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Confirmation of cannabis abuse by GC-MS
Abstract
Cannabis and cannabis resins contain the substance " 9-tetrahydrocannabinol (THC). The plant and drug are both regulated by the Drug Enforcement Administration (DEA) of Jordan. The regulations require testing for THC and its metabolites, which can be found in urine. 11-nor-9-carboxy" 9-THC, also known as THC-COOH, is the primary urinary metabolite of THC. In this study, a method for confirmation analysis of large number of urine samples previously screened positive for THC using solid-phase extraction (SPE) and Gas Chromatography-Mass Spectrometry (GC-MS) analysis is developed. The results of the analysis of standard reference material and actual samples are presented and discussed. The developed method is sensitive enough to assess relevant THC-COOH levels in human urine for forensic investigations.
... The presence of illicit drugs or their metabolites in urine is an evidence of the substance intake. 1,2 Heroin as one of the most widely abused drugs, rapidly metabolized to 6-monoacetylmorphine (6-MAM) once inside the human body. This specific heroin metabolite 6-MAM is detected at a higher concentration usually within 2 to 4 hours, and after six hours, has not been detected in the urine. ...
A method for the analysis of urine for the heroin abuse is described. The analytical
procedure uses solid-phase extraction (SPE), gas chromatography/mass spectrometry
(GC/MS). It allows extraction, derivatization and analysis of 6-MAM and Morphine from
urine. After solid-phase extraction was complete, the eluate was selectively derivatized
with N-Methyltrimethylsilyltrifluoroacetamide (MSTFA). Analysis was performed using a
GC/MS system operating in full scan mode. The mass spectrum of the derivatized
metabolites was searched manually against reference libraries for positive identification
and the retention time checked against that of the standard. This procedure has increased
both the amount and the reliability of information given to analyst. The system has been
in routine operation processing 100-150 urine samples per week. The results of the
analysis of standard reference material and actual samples are presented and discussed.
The developed method is sensitive enough to assess relevant 6-MAM and morphine levels
in urine for forensic investigations.
Cannabis is one of the most commonly abused drugs worldwide. There is a distinct clinical correlation between cannabis abuse and mental disorders. However, it is essential to establish cannabis intake in the abusers in order to establish causality between cannabis and psychiatric illness. The limitations of current detection methods using commercial cassettes prompted us to standardize the method of extraction and detection of cannabinoids in the urine samples of cannabis abusers attending a de-addiction centre in south India.
In this study, diagnostic tests on 102 male patients suspected with cannabis abuse were done. Liquid-liquid extraction of cannabinoids from urine was done and screened by Duquenois-Levine, fast blue B salt and p-dimethylaminobenzaldehyde (p-DMAB) tests. All the results were confirmed by high performance thin layer chromatography (HPTLC). Samples were considered positive for cannabis based on the positive indication in colour test and by detection of 11-nor-delta(9) tetrahydrocannabinol-9-carboxylic acid (THC-COOH) on HPTLC.
Based on the colour tests and HPTLC, cannabis abuse was detected in 64 of 102 patients tested. HPTLC method was found to be sensitive for detection and possible quantitation of THC-COOH.
We report the standardization and utility of cannabinoid extraction, screening and detection by HPTLC in the urine samples of cannabis abusers. The HPTLC method was found to be high throughput, sensitive, reproducible and cost-effective compared to commercial kits.
A specimen pretreatment procedure, combining solid-phase extraction and methylation, was developed for the analysis of 11-nor-9-carboxy-Δ9- tetrahydrocannabinol (9-THC-COOH) in urine for monitoring marijuana exposure. d3-11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (d3-9-THC-COOH) was used as an internal standard, while the now universal selected ion monitoring gas chromatography-mass spectrometry approach was used for detection (m/z 313, 357, 372 and 316, 360, 375 for 9-THC-COOH and d3-9-THC-COOH, respectively) and quantitation (m/z 372 and 375 for 9-THC-COOH and d3-9-THC-COOH, respectively). The effectiveness of the overall procedure was evaluated. One of the extraction columns evaluated achieved > 90% recovery of the analyte in 5-mL drug-free urine fortified with 15 ng/mL 9-THC-COOH. Using the one-point calibration approach, the overall protocol achieved the following analytical parameters: detection limit: 2.09 ng/mL; limit of quantitation: 4.18 ng/mL; upper end of linear range: 376 ng/mL (or better); interday precision: 1.49- 8.03% CV in the 4.18-376 ng/mL concentration range studied.
An extraction and concentration procedure using a unique solid-phase extraction tube was developed for the analysis of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in urine. Absolute and relative recoveries of the drug metabolite were found to be
greater than 85% and 92%, respectively. The extraction procedure allows quantitation of the drug metabolite by GC/MS, HPLC
with UV detection, or GC with flame ionization detection. The extraction procedure was verified with urine samples obtained
from marijuana users as well as spiked urine samples.
A procedure for detection and quantitation of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid, a major metabolite of
delta-9-tetrahydrocannabinol in urine, has been described. Since the metabolite is present in both conjugated and unconjugated
forms, hydrolysis of urine was carried out to increase the sensitivity of detection. The acidic metabolite was isolated by
strongly basic anion exchange resin, and subsequently derivatized to methyl 1-dehydroxy-1-methoxy-11-nor-delta-9-tetrahydrocannabinol-9-carboxylate
(1a) by methyliodide in the presence of tetramethylammonium hydroxide. The derivatized product was separated in a capillary column
gas chromatograph, and finally detected by a mass spectrometer under electron impact mode. Confirmation of the product was
carried out by monitoring three ions that represent the major portions of the molecule and comparing their relative abundances
to that of a standard. Quantitation was based on 5′-2H3-11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid as internal standard. Excellent linearity was obtained over the range
of 2 to 1000 ng/mL. The overall yield of extraction using the anion exchange resin was 50 to 60%. This extraction process
is rapid and suitable for a large number of sample analyses. The methylated product (1a) is stable for at least 72 hr at room temperature.
A rapid and sensitive GC/MS method for the determination of Δ9tetrahydrocannabinol-11-oic in urine is presented. Enzymically hydrolyzed urine was extracted with hexane/ethyl ether followed
by a one-step derivatization procedure with a mixture of pentafluoropropionic anhydride and pentafluoropropanol. By using
negative ion chemical ionization and selected ion monitoring, a 200-fold higher detectability was observed, compared with
El or positive ion chemical ionization under the conditions used. Samples were analyzed with split injection on a fused silica
capillary column with a total analysis time of less than four minutes. A deuterium labelled analogue was used as internal
standard and the precision for the overall method was measured to 3.4% for a sample concentration of 230 ng/mL, and to 6.7%
for 3 ng/mL. The minimum detectable amount was below 1 ng/mL.
A fast method using liquid-liquid extraction and HPLC/tandem-mass spectrometry (LC/MS/MS) was developed for the simultaneous detection of 11-Nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid beta-glucuronide (THC-COOH-glucuronide) and 11-Nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. This highly specific method, which combines chromatographic separation and MS/MS analysis, can be used for the confirmation of positive immunoassay results even without hydrolysis of the sample or derivatisation of extracts. Liquid-liquid extraction was optimised: with ethylacetate/diethylether (1:1, v/v) THC-COOH-glucuronide and THC-COOH could be extracted in one step. Molecular ions of the glucuronide (MH(+), m/z 521) and THC-COOH (MH(+), m/z 345) were generated using a PE/SCIEX turboionspray source in positive ionisation mode; specific fragmentation was performed in the collision cell of an API 365 triple-quadrupole mass spectrometer and yielded major fragments at m/z 345 (for THC-COOH-glucuronide) and m/z 327 as well as m/z 299 for both cannabinoids. Chromatographic separation was performed using a reversed-phase C8 column and gradient elution with 0.1% formic acid/1 mM ammonium formate and acetonitrile/0.1% formic acid. Retention times were 22.2 min for the glucuronide and 26.8 min for THC-COOH. After enzymatic hydrolysis of urine samples with beta-glucuronidase/arylsulfatase (37 degrees C, 5 h), THC-COOH-glucuronide was no longer detectable by LC/MS/MS in urine samples. However, the THC-COOH concentration was increased. For quantitation of THC-COOH, THC-COOH-D(3) was added to the urine samples as internal standard prior to analysis. From the difference of THC-COOH in the native urine and urine after enzymatic hydrolysis, molar concentration ratios of THC-COOH-glucuronide/THC-COOH in urine samples of cannabis users were determined and found to be between 1.3 and 4.5.
Delta9-tetrahydrocannabinol (THC), the main psychologically active ingredient of the cannabis plant (marijuana), has been prepared synthetically and used as the bulk active ingredient of Marinol, which was approved by the FDA for the control of nausea and vomiting in cancer patients receiving chemotherapy and as an appetite stimulant for AIDS patients. Because the natural and the synthetic THC are identical in all respects, it is impossible to determine the source of the urinary metabolite of THC, 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), in a urine specimen provided in a drug-testing program. Over the last few years there has been a need to determine whether a marijuana positive drug test is the result of the ingestion of marijuana (or a related product) or whether it results from the sole use of Marinol. We have previously proposed the use of delta9-tetrahydrocannabivarin (THCV, the C3 homologue of THC) as a marker for the ingestion of marijuana (or a related product) because THCV is a natural component of most cannabis products along with THC and does not exist in Marinol. We have also reported that THCV is metabolized by human hepatocytes to 11-nor-delta9-tetrahydrocannabivarin-9-carboxylic acid (THCV-COOH); therefore, the presence of the latter in a urine specimen would indicate that the donor must have used marijuana or a related product (with or without Marinol). In this study, we provide clinical data showing that THCV-COOH is detected in urine specimens collected from human subjects only after the ingestion of marijuana and not after the ingestion of Marinol (whether the latter is ingested orally or by smoking). Four subjects (male and female) participated in the study in a three-session, within-subject, crossover design. The sessions were conducted at one-week intervals. Each subject received, in separate sessions and in randomized order, an oral dose of Marinol (15 mg), a smoked dose of THC (16.88 mg) in a placebo marijuana cigarette, or a smoked dose of marijuana (2.11% THC and 0.12% THCV). Urine samples were collected and vital signs were monitored every 2 h for a 6-h period following drug administration. Subjects were then transported home, were given sample collection containers and logbooks, and were instructed to record at home the volume and time of every urine collection for 24 h, and once a day for the remainder of a week (6 days). Subjects were also instructed to freeze the urine samples until the next session. All urine samples were analyzed by GC-MS for THC-COOH and THCV-COOH using solid-phase extraction and derivatization procedure on RapidTrace and TBDMS as the derivative. The method had a limit of detection of 1.0 ng/mL and 1.0 ng/mL for THCV-COOH and THC-COOH, respectively.
In order to facilitate the confirmation analysis of large numbers of urine samples previously screened positive for Δ9-tetrahydrocannabinol (THC), an extraction, derivitization, and GC-MS analysis method was developed. This method utilized
a positive pressure manifold anion-exchange polymer-based solid-phase extraction followed by elution directly into the automated
liquid sampling (ALS) vials. Rapid derivitization was accomplished using pentafluoropropionic anhydride/pentafluoropropanol
(PFPA/PFPOH). Recoveries averaged 95% with a limit of detection of 0.875 ng/mL with a 3-mL sample volume. Performance of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)-d3 and THC-COOH-d9 internal standards were evaluated. The method was linear to 900 ng/mL THC-COOH using THC-COOH-d9 with negligible contribution from the internal standard to very weak samples. Excellent agreement was seen with previous
quantitations of human urine samples. More than 1000 human urine samples were analyzed using the method with 300 samples analyzed
using an alternate qualifier ion (m/z 622) after some interference was observed with a qualifier ion (m/z 489). The 622 ion did not exhibit any interference even in samples with interfering peaks present in the 489 ion. The method
resulted in dramatic reductions in processing time, waste production, and exposure hazards to laboratory personnel.
Cannabinoids are the natural constituents of marihuana (cannabis). The main of them are delta9-tetrahydrocannabinol (9THC)--psychoactive agent, cannabinol (CBN) and cannabidiol (CBD). Cannabis is administered either by smoking or orally. 9THC potency and duration of action as well as its and two of its major metabolites concentrations in organism highly depend on the route of administration. A single active dose of 9THC is estimated on 520 mg. 9THC is rapidly metabolised. It is hydroxylated to an active metabolite, I1 -hydroxy-delta9-tetrahydro-cannabinol (11-OH-THC), then oxidised to an inactive 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCCOOH), which is conjugated with glucuronic acid and predominantly excreted in the urine. The maximum psychological effect persists for 4-6 h after administration despite of very low 9THC blood concentrations. 9THC plasma concentration declined to values of 2-3 ng/ml during 3-4 h after smoking. Such a low concentration of the active compound in human organism create a demand for use of sensitive analytical methods for detection and determination of 9THC and its metabolites. The most effective techniques for 9THC and related compounds determination in biological material are chromatographic ones (gas and liquid) with mass spectrometric detection and different ionization modes. 9THC and its two metabolites (11-OH-THC and THCCOOH) are present in blood and hair, 9THC in saliva, and THCCOOH in urine. 9THC and related compounds are determined in autopsy material, although deaths by overdose of cannabis are exceptionally rare. Fatalities happen most often after intravenous injection of hashish oil. 9THC and its metabolites determination in different biological materials gives the basis for a wide interpretation of analytical results for clinical and forensic toxicology purposes.