Study on the determination and chiral inversion of R-salbutamol in human plasma and urine by liquid chromatography–tandem mass spectrometry

• Chiral analysis of salbutamol enantiomers in human plasma and urine.
• The chiral inversion of R-salbutamol was fully investigated.
• A fitted model for chiral inversion ratio of R-salbutamol was established.
• The chiral inversion of R-salbutamol resulted from the gastric acid environment.
• Application to a clinical pharmacokinetics study of R-salbutamol.

The chiral inversion has been a concerned issue during the research and development of a chiral drug. In this study, a sensitive chiral liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for determination of salbutamol enantiomers in human plasma and urine. The chiral inversion mechanism of R-salbutamol was fully investigated for the first time by studying the effects of physicochemical factors, including pH, temperature and time. A fitted model to predict the chiral inversion ratio of R-salbutamol was proposed using a Box–Behnken design. All the samples were separated on an Astec Chirobiotic T column and detected by a tandem mass spectrometer in multiple reaction monitoring mode. Lower limit of quantification of 0.100 ng/mL was achieved under the optimized conditions. The method was fully validated and successfully applied to the clinical pharmacokinetic study of R-salbutamol in healthy volunteers. Chiral inversion of R-salbutamol to S-salbutamol has been detected in urine samples. The results indicated that pH and temperature were two dominant factors that caused the chiral inversion of R-salbutamol, which should be taken into consideration during the analysis of chiral drugs. The chiral inversion of R-salbutamol determined in this study was confirmed resulted from the gastric acid in stomach rather than caused by the analysis conditions. Moreover, the calculated results of the fitted model matched very well with the enantioselective pharmacokinetic study of R-salbutamol, and the individual difference of the chiral inversion ratio of R-salbutamol was related to the individual gastric environment. On the basis of the results, this study provides important and concrete information not only for the chiral analysis but also for the metabolism research of chiral drugs.