FORENSIC INVESTIGATION OF KERATINMATRIX: DEVELOPMENT OF A METHODBY TURBOFLOW™ HPLC-MS/MSFOR CANNABINOIDS QUANTITATIVE ANALYSIS

The products of cannabis are the most frequently used illicit drugs of abuse detected inworkplace drug-testing programs or in cases of driving under the influence of drugs. Forthis reason, there is a great need for sensitive and specific methods which can confirm thechronic exposure and which take account of the cut-off values that have been proposed inItaly by the GTFI Guidelines (Group of Italian Forensic Toxicologists). The methodcurrently more appropriate for quantification of Δ9-Tetrahydrocannabinol (THC) and itsmajor metabolite 11-nor-9-Carboxy-Δ9-THC (THCA) appears to be one that involvesHPLC-MS/MS and keratin matrix. The purpose of this work, performed at the MassSpectrometry Laboratory for Clinical Risk and Quality Control (CQRC) in A.O.U.P.“Paolo Giaccone” of Palermo, is the developing of an accurate method equally or moresensitive than those used routinely in forensic toxicology laboratories. A pre-analyticalphase has seen the preparation of hair samples, comprising basic hydrolysis, and asubsequent Liquid-Liquid Extraction (LLE). In the analytical phase, the step of detectionwas performed using the Thermo Scientific™ Transcend™ II system which combines theonline sample preparation technology TurboFlow™ with the chromatographic separationtechnique of High Performance Liquid Chromatography (HPLC) and the detectiontechnique of tandem Mass Spectrometry (MS/MS) using a high sensitive ThermoScientific™ TSQ Quantiva™ Triple Quadrupole Mass Spectrometer. During the phase ofthe developing method a series of experimental tests regarding the analytical conditionshave been performed, on six different chromatographic columns. This has highlighted themain advantages of TurboFlow™, the adequate selectivity in the sample cleaning and thepossibility of injecting more volumes of sample than the normal LC-MS techniques,leading to an appreciable increase in the sensitivity. According to the aim of the work, thelatter steps of sample preparation, including LLE, were replaced with a simple hydrolyzatefiltration and direct injection into TurboFlow™ system. In the light of the results weobtained it can be said that the method developed by TurboFlow™ -HPLC-MS/MS thatinvolves the short phase of sample preparation procedure allows accurate quantification ofcannabinoids in the keratin matrix. This technique, showing an adequate sensitivity, can be compared and preferred to used routine techniques. The short phase of preparation of thesample, also, results in a considerable saving of time and materials and in the reduction ofpossible errors caused by the operator.