Simultaneous determination of THC-COOH and THC-COOH-glucuronide in urine samples by LC/MS/MS.
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.
Weinmann
W
W
Institute of Forensic Medicine, Klinikum der Albert-Ludwigs-Universität Freiburg, Albertstrasse 9, D-79104, Freiburg, Germany. weinmann@sun11.ukl.uni-freiburg.de
Vogt
S
S
Goerke
R
R
Müller
C
C
Bromberger
A
A
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D013759:Dronabinol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000652:urine*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D009994:Osmolar Concentration; D011619:Psychotropic Drugs / Q000378:metabolism / Q000652:urine*; D012680:Sensitivity and Specificity; D015813:Substance Abuse Detection / Q000379:methods*
D011619:Psychotropic Drugs; C016780:11-nor-delta(9)-tetrahydrocannabinol-9-carboxylic acid; C026502:delta(9)-tetrahydrocannabinol-11-oic acid glucuronide; D013759:Dronabinol