Quantitative analysis of Δ9-THC-COOH in Human Urine by the Liquid-Liquid Extraction technique and Gas Chromatography-Mass Spectrometry: Adaptation, Optimization and Validation (original) (raw)
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Forensic Science International, 2005
An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography-mass spectrometry (LC-MS) has been developed and validated for the confirmation of D 9 -tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm 3 , 300 mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC + H + ], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d 3 -THC + H + ]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100 ng/mL showed a linear relationship (r 2 = 0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette 1 . In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552 ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937 ng/mL. However, using Salivette 1 collection, 26 of the 40 cases had an undetectable THC. #
Determination of Δ9-THC in whole blood using gas chromatography-mass spectrometry
Journal of analytical toxicology, 2002
A simple and reliable liquid-liquid extraction method for the determination of A%tetrahydrocannabinol (THC) in whole blood utilizing gas chromatography-mass spectrometry in electron impact mode is described. The substance is derivatized with pentafluoropropionic anhydride in pentafluropropanol. The limit of detection is 0.5 ng/mL for a 1-mL specimen, with recovery greater than 70%. The intra-assay coefficient of variation (CV) is 3.1% to 5.2%, and the interassay CV is 6.4% to 9.5%, calculated at THC concentrations of 1, 5, and 25 ng/mL. The accuracy is between 95 and 97%. The optimization of extraction and derivatization conditions is detailed.
New extraction method of THC and its metabolites, 11-OH-THC and THC-COOH, in plasma
Annales de Toxicologie Analytique, 2013
Objectives: A liquid/liquid extraction technique on solid support of Δ 9 -tetradydrocannabinol (THC), 11-hydroxy-Δ 9 -tetradydrocannabinol (11-OH-THC) and 11-nor-Δ 9 -tetradydrocannabinol-9-carboxylic acid (THC-COOH) in plasma was developed in order to be assayed by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS). Methods: The samples were extracted by liquid/liquid extraction over solid support of an extraction cartridge. The extracts were thereafter dried down and injected into the HPLC-MS/MS system set with a positive electrospray mode using a Waters XTerra MS C18 3.5-μm 2.1 × 150 mm column. Results: The extraction recovery levels were 66%, 70% and 71% for THC, and 75%, 93% and 101% for 11-OH-THC at concentrations of 2.5, 5 and 10 ng/mL, respectively. They were 86% and 78% for THC-COOH at concentrations of 5 and 10 ng/mL. The limits of detection (LOD) were 0.09, 0.08 and 0.91 ng/mL for THC, 11-OH-THC and THC-COOH, respectively. The limits of quantification (LOQ) were 0.16, 0.15 and 3.24 ng/mL for THC, 11-OH-THC and THC-COOH, respectively. The inter-series incertitude CV determined for concentrations of 1, 2.5 and 10 ng/mL were 12.1%, 12.0% and 6.4% for THC, 14.5%, 11.1% and 7.2% for 11-OH-THC, and 14.9%, 26.2% and 11.3% for THC-COOH. Conclusion: The novel extraction method for THC, 11-OH-THC and THC-COOH developed in this work is rapid, sensitive and specific. It may be a valuable tool for predictive toxicology, high-throughput metabolism and pharmacokinetic studies of cannabinoids.
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.
Determination of 9-THC in Whole Blood using Gas Chromatography-Mass Spectrometry
Journal of Analytical Toxicology, 2002
A simple and reliable liquid-liquid extraction method for the determination of A%tetrahydrocannabinol (THC) in whole blood utilizing gas chromatography-mass spectrometry in electron impact mode is described. The substance is derivatized with pentafluoropropionic anhydride in pentafluropropanol. The limit of detection is 0.5 ng/mL for a 1-mL specimen, with recovery greater than 70%. The intra-assay coefficient of variation (CV) is 3.1% to 5.2%, and the interassay CV is 6.4% to 9.5%, calculated at THC concentrations of 1, 5, and 25 ng/mL. The accuracy is between 95 and 97%. The optimization of extraction and derivatization conditions is detailed.
Journal of Analytical Toxicology, 2014
Recently, the estimation of the measurement uncertainty has become a significant issue in the quality control of forensic drug testing. In the present study, the uncertainty of the measurement was calculated for the quantification of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) and its glucuronide conjugate (THC-COOH-glu) in urine using liquid chromatography-tandem mass spectrometry. The procedure was based on liquid-liquid extraction of a volume of urine (800 mL) with ethyl acetate. The sources of uncertainty were identified and classified into four major categories as follows: standard preparation, calibration curve, method precision and bias. The overall contribution of combined standard uncertainty on THC-COOH increased in the order of standard preparation (0.9%), method precision (10.4%), calibration curve (30.3%) and bias (58.4%) and, while calibration curve (53.0%) and bias (40.4%) gave the bigger contributions to the combined standard uncertainty for THC-COOH-glu than method precision and standard preparation, which accounted for 6.3 and 0.3%, respectively. The reliability of a measurement was expressed by stating the expanded uncertainty of the measurement result at 95% confidence level. The concentrations of THC-COOH and THC-COOH-glu in the urine sample with their expanded uncertainties were 10.20 + + + + + 1.14 ng/mL and 25.42 + + + + + 5.01 ng/mL, respectively. Experimental Chemicals The reference compounds of THC-COOH and THC-COOH-glu were obtained from Cerilliant (Austin, TX, USA) in a vial at a concentration of 100 mg/mL in methanol. The deuterated internal
Journal of Chromatography B, 2008
Cannabis is considered to be the most widely abused illicit drug in Europe. Consequently, sensitive and specific analytical methods are needed for forensic purposes and for cannabinoid pharmacokinetic and pharmacodynamic studies. A simple, rapid and 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 (THC-COOH) in blood is presented. The method was fully validated according to international guidelines and comprises simultaneous liquid-liquid extraction (LLE) of the three analytes with hexane:ethyl acetate (90:10, v/v) into a single eluant followed by separation and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was achieved using a XBridge C 18 column eluted isocratically with methanol:0.1% formic acid (80:20, v/v). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. The use of the LLE 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 250 L of blood. The method was linear over the range investigated (0.5-40 g/L for THC, 1-40 g/L for 11-OH-THC, and 2-160 g/L for THC-COOH) with excellent intra-assay and inter-assay precision; relative standard deviations (RSDs) were <12% for THC and 11-OH-THC and <8% for THC-COOH for certified quality control samples. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments. No instability was observed after repeated freezing and thawing or in processed samples. The method was subsequently applied to 63 authentic blood samples obtained from toxicology cases. The validation and actual sample analysis results show that this method is rugged, precise, accurate, and well suited for routine analysis.
Biological Mass Spectrometry, 1988
A gas chromatographic/mass spectrometric electron impact method is presented for the detection and quantification of 1 l-nor-9-carboxy-A9-tetrahydrocannabinol (THC-COOH) in urine, for use in the confirmation of presump tive results obtained by other techniques. Four extraction procedures, two solid-liquid and two liquid-liquid, have been compared. A comparison of two trimethylsilylating methods demonstrates that the best results are obtained by the use of a mixture containing N-methyl-N-trimethylsilyl-trifluoroacetamide, trimethyliodosilane and dithioerithritol (100:0.2:1) v/v/w. The use of ketoprofen as a new internal standard for the quantification of THC-COOH has proved to be very effective. Both spiked samples and samples from cannabis users have been successfully analysed. It has also been demonstrated that the presence of other drugs of abuse in urine samples do not interfere with cannabis quantification by the method reported here.
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 Analytical Toxicology, 1997
A sensitive and reliable method was developed for the identification and quantitation of 11-nor-9-carboxy-Agtetrahydrocannabinol (THCCOOH) in urine using a microbed solid-phase extraction (SPE) column. Extensive method validation is presented that evaluates the recovery, linearity, precision, limit of quantitation, limit of detection, and capacity of THCCOOH in urine extracts using CLEAN SCREEN | reduced solvent volume (RSV) SPE columns followed by gas chromatographic-mass spectrometric analysis. The mean recovery of THCCOOH at concentrations of 2, 3, 5, 15, 50, and 150 ng/mL was found to be 91% with coefficients of variation of 7.3% or less. Linearity of the method ranged from 1.95 to 1000 ng/mL with sensitivity at 1 ng/mL for THCCOOH. The sorbent was found to retain at least 1000 ng/mL of THCCOOH with no analyte breakthrough using the described method. Reduction in both processing times and total solvent volume is shown. The RSV SPE columns showed excellent efficiency and performance while demonstrating recoveries, cleanliness, and dynamic ranges comparable with standard SPE products.