Segmental ion spray LC-MS-MS analysis of benzodiazepines in hair of psychiatric patients

The aim of this study was to develop a liquid chromatographytandem mass spectrometry (LC-MS-MS) method for the analysis of benzodiazepines in human hair. The method was tested by analyzing hair samples from forensic and clinical psychiatric patients where benzodiazepines had been prescribed during hospitalization and after care. Hair samples were obtained at discharge from the clinic and then after six months. Two-centimeter segments of the hair samples (10-30 mg) were washed once with isopropanol, three times with phosphate buffer, and again with isopropanol, dried, weighed, and digested with proteinase K before solid-phase extraction with BondElut Certify columns. Diazepam, nordiazepam, oxazepam, alprazolam, OH-alprazolam, nitrazepam, 7-aminonitrazepam, flunitrazepam, 7-aminoflunitrazepam, clonazepam, and 7-aminoclonazepam were quantitated in MRM mode using one transition for each analyte and deuterated internal standard. The calibration range was 0.125-5 ng/mg for diazepam, nordiazepam, and oxazepam and 0.025-1.0 ng/mg for the other compounds. In the hair samples analyzed, diazepam, flunitrazepam, nitrazepam, and clonazepam was detected together with their metabolites. Alprazolam was not detected in any sample. Segmental hair analysis revealed differences in drug deposition in hair before and after release from psychiatric treatment. Both increases and decreases of hair drug concentrations were seen after release even though the prescribed dose was the same. This was taken as an indication of noncompliance during the after-care period. We conclude that the extraction and LC-MS-MS procedures were adequate to detect benzodiazepines in hair and that the results indicated that segmental hair analysis might provide retrospective information about medication intake.