Three different derivatisation procedures were evaluated for their general applicability to systematic toxicological analysis (STA) involving (a) acylation with pentafluoropropionyl anhydride (PFPA) and pentafluoropropanol (PFP-OH), (b) acylation/esterification (methylation) with pentafluoropropionyl anhydride (PFPA) and a novel methylating agent trimethylsilyldiazomethane (TMS-diazomethane), used as a chromatographic derivatisation reagent for the first time in this study, and (c) silylation with tertiary-butyldimethylsilyl-trifluoroacetamide (MTBSTFA). Model compounds were selected for the evaluation process including a primary amine (amphetamine), a secondary amine (methamphetamine), alicyclic and aromatic hydroxy compounds (morphine, tetrahydrocannabinol), and carboxylic acids (benzoylecgonine, 11-nor tetrahydrocannabinol-9-carboxylic acid).
For method (a) derivatisation was successful for all of the test compounds and mass spectra were obtained for each of them. For method (b), the novel methylating agent trimethylsilyl-diazomethane was used to convert carboxylic acids into the corresponding methyl esters. This reaction was found to proceed rapidly and quantitatively at room temperature and holds potential for future use in toxicology to replace diazomethane, a hazardous and toxic material. Method (c) gave derivatives with all test compounds except the secondary amine, methamphetamine, and the alcohol, morphine. The gas chromatographic behaviour of these derivatives was good and the mass spectra had prominent ions suitable for GC-MS-SIM.
The extraction of multiple drugs from blood was evaluated using the novel polymeric SPE sorbent Strata-X. The same test compounds were used to assess the extraction step in terms of recovery and variation (within day and between days). The extracts were analysed by GC-MS-SIM using each of the three types of derivative. Recoveries of the test compounds were in the range of 50-100 percent depending on the analyte and its concentration in blood. All calibration curves were linear and had correlation coefficients higher than 0.99. Within day variations and between day variations were in the range of 2-22% relative standard deviation. Limits of detection and quantitation were measured for the model compounds and were found to be in the ranges of 0.4-7.3 ng/ml and 1.1-24.4 ng/ml respectively. The full method, combining extraction with each of the derivatisation reactions was finally evaluated for the presence of interferences with real case blood samples.
The three derivatisation procedures were evaluated using four test compounds comprising diazepam plus its three metabolites nordiazepam, temazepam and oxazepam. The hydroxylated metabolites (temazepam and oxazepam) formed derivatives readily with all three reagent mixtures but nordiazepam (secondary aromatic amine) did not react except with MTBSTFA.
Based on the work of this study it is concluded that a method is possible for STA based on a polymeric sorbent, to give a general extract, followed by a generalised derivatisation procedure such as acylation, with PFPA/PFP-OH prior to GC-MS
