Abstract

The method to analyse hair samples consists of the following steps: decontamination of the hair strand, extraction of the pulverised material with methanol, purification of the raw extract using LL-extraction and HPLC-Clean up, formation of TMS-derivatives, detection by GC-HRMS. Anabolic compounds included in the standard procedure are clenbuterol, 19-nortestosterone (NT), testosterone (T) and several esters of both of these steroids. Investigations have been carried out to optimise the initial extraction of the hair material. An ideal procedure is characterised by the complete release of incorporated substances combined with the separation of interfering compounds. Several procedures are applicable performed either by extraction (organic solvents, buffer solutions) or by digestion of hair samples (alkaline, acidic, enzymatic, reductive pulping). The suitability of different treatments depends on the substance-specific properties and the kind of binding in hair. Therefore, the stability of the analytes under conditions required for digestion and extraction, respectively, has to be taken into consideration. Due to the usage of methanol for the standard procedure, incorporated steroids are extracted unchanged from hair. Therefore, it is possible to analyse the applied patent compounds itself, e. g. the entire esters of NT and T alter intramuscular injection. However, the resulting methanol extract contains fatty impurities, and a time-consuming sample preparation is necessary. In contrast, the alkaline digestion of hair results in cleaner solutions, but the detection of the esters of steroids is prevented due to the hydrolysis under alkaline conditions. Different methods were tested and compared with the methanol extraction, concerning sensitivity, recovery, feasibility and efficiency. Disintegration utilising the reductive agent tris(2-carboxyethyl)phosphine hydrochloride (TCEP) has been described for hair analysis and was therefore of particular interest. The stability of several 19-norsteroids and esters of NT and T against TCEP solution was confirmed (25 mM TCEP, at 50°C for 2 hours). The TCEP digest of hair samples contained less fatty impurities compared to the methanol extract, resulting in improved S/N ratios. Separation of lipophilic analytes was incomplete using only the TCEP disintegration, caused by an adsorption of the released substances to the hair residue. To solubilise the complete amount and therefore exceed the recovery of the standard procedure, a subsequent extraction of the remaining hair material with methanol was necessary. Aus dem Text (geändert)