Analysis of Cannabinoids in Post-mortem Blood Samples

Abdul Rahman, Mohamed Zaini (1991) Analysis of Cannabinoids in Post-mortem Blood Samples. MSc(R) thesis, University of Glasgow.

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This thesis is concerned with the analysis of cannabinoids, delta-9-tetrahydrocannabinol (THC) and its major metabolite, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THCCOOH), in post-mortem blood samples. It concentrates on sample preparation procedures and end-step detection techniques by High Pressure Liquid Chromatography (HPLC) and Gas Chromatography-Mass Spectrometry (GC-MS). A comparative evaluation was made of solid-phase (diatomaceous earth, Extrelut) and solvent extraction procedures. When extracted with hexane, mixtures of hexane and more polar solvents, or acetonitrile, the former gave low extraction yields, for example, with acetonitrile the recoveries were 70.4% for THC and 43.9% for THCCOOH. It was observed that clogging of the extrelut columns by precipitated blood proteins might have resulted in the poor results obtained. On the other hand, solvent extraction procedures using acetonitrile gave better results. Recoveries obtained for THC and THCCOOH were 82.1% and 82.7% respectively. These values were found to be higher when deionised water was added to dilute the blood samples (83.2% for THC and 86.9% for THCCOOH). However, addition of buffers at pH 5.0 and 7.4 did not improve the recoveries obtained by addition of water. Calibration curves of the solvent extraction procedure produced linear plots within the 0-80ng/ml range for both THC and THCCOOH. The solid-phase extraction material Bond Elut Certify II was developed recently for the extraction and purification of cannabinoids from biological matrices. It was incorporated into this study as a clean-up procedure for HPLC analysis of the solvent extracts. Therefore, extraction procedure selected for further analysis involved deproteinization and extraction with acetonitrile, supernatant clean-up using the Bond Elut Certify II columns, evaporation to dryness and either reconstitution of the residue in mobile phase for HPLC analysis or derivatization of the residue prior to GC-MS analysis. For HPLC analysis, acetonitrile-methanol-0. 02N sulphuric acid (65:15:50, v:v:v) was chosen from four mobile phases examined. Detection of analytes was carried out using ultra-violet (UV) and electro-chemical (ECD) detectors at wavelengths 212nm, 220nm and at an applied potential of +1.10V respectively, after detector optimisation procedures. The clean-up procedure separated the cannabinoids into THC and THCCOOH fractions. Thus, two internal standards were needed for quantitation purposes. N-octyl p-hydroxy benzoate and phenylbutazone were found to be the most suitable choices for the two fractions, respectively. It was also observed that the THC fraction gave clean chromatograms while the THCCOOH fraction contained co-extracted interferences which eluted closed to the metabolite. Average recoveries for THC, cannabidiol (CBD), cannabinol (CBN) and THCCOOH by this HPLC system were 91.1%, 93.6%, 90.1% and 91.4% detected by UV 212nm; 88.9%, 93.5%, 88.9% and 90.7% by UV 220nm and 93.4%, 94.7%, 98.7% and 89.1% by ECD +1.10V, respectively. Calibration curves were linear from 0-100ng/ml for both UV detection wavelengths, and within the range 0-200ng/ml for ECD detection. When eight test samples were analysed, which were positive when screened for cannabinoids by radioimmunoassay, none of the samples gave positive results for THC and CBN. Electrochemical detection but not UV detection gave five samples positive for CBD, while THCCOOH was found to be present in all of the test samples. GC-MS analysis was performed under full scan, selected ion recording (SIR), multiple reaction monitoring (MRM) and negative ion chemical ionisation (NICI) modes. Extracted samples were not cleaned up with Bond Elut Certify II in order to evaluate the sensitivity and selectivity of the different GC-MS modes. Full scan mass spectra of THC, THCCOOH and their derivatives were compared. They were found to produce parallel series of ion fragments through similar fragmentation mechanisms. Analyses by SIR-MS were performed to confirm the identity of the analytes in blood extracts. Calibration curves for THC and THCCOOH were linear from 0-100ng/ml. When the same eight test samples were analysed, three of them were found to be positive for THCCOOH, but only two of the samples gave positive results for THC. In the MRM mode, the transitions to the most prominent daughter fragments [M-15]+ formed by the parent analytes, THC and THCCOOH, were monitored. Linear calibration curves were obtained within the range 0-100ng/ml. In conclusion, the optimum method established in this study was acetonitrile extraction of blood samples followed by GC-MS in El(+) selected ion recording mode used as the end-step analysis. Sample clean-up is not essential but would help in prolonging the column life.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Keywords: Analytical chemistry, Toxicology, Forensic anthropology
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78314
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:33
Last Modified: 30 Jan 2020 15:33

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