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Thursday, August 28 Analytical Procedures

Polettini A., Marrubini Bouland G., Valli A., Montagna M.
Department of Legal Medicine and Public Health, University of Pavia, Via Forlanini, 12, 27100 Pavia, Italy

Among the increasing difficulties the analytical toxicologist has to face, one of the most challenging is the selective analysis of drugs in biological samples at the trace level (ppb or lower) in order (a) to detect toxic compounds active at low doses, (b) to improve the retrospectivity of results (e.g. to ascertain the past abuse of drugs for recreational purposes, or of drugs enhancing performance in sports), (c) to exclude the presence of drugs in the organism (e.g. in the case of organ donation). The selective detection of drugs at trace level is usually achieved with extensive sample cleanup and derivatization procedures followed by chromatographic separation and mass spectrometric detection. Besides being time-consuming and expensive, this approach may lead to sample contamination, loss of analyte through adsorption phenomena, hydrolytic or oxidative degradation, etc.

The direct analysis of the sample with no or minimal manipulation using hyphenated analytical techniques may be an effective alternative strategy. The coupling on-line of different analytical stages, in fact, improves the overall selectivity of the method and allows a substantial reduction in the limits of detection provided that at the final stage there is a detectable signal above the electrical noise. In fact, the loss of analyte's signal occurring at each stage is usually compensated by a greater decrease in chemical noise, with an overall improvement in the signal/noise ratio.

The authors have investigated the potential of coupled column liquid chromatography (LC/LC) for both cleanup and sample enrichment purposes, combined with the selectivity of detection of tandem mass spectrometry (MS/MS) in the analysis of different drugs (i.e. ß2-agonists, corticosteroids, cardiac glycosides) in biological matrices. After minimal treatment (filtration in the case of urine or serum; deproteinization and filtration in the case of whole blood), a large volume of sample (0.1-0.5 ml) is injected in the first LC column (C1). Just before the elution of the analyte(s), C1 is switched on-line with the second LC column (C2). When the transfer of the analyte(s) is completed the two columns are switched off-line, while the eluent of C2 is analysed by thermospray MS/MS in the selected reaction monitoring (one parent - one daughter) mode. By optimizing the critical thermospray parameters (vaporizer temperature, repeller voltage) and MS/MS parameters (collision energy, collision gas pressure), quantitation limits in the range 0.1-1 ppb can be easily reached. The lack of extensive sample preparation allows an easy and rapid method development, which is limited to the optimization of the chromatographic parameters with a traditional UV detector on one side, and to the optimization of the mass spectrometric parameters by flow injection analysis on the other. Furthermore, a sample throughput of 4 to 8 h-1 can be achieved, yielding a favorable cost/benefit ratio for the expensive equipment used.

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