Qualitative analysis of zolpidem and its metabolites M-1 to M-4 in human blood and urine using liquid chromatography–tandem mass spectrometry

The aim of this study was to develop the method for the qualitative analysis of zolpidem and its metabolites M-1 to M-4 using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in human blood and urine. To obtain analytical standards, a method for synthesizing zolpidem metabolites M-1 to M-4 was developed. A combination of a copper-catalyzed three-component coupling reaction and pyrrolidine-catalyzed imine formation was adopted to synthesize the methyl esters of M-1 (M-1-Me) and M-2 (M-2-Me), which were hydrolyzed to afford M-1 and M-2, respectively. M-3 and M-4 were also synthesized. The LC–MS/MS conditions were optimized using authentic standards. M-1 to M-4 were analyzed in human blood and urine. M-1 to M-4 were successfully synthesized. The mass spectra of M-1 and M-2 were almost identical, but their peaks were chromatographically separated using a octadecyl silica column. Likewise, mass spectra of M-3 and M-4 were similar, but their peaks were chromatographically separated. The peak intensities of the metabolites in human blood and urine were M-1 > M-2, and M-4 > M-3; M-3 was detected only in urine. The presence of other hydroxyzolpidems was also revealed. Chromatographic separation of M-1 and M-2 and that of M-4 and other hydroxyzolpidems is necessary to analyze zolpidem metabolites in biological matrices because their mass spectra are quite similar. This study is the first to analyze zolpidem and M-1 to M-4 simultaneously using LC–MS/MS, which can provide more compelling evidence of zolpidem intake.