. Specimens were adjusted to pH 5-6.5, treated with acetonitrile to precipitate protein, and centrifuged, and the supernatants were subjected to solid-phase extraction. Extracted analytes were derivatized with BSTFA and quantified by gas chromatography-mass spectrometry with electron impact ionization. Standard curves were linear from 2.5 to 300 ng/mL. Extraction efficiencies were 57.0-59.3% for THC, 68.3-75.5% for 11-OH-THC, and 71.5-79.7% for THCCOOH. Intra- and interassay precision across the linear range of the assay ranged from 0.1 to 4.3% and 2.6 to 7.4%, respectively. Accuracy was within 15% of target concentrations. This method was applied to the analysis of urine specimens collected from individuals participating in controlled administration cannabis studies, and it may be a useful analytical procedure for determining recency of cannabis use in forensic toxicology applications.

Oral fluid is considered to be an alternative to urine testing for the detection of acute ingestion of drugs. The OraSure Intercept DOA Oral Specimen Collection Device (OSCD) has been used in studies for the quantitation of Delta9-tetrahydrocannabinol (THC), but concerns have been raised. In the present study, we investigated whether the volume of oral fluid can be determined and how much THC remains adsorbed on the device. It was found that THC is markedly adsorbed onto the absorptive pad. The recovery using the standard elution procedure was only 37.8 +/- 9.4% for 10 ng/mL and 55.6 +/- 1.0% for 100 ng/mL of THC in oral fluid (n = 5 each). With an additional methanol wash, a further 25% could be eluted. Therefore, a modification of the procedure was evaluated, consisting of the addition of 2 mL of methanol to the elution buffer. THC could be completely recovered over a range of concentrations (1 to 1000 ng/mL). For the determination of the amount of oral fluid absorbed, a gravimetric approach was evaluated as the weights of the devices vary only by 0.6% relative standard deviation. After application of 0.5 mL oral fluid to pads and evaluation of the weight differences, the applied amount could be estimated with a precision of 7.5% (n = 8) and an accuracy of 6.1%. From these results it can be concluded that the OraSure OSCD is useful to collect oral fluid for reliable quantitative THC assay applying a modified elution procedure and gravimetric determination of the amount of oral fluid.

Foods containing seeds or oil of the hemp plant (Cannabis sativa L.) are increasingly found in retail stores in the U.S. The presence of delta9-tetrahydrocannabinol (THC) in these foods has raised concern over their impact on the results of workplace drug tests for marijuana. Previous studies have shown that eating hemp foods can cause screening and confirmed positive results in urine specimens. This study evaluated the impact of extended daily ingestion of THC via hemp oil on urine levels of its metabolite 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THC-COOH) for four distinct daily THC doses. Doses were representative of THC levels now commonly found in hemp seed products and a range of conceivable daily consumption rates. Fifteen THC-naïve adults ingested, over four successive 10-day periods, single daily THC doses ranging from 0.09 to 0.6 mg. Subjects self-administered THC in 15-mL aliquots (20 mL for the 0.6-mg dose) of four different blends of hemp and canola oils. Urine specimens were collected prior to the first ingestion of oil, on days 9 and 10 of each of the four study periods, and 1 and 3 days after the last ingestion. All specimens were screened for cannabinoids by radioimmunoassay (Immunalysis Direct RIA Kit), confirmed for THC-COOH by gas chromatography-mass spectrometry (GC-MS), and analyzed for creatinine to identify dilute specimens. None of the subjects who ingested daily doses of 0.45 mg of THC screened positive at the 50-ng/mL cutoff. At a daily THC dose of 0.6 mg, one specimen screened positive. The highest THC-COOH level found by GC-MS in any of the specimens was 5.2 ng/mL, well below the 15-ng/mL confirmation cutoff used in federal drug testing programs. A THC intake of 0.6 mg/day is equivalent to the consumption of approximately 125 mL of hemp oil containing 5 microg/g of THC or 300 g of hulled seeds at 2 microg/g. These THC concentrations are now typical in Canadian hemp seed products. Based on our findings, these concentrations appear to be sufficiently low to prevent confirmed positives from the extended and extensive consumption of hemp foods.

The loss of delta9-tetrahydrocannabinol (THCCOOH) from urine specimens stored in polypropylene and polyethylene containers at 4 degrees C and 25 degrees C was examined. All specimens were analyzed by GC-MS after sampling at various times over a one-week period. Data were analyzed by one-way analysis of variance and fitted with a first order kinetic equation. Rapid loss of THCCOOH was seen at 4 degrees C for both polypropylene (14% maximal loss, t(1/2) = 0.53 min) and polyethylene (17% maximal loss, t(1/2) = 5.77 min) bottles. At 25 degrees C, a small loss (< 5%) was observed in polypropylene and no significant loss was seen for urine in polyethylene. All losses stabilized within 1 h, and no further losses were seen over one week. The results indicate that THCCOOH binding may be due to decreased solubility of THCCOOH at lower temperatures and subsequent association of THCCOOH with the more lipophilic plastic. The results also indicate that polypropylene and polyethylene do not bind THCCOOH to such an extent as to compromise the integrity of specimens.

The loss of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) from solution was studied using fluorescence polarization immunoassay (FPIA) technology and x-ray photoelectron spectroscopy (XPS). Several materials (glass, silylated glass, high density polyethylene, polypropylene, polystyrene, polymethylmethacrylate, Teflon, and Kynar) were studied along with three solvents (water, urine, and Abbott cannabinoids diluent). THC-COOH losses ranging from 0 to 9.7 ng/cm2 and concentration reductions to 46% of starting values were measured. XPS indicated the presence of fluorine-labeled THC-COOH at materials surfaces. A half-life of 10 min was calculated for THC-COOH loss from urine stored in high density polyethylene at room temperature. Sample handling losses during pipetting were determined and ranged from 1.1 to 7.9 ng per aliquot. The effects of sample volume and sample handling on the THC-COOH concentrations of controls were also investigated.

delta 9-Tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) are three constituents of the 16 that can be currently isolated from some Cannabis spp plants. Their identification in decontaminated hair can indicate exposure to cannabis. In this study, we propose a rapid, simple, and direct (without derivatization) screening procedure for the simultaneous identification and quantitation of CBD, CBN, and THC in hair of chronic cannabis abusers. Hair samples were washed with methylene chloride, hydrolyzed with sodium hydroxide, extracted with n-hexane-ethyl acetate (9:1, v/v), evaporated to dryness, and injected directly on a gas chromatographic-mass spectrometric system operating in electron-impact mode. THC-d3 was used as the internal standard. Thirty hair samples were tested. CBD was detected 23 times, CBN was detected 22 times, and THC was detected five times. Concentrations ranged from 0.03 to 3.00 ng/mg (mean, 0.44 ng/mg), from 0.01 to 1.07 ng/mg (mean, 0.13 ng/mg), and from 0.1 to 0.29 ng/mg hair (mean, 0.15 ng/mg) for CBD, CBN, and THC, respectively. These results show that this new screening procedure is suitable for the detection of CBD and CBN in the hair of cannabis abusers.

The metabolic formation of an oxepin derivative, 3-pentyl-6,7,7a,8,9,11a-hexahydro-1,7-dihydroxy-7,10- dimethyldibenzo-[b,d]-oxepin, from cannabidiol was studied in-vitro using guinea-pig hepatic microsomes. The hepatic microsomes catalysed the formation of the metabolite from cannabidiol and 8S, 9-epoxycannabidiol in the presence of an NADPH-generating system and 3, 3, 3-trichloropropene-1, 2-oxide. 8S, 9-Epoxycannabidiol was thought to be an intermediate in the formation of the metabolite, which was identified by gas chromatography-mass spectrometry. The metabolite synthesized from 8S, 9-epoxycannabidiol diacetate exhibited catalepsy, hypothermia and pentobarbitone-induced sleep prolongation in mice, although the pharmacological effect was less potent than that of delta 9-tetrahydrocannabinol.