Optimization of the quantitative analysis of the major cannabis metabolite (11-nor-9-COOH-Δ9-tetrahydrocannabinol) in urine by gas chromatography/mass spectrometry (original) (raw)
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Cannabis is the most consumed illicit drug worldwide, and its legal status is a source of concern. This study proposes a rapid procedure for the simultaneous quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cannabidiol (CBD), and cannabinol (CBN) in urine samples. Microextraction by packed sorbent (MEPS) was used to pre-concentrate the analytes, which were detected by gas chromatography–mass spectrometry. The procedure was previously optimized, and the final conditions were: conditioning with 50 µL methanol and 50 µL of water, sample load with two draw–eject cycles, and washing with 310 µL of 0.1% formic acid in water with 5% isopropanol; the elution was made with 35 µL of 0.1% ammonium hydroxide in methanol. This fast extraction procedure allowed quantification in the ranges of 1–400 ng/mL for THC and CBD, 5–400 ng/mL for CBN and 11-OH-THC, and 10–400 ng/mL for THC-COOH with coeffic...
Journal of Chromatography B, 2003
A fully validated, highly sensitive and specific method for the extraction and quantification of 9 -tetrahydrocannabinol (THC), 11-hydroxy-9 -tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-9 -tetrahydrocannabinol (THCCOOH) in plasma is presented. This method incorporates Escherichia coli -glucuronidase hydrolysis to cleave glucuronic acid moieties to capture total analyte concentrations, and simultaneous solid phase extraction (SPE) of the three analytes in a single eluant with separation and quantification on a bench-top positive chemical ionization (PCI) gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode. Quantitation was achieved by the addition of deuterated analogues for each analyte as internal standards (IS). Limits of quantitation (LOQ) were 0.5, 0.5 and 1.0 for THC, 11-OH-THC and THCCOOH, respectively, with linearity ranging up to 50 ng/ml for THC and 11-OH-THC, and 100 ng/ml for THCCOOH. Absolute recoveries ranged from 67.3 to 83.5% for all three analytes. Intra-assay accuracy and precision ranged from 1.2 to 12.2 and 1.4 to 4.7%, respectively. Inter-assay accuracy and precision ranged from 1.4 to 12.2 and 3.1 to 7.3%, respectively. This method was used to analyze plasma samples collected from individuals participating in a controlled oral THC administration study. Statistically significant (P ≤ 0.05) increases of 40% for 11-OH-THC and 42% for THCCOOH concentrations were found between hydrolyzed and non-hydrolyzed results. This method will be utilized in ongoing controlled cannabinoid administration studies and may be a useful analytical procedure for the fields of forensic toxicology and cannabinoid pharmacology.
Journal of Chromatography A, 2007
A two-dimensional (2D) gas chromatography/electron impact-mass spectrometry (GC/EI-MS) method for simultaneous quantification of 9 -tetrahydrocannabinol (THC), 11-hydroxy-9 -tetrahydrocannabinol (11-OH-THC), and 11-nor-9 -tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in human plasma was developed and validated. The method employs 2D capillary GC and cryofocusing for enhanced resolution and sensitivity. THC, 11-OH-THC, and THCCOOH were extracted by precipitation with acetonitrile followed by solid-phase extraction. GC separation of trimethylsilyl derivatives of analytes was accomplished with two capillary columns in series coupled via a pneumatic Deans switch system. Detection and quantification were accomplished with a bench-top single quadrupole mass spectrometer operated in electron impact-selected ion monitoring mode. Limits of quantification (LOQ) were 0.125, 0.25 and 0.125 ng/mL for THC, 11-OH-THC, and THCCOOH, respectively. Accuracy ranged from 86.0 to 113.0% for all analytes. Intra-and inter-assay precision, as percent relative standard deviation, was less than 14.1% for THC, 11-OH-THC, and THCCOOH. The method was successfully applied to quantification of THC and its 11-OH-THC and THCCOOH metabolites in plasma specimens following controlled administration of THC.
Journal of Pharmaceutical and Biomedical Analysis, 2008
Recently, electrospray ionization mass spectroscopy (ESI-MS) has been widely used for the identification of drugs of abuse and their metabolites in biological samples. However, the sensitivity and selectivity of this technique are commonly inadequate for the analysis of tetrahydrocannabinol (THC) and its metabolites at very low levels, such as those sometimes required in forensic and clinical-legal applications. We coupled electrospray ionization and surface-activated chemical ionization (ESI-SACI) to various types of mass analyzers (ion trap, triple quadrupole and orbitrap) (ESI-SACI-MS) to improve the detection of 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH), the most common marker of THC abuse. The benefits of this approach in terms of sensitivity and selectivity compared with a common ESI-MS approach are clearly demonstrated.
Analytical and Bioanalytical Chemistry, 2010
A sensitive analytical method for simultaneous quantification of cannabidiol (CBD), Δ 9tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) in plasma is presented for monitoring cannabinoid pharmacotherapy and illicit cannabis use. Analytes were extracted from 1 mL plasma by solid phase extraction, derivatized with N, O,-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane, and analyzed by two-dimensional gas chromatography mass spectrometry (2D-GCMS) with cryofocusing. The lower calibration curve was linear from 0.25-25 ng/mL for CBD and THC, 0.125-25 ng/mL for 11-OH-THC and 0.25-50 ng/mL for THCCOOH. A second higher linear range from 5-100 ng/ mL, achieved through modification of injection parameters, was validated for THC, 11-OH-THC and THCCOOH and was only implemented if concentrations exceeded the lower curve upper limit of linearity. This procedure prevented laborious re-extraction by allowing the same specimen to be re-injected for quantification on the high calibration curve. Intra-and inter-assay imprecision, determined at four quality control concentrations, were <7.8% CV. Analytical bias was within ±9.2% of target and extraction efficiencies were >72.9% for all analytes. Analytes were stable when stored at 22°C for 16h, 4°C for 48h, after three freeze-thaw cycles at −20°C and when stored on the autosampler for 48h. This sensitive and specific 2D-GCMS assay provides a new means of simultaneously quantifying CBD, THC and metabolite biomarkers in clinical medicine, forensic toxicology, workplace drug testing, and driving under the influence of drugs programs.
Forensic Toxicology, 2014
Marijuana abuse can be detected by means of toxicological analysis of the most important metabolite 11-nor-9-carboxy-D 9 -tetrahydrocannabinol (THC-COOH) in urine samples. The aim of this study is the establishment of the detailed procedure for analysis of THC-COOH in urine by combination of hollow fiber-liquid phase microextraction (HF-LPME) and gas chromatography-mass spectrometry (GC-MS). The conditions of hydrolysis and extraction were optimized. The method was shown to be very simple and rapid, and a low amount of organic solvent was necessary for extraction. The limit of detection was 1.5 ng/ml. The calibration curves were linear over the specified range (2.0-170 ng/ml; r 2 [ 0.99). The main sources of uncertainty were found to be analyte concentration, accuracy, method precision and sample volume. The effect of the analyte concentration on the overall combined uncertainty was most significant. The developed method was successfully applied to a human urine standard reference material at two levels of concentration. The obtained relative combined uncertainty was 8 %, which can be considered acceptable according to international guidelines. The present method seems very useful in clinical and forensic toxicology, because of its simplicity, rapidness and inexpensiveness.
Journal of Analytical Toxicology, 2001
Ag.Tetrahydrocannabinol (THC) and 11-nor.9-carboxy-A9tetrahydrocannabinol (THCA) in human plasma can be simultaneously detected using solid.phase extraction with gas chromatography and negative ion chemical ionization mass spectrometry. THC-d3 and THCA-d3 are added as internal standards; protein is precipitated with acetonitrile and the resulting supernatants diluted with 0.1M sodium acetate (pH 7.0) prior to application to the solid-phase extraction columns. THC and THCA were eluted separately and then pooled, dried under air, and derivatized with trifluoroacetic anhydride and hexafluoroisopropanol. The derivatized THC-d0 gives abundant molecular anions (m/z 410), and the derivatized THCA-do gives abundant fragment ions (m/z 422) formed by loss of (CF3)2CHOH from its molecular anion. The recoveries of THC and THCA were 74% and 17%, respectively. The lower and upper limits of quantitation were 0.5 and 100 ng/mL for THC and 2.5 ng/mL and 100 ng/mL for THCA. The within-run accuracy and precision for THC (measured at 0.5, 1, 10 and 75 ng/mL) ranged from 98 to 106% (% target) and 4.1 to 9.5 (%CV), respectively. For THCA, the within-run accuracy and precision (measured at 2.5, 5, 10, and 75 ng/mL) ranged from 89 to 101% and 4.3 to 7.5%, respectively. The between-run accuracy and precision for THC ranged from 92 to 110% and 0.4 to 12.4%, respectively. The between-run accuracy and precision for THCA ranged from 97 to 103% and 6.5 to 12.3%, respectively. In processed samples stored in reconstituted form at -20~ THC and THCA were stable for at least three days. THC and THCA stored in plasma were stable following three freeze/thaw cycles. THC and THCA in whole blood at room temperature for 6 h, or in plasma stored at room temperature for 24 h, did not show significant change. Storage in polypropylene containers for 7 days at -20~ and the presence of 1% sodium fluoride or the cannabinoid receptor antagonist, SR141716, at I pg/mL did not interfere with the quantitation of THC and THCA. In three individuals who smoked marijuana under controlled dosing conditions, peak THC concentrations of 151, 266, and 99 ng/mL were seen in the first plasma samples drawn immediately after the end of smoking, and corresponding peak THCA concentrations of 41, 52, and 17 ng/mL occurred at 0.33 to 1 h after cessation of smoking.
Journal of Chromatography A, 2005
A rapid and sensitive method for the analysis of 9 -tetrahydrocannabinol (THC) in preserved oral fluid was developed and fully validated. Oral fluid was collected with the Intercept, a Food and Drug Administration (FDA) approved sampling device that is used on a large scale in the U.S. for workplace drug testing. The method comprised a simple liquid-liquid extraction with hexane, followed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis. Chromatographic separation was achieved using a XTerra MS C 18 column, eluted isocratically with 1 mM ammonium formate-methanol (10:90, v/v). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. The use of the liquid-liquid extraction was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Validation of the method was performed using both 100 and 500 L of oral fluid. The method was linear over the range investigated (0.5-100 ng/mL and 0.1-10 ng/mL when 100 and 500 L, respectively, of oral fluid were used) with an excellent intra-assay and inter-assay precision (relative standard deviations, RSD <6%) for quality control samples spiked at a concentration of 2.5 and 25 ng/mL and 0.5 and 2.5 ng/mL, respectively. Limits of quantification were 0.5 and 0.1 ng/mL when using 100 and 500 L, respectively. In contrast to existing GC-MS methods, no extensive sample clean-up and time-consuming derivatisation steps were needed. The method was subsequently applied to Intercept samples collected at the roadside and collected during a controlled study with cannabis.
Journal of Chromatography A, 2013
a b s t r a c t 9 -Tetrahydrocannabinol (THC) is the primary target in oral fluid (OF) for detecting cannabis intake. However, additional biomarkers are needed to solve interpretation issues, such as the possibility of passive inhalation by identifying 11-nor-9-carboxy-THC (THCCOOH), and determining recent cannabis smoking by identifying cannabidiol (CBD) and/or cannabinol (CBN). We developed and comprehensively validated a microflow liquid chromatography (LC)-high resolution mass spectrometry method for simultaneous quantification of THC, THCCOOH, CBD and CBN in OF collected with the Oral-Eze ® and Quantisal TM devices. One milliliter OF-buffer solution (0.25 mL OF and 0.5 mL of Oral-Eze buffer, 1:3 dilution, or 0.75 mL Quantisal buffer, 1:4 dilution) had proteins precipitated, and the supernatant subjected to CEREX TM Polycrom TM THC solid-phase extraction (SPE). Microflow LC reverse-phase separation was achieved with a gradient mobile phase of 10 mM ammonium acetate pH 6 and acetonitrile over 10 min. We employed a Q Exactive high resolution mass spectrometer, with compounds identified and quantified by targeted-MSMS experiments. The assay was linear 0.5-50 ng/mL for THC, CBD and CBN, and 15-500 pg/mL for THCCOOH. Intra-and inter-day and total imprecision were <10.8%CV and bias 86.5-104.9%. Extraction efficiency was 52.4-109.2%, process efficiency 12.2-88.9% and matrix effect ranged from −86 to −6.9%. All analytes were stable for 24 h at 5 • C on the autosampler. The method was applied to authentic OF specimens collected with Quantisal and Oral-Eze devices. This method provides a rapid simultaneous quantification of THCCOOH and THC, CBD, CBN, with good selectivity and sensitivity, providing the opportunity to improve interpretation of cannabinoid OF results by eliminating the possibility of passive inhalation and providing markers of recent cannabis smoking.
Journal of Chromatography B, 2008
A direct liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for measurement of urinary Delta(9)-tetrahydrocannabinol carboxylic acid (THCA) was developed. The method involved dilution of the urine sample with water containing (2)H(9)-deuterated analogue as internal standard, hydrolysis with ammonia, reversed phase chromatography using a Waters ultra-performance liquid chromatography (UPLC) equipment with gradient elution, negative electrospray ionization, and monitoring of two product ions in selected reaction monitoring mode. The measuring range was 2-1000 ng/mL for THCA, and the intra- and inter-assay imprecision, expressed as the coefficient of variation, was below 5%. Influence from urine matrix on ionization efficiency was noted in infusion experiments, but was compensated for by the internal standard. Comparison with established gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry methods in authentic patient samples demonstrated accuracy in both qualitative and quantitative results. A small difference in mean ratios (~15%) may be explained by the use of different hydrolysis procedures between methods. In conclusion, the high efficiency LC-MS/MS method was capable of accurately identify and quantify THCA in urine with a capacity of 14 samples per hour.