*3.2.3 Confirmatory analysis*

Confirmatory techniques for drugs in oral fluid [20] are mostly adapted from those used in the analyses of blood or plasma/serum specimens. Recovery of drugs is not typically a limiting factor, considering the higher water content and lower protein levels of oral fluid compared to blood. However, the sample volume of oral fluid will be smaller, with potentially lower concentrations, which means that more adjustments are required to analytical techniques. Indeed, in saliva analysis the detection or quantification limit for drugs is very much determined by the type of screening test and its application. The confirmation method must be able to produce an analytical result that is optimally independent from that of the screening. Therefore, it must be based on different physico-chemical principles and have superior analytical selectivity and sensitivity. In this regard, a quantitative confirmatory method capable of reaching a lower limit of quantification (LLOQ ) equal to at least half the cut-off of the screening method is considered acceptable. The use of a confirmatory method which is based on the measurement of a similar analytical signal is not acceptable since it is highly correlated to that of the screening (e.g. confirmation of a given immunochemical with another immunochemical method). The use of an identical chromatographic technique to confirm a set of data obtained by chromatography is acceptable if the detection technique combined with chromatography changes.

The use of a chromatographic technique to confirm screening data obtained by chromatography with the same detection system is allowed only if the two separation techniques produce poorly correlated results (for example, two series of significantly different retention times, with the use of columns of different polarity or selectivity, etc). However, in the forensic toxicological field, chromatographic separation is always necessary in a confirmatory method; the general consensus of the international scientific community is that mass spectrometry (MS) with its many methodological possibilities can be combined with a chromatographic separation technique such as gas chromatography (GC), high pressure liquid chromatography (HPLC) or capillary electrophoresis (EC) for confirmatory analysis (**Figure 3**). Many methods

**Figure 3.** *Ultra-high performance liquid chromatography.*

use LC–MS as distinct from GC–MS to cater for the lower sample volumes and low detection limits, although a number of GC–MS techniques have exhibited adequate sensitivity [34].
