14.2. Confirmatory methods of analysis:
In forensic settings, a positively screened sample by IAs should be confirmed by another independent method such as GC and GC-MS.GC is used to separate the analyzed components by partition between a gas as a mobile phase and nonpolar liquid on solid stationary phase, of a vaporized sample using GC- capillary column at elevated temperature. The analyzed sample should be readily volatile, nonpolar and thermostable. GC is not appropriate for the analysis of non-volatile or thermo sensitive compounds. GC-separation depends on the degree of polarity of the analytes, where compounds of relatively high polarity will be firstly eluted and detected. Detection of the separated compounds is done using flame ionization (FID) detector. (Figure 9)
Figure 9: A diagram of GC, Adapted from (16)
Highly lipophilic BZD could be directly analyzed by GC, however hydroxy metabolites are not. Therefore, an initial derivatization step should be done, where the sample is extracted and derivatized using silyl mixture. The derivatization reaction should be done at 80 ͦ C for 30 min under closed condition, the silyl derivative is volatile, of high molecular weight and can be detected. The obtained GC spectra should be compared with spectra of reference substances.
GC-MS, on the other hand, is considered more specific for confirmation of BZD in the sample. In this technique GC separation with MS detection is applied. .In GC-MS, a full MS scan of BZD together with GC spectrum, are obtained to identify BZD compound. In order to identify the BZD compound, the retention time (Rt) from total ion chromatogram and mass spectrum, are compared with a reference standard or the MS spectrum is matched with the mass spectra of reference BZD compounds in the instrument library (Figure 10), shows the GC-MS of sample containing diazepam and tramadol. Diazepam is detected at retention time 8.6 min at a molecular mass of m/z 283, whereas tramadol is detected at Rt 7.07 min. at a molecular mass m/z 261.
Figure 10: GC-MS of diazepam (Rt: 8.6 min, m/z 283) in a mixture of diazepam and tramadol
GC-MS is usually used to detect and identify free and unchanged BZD in the sample. Therefore, for the detection of BZD metabolites in urine samples, a hydrolysis step of the glucuronide conjugates is required before derivatization and detection by GC-MS.
If BZD get exposed to strong acid hydrolysis, BZD forms the corresponding stable benzophenone compounds. Benzophenones can also be formed from the hydroxy metabolites and their conjugates. Benzophenones could be used to identify BZD and their metabolites using GC or GC-MS without a need of derivatization. Yet due to the fact that unchanged BZD, hydroxy and glucuronide metabolites can yield the same benzophenone compound, therefore it’s difficult to distinguish between them. In case of BZD e.g. alprazolam, triazolam, and midazolam, they don’t produce benzophenone compounds when treated with...