LC-MS/MS method for determining picogram-level of zolpidem and its main metabolites in hair using a zirconia-based sorbent

Abstract

Although urine and blood samples have been conventionally used for testing zolpidem (ZPD), a sedative-hypnotic, these matrices have limited application because they have a relatively short detection period and can be used only in case of recent drug exposure. Therefore, it is necessary to use an alternative biological sample to obtain the evidence of ZPD misuse. Herein, a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of ZPD and its metabolites, zolpidem phenyl-4-carboxylic acid (ZPCA) and zolpidem 6-carboxylic acid (ZCA), in hair to resolve the above-mentioned problems. Mechanical pulverization of hair, methanol extraction with sonication, and the zirconia-based hybrid solid-phase extraction technique were used for obtaining improved extraction efficiency and effective sample purification. The treated hair sample was analyzed using the LC-MS/MS method with the electrospray ionization source in positive and multiple-reaction monitoring modes. The target analytes were separated and detected within 8 min using an Xselect HSS T3 column. Gradient elution was performed using 5 mM ammonium formate and acetonitrile. The lower limit of quantification of ZPD, ZPCA, and ZCA were 1.0, 0.5, and 1.0 pg mg^-1, respectively. The calibration ranges were 1.0-1000.0 pg mg^-1 for ZPD, 0.5-200.0 pg mg^-1 for ZPCA, and 1.0-200.0 pg mg^-1 for ZCA, with the determination coefficients (r² ≥ 0.9986). The intraday accuracy and precision ranged from -7.1 to 9.0% and within 6.5%, respectively, and the interday accuracy and precision ranged from -6.1 to 7.9% and within 5.4%, respectively. The recovery, matrix effect, and process efficiency were 65.2-96.6%, 64.6-106.5%, and 44.3-100.5%, respectively, with the relative standard deviation of 4.0-5.0%. The developed method was successfully applied to analyze 13 forensic hair samples of ZPD abusers, and the concentration ratios of ZPD and its two main metabolites (ZPCA and ZCA) in the ZPD-positive samples were also presented. These results revealed that ZPCA and ZCA were not easily incorporated into hair, and demonstrated that their analysis in hair samples requires the employed method to have picogram-level sensitivity. Therefore, the developed method was suitable for simultaneous analysis of ZPD, ZPCA, and ZCA in hair samples, and it could provide clear evidence for illegal ZPD administration, including ZPD-facilitated sexual assault.

Keywords: Hair analysis; LC–MS/MS; Metabolite; Zirconia-based HybridSPE; Zolpidem.