Hair analysis is a widely applied retrospective monitoring tool. One major challenge is to monitor a wide range of analytes including e.g. opioids, stimulants, benzodiazepines, Z-drugs, antidepressants and neuroleptics and some metabolites within the same sample workup followed by a single analytical measurement. Analytes were selected according to availability, requirements for our routine casework and local prevalence. The aim of this study was to establish a routine compatible multi-analyte method considering that expected drug concentrations in hair are strongly substance-dependent and usually vary over a wide range. Based on the expected hair concentrations, each drug was assigned to one out of three analyte groups covering different concentration ranges: 0.5 pg/mg–600 pg/mg (group 1), 10–12,000 pg/mg (group 2), and 50–60,000 pg/mg (group 3). The internal standard solution contained 44 deuterated standards. Drugs were extracted from 20 mg washed and pulverized hair by a 2-step protocol [step 1: methanol; step 2: 1 mM aqueous ammonium formate containing 0.1% formic acid/methanol = 1/1 (v/v) by shaking at 10 Hz for 90 min]. Combined extracts were dried, reconstituted and injected into a LC-MS/MS system using a Sciex QTrap 5500 mass spectrometer (positive advanced scheduled MRM mode). Separation was achieved using a Kinetex ® F5 column (100 mm × 2.1 mm, 100 Å, 2.6 μm, Phenomenex) which operated in a Shimadzu Prominence high performance liquid chromatography system (Shimadzu, Duisburg, Germany) at a flow rate of 0.6 mL/min with a total run time of 12 min (mobile phase A: 1 mM ammonium formate buffer with 0.1% formic acid and acetonitrile containing 1 mM ammonium formate buffer and 0.1% formic acid). A method for the quantification of 116 analytes in hair by a 2-step extraction was established. In total, 276 MRM transitions including deuterated standards were measured. Three substance-depending calibration ranges were successfully implemented. Calibration ranges covered up to three orders of magnitude. In order to avoid saturation effects, parent mass ions containing one or two 13C-isotopes were used for selected transitions (14%) and detuning of the declustering potential (DP) was applied for 41% of the transitions. The method has been fully validated according to international guidelines. The matrix effects and recoveries were within the allowed ranges for 89% and 97% of the analytes, respectively. The lower limits of quantification (LLOQs) were for the majority of the analytes in the low-pg/mg-range (0.5–5 pg/mg) and for approx. 23% of the analytes between 10 and 50 pg/mg. These LLOQs considered cut-offs by the SoHT, if recommended. The presented multi-analyte method can be applied e.g. in assessments for abstinence controls (e.g. regranting the driving license). Due to the low LLOQs, the method is also applicable for the detection of single dose drug exposure, e.g. for DFC or DFSA cases. The detection of metabolites even in the low concentration range allows for determination of metabolic ratios, which can be used as indicators to differentiate between drug use and external contamination. The method was successfully applied for international proficiency tests. The herein established multi-analyte approach can be applied in routine hair testing for the rapid and robust measurement of a wide range of psychoactive substances. The analyte-specific wide concentration ranges open up a wide range of application fields.
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