Development of a new liquid phase micro-extraction method for the determination of drugs in oral fluids by HPLC-MS / MS analysis

The use of psychoactive substances is increasing worldwide and remains an important social concern. Even if instruments with high sensitivity and precision are available, innovative methodologies to isolate the molecules is essential [1]. The purpose of this work is to develop an analysis method for the simultaneous determination of illicit substances, both natural and synthetic, belonging to different pharmacological classes (synthetic cannabinoids, synthetic cathinones, amphetamines, arylcyclohexylamines, alkaloids, opiates and related metabolites, fentanyl and derivatives and benzodiazepines) in the oral fluid (OF) or saliva (S), an unconventional matrix, alternative to or complementary to plasma and urine in the fields of forensics and therapeutic drug monitoring, as well as for drug-related examination procedures and on-site testing [2]. Liquid chromatography coupled with tandem mass spectrometry (LCMS/MS) was used for the identification and quantification of the targeted drugs. Before the analysis with these techniques, a pre-treatment of the sample is necessary to remove any interferences, while a concentration step can be useful to detect parent drugs and/or their metabolites which cn be found at very low concentration in OF. These steps were performed simultaneously, by means of the same technique, the parallel extraction with artificial liquid membrane (PALME), i.e. a type of alternative liquid phase microextraction based on the use of a liquid support membrane which was first described and used in 2013 by Gjelstad et al. for the determination, in human plasma samples, of any basic compounds [3]. This technique uses a pH gradient to facilitate the mass transfer of uncharged analytes across a liquid membrane with organic support and this allows to extract, from an aqueous biological sample, acid, basic or neutral molecules. For this reason it is essential to know the chemical properties of the target analytes [4]. The greatest challenge is the identification of the right compromises, for each parameter (extraction time, extraction pH, the composition of the extracting solvent and acceptor solution, the effect of adding salt and its concentration) which could be adapted to the large number of analytes to be recognized and quantified. The method proved to be suitable for analytes of different classes and since a very small quantities of organic solvent (only 3 µL for each sample) is required, it is an example of green chemistry. The method was subsequently validated following the international guidelines of Scientific Working Group for Forensic Toxicology (SWGTOX) by evaluating a series of parameters, such as precision and accuracy, LOD and LOQ, linear dynamic range, matrix effect and recoveries. Results obtained demonstrate the applicability of the presented method in clinical, toxicological and forensic investigation.