Specimens and analytical methods
One of the most important issues that needs to be considered in a DFSA investigation is the sensitivity of both screening and confirmatory techniques, since some of the compounds, e.g. benzodiazepines, are typically used in a single low dose. There is the added complication that victims of DFSA often do not report the incident until sometime after the event owing to amnesia and doubt about what may have happened, and possibly other psychological reasons. Sufficient time may have elapsed for most, if not all, of the drug to have been metabolised and eliminated from the more usual matrices sampled for toxicological testing (e.g. blood and urine). Preliminary screening techniques for drugs in urine include enzyme multiplied immunoassay (EMIT), fluorescence polarisation immunoassay (FPIA), and Abuscreen On Trak and Online imunoassays (Roche Diagnostics). Enzymatic hydrolysis followed by the extraction of benzo diazepines, including flunitrazepam and 7 amino flunitrazepam, is frequently performed in order to increase the sensitivity of the assay. Confirmatory methodologies include gas chromatography–mass spectrometry with electron ionisation (EI-GC-MS) and both positive (PCI-GC-MS) and negative (NCI-GC-MS) chemical ionisation detection. In developing back ground information for the construction and optimisation of analytical methods for the drugs and their metabolites, one important compo nent is a controlled, clinical dosing study with the drugs. In such a study, healthy volunteers are given single doses of a drug under careful medical supervision, then monitored for drug and metabolite concentrations in urine and hair for several days afterwards. In recent years, substantial analytical progress has been achieved in the USA and in France, allowing the detection for extended periods of selected benzodiazepines and GHB in urine and hair after a single low dose. As noted above, this is important because DFSA victims often do not report the incident immediately after the assault. Thus, it would not be unusual for a urine or blood analysis to provide a negative result when the drug had in fact been ingested, because of the likelihood of a single low dose, urine volume and the length of time since the drug was taken. Many drugs are deposited in hair and hence it may be possible to detect traces of drug in hair sometime after an alleged DFSA.
As noted above, GHB is produced endogenously in the body. However, because of this endogenous production, it has been proposed that exogenous sources of GHB should only be considered when levels in blood and urine exceed 5 and 10 lg/mL, respectively. GBL is hydrolysed to GHB, particularly at high pH (Del Signore et al. 2005) and hence, if an analysis is carried out to differentiate between GBL and GHB, attention must be paid to minimising hydrolysis. It has also been reported that there can be site-dependent production of GHB in the early postmortem period (Moriya and Hashimoto, 2005) such that higher levels of endogenous GHB may occur in postmortem samples. This aspect should be taken into account when analysing for GHB in post-mortem samples. Shima et al. (2005) noted that the alpha-hydroxybutyric acid (AHB) and beta hydroxybutyric acid (BHB), isomers of GHB, are also produced endogenously in the body and that levels of BHB in urine are considerably higher than levels of GHB. As the mass spectra of the various isomers are similar, there needs to be good chromatographic separation of the various isomers when using GC-MS or LC-MS to avoid inaccurate identification and quantification. Saudan et al. (2005) applied for the first time a method using continuous flow gas chromatography–combustion–isotope ratio mass spectrometry (GC-C-IRMS), described in Chapter 3, to discriminate between endogenous and exogenous (synthetic) GHB in blood. Significant differences in the carbon isotope ratio (13C/12C) were found between endogenous and synthetic GHB. The method can presumably be applied to other biological specimens including urine.
