Study of On-Line Fluorimetric Determination of Oxazepam. Application to the Screening of 1,4-Benzodiazepines Giving Oxazepam as Metabolite in Urine

Abstract

An automated, rapid, and sensitive flow injection method was developed for the fluorimetric determination of oxazepam and afterward applied for the screening of benzodiazepines with oxazepam as a common metabolite in urine. This method is based on the hydrolysis of oxazepam to the corresponding benzophenone and subsequent cyclization to form the fluorescent molecule 2-chloro-9(10H)-acridinone. Both reactions were allowed to proceed in a two channel flow injection manifold. The physical and chemical variables affecting the method were optimized and a linear calibration graph obtained. Oxazepam was detectable in the 40–700 ng mL⁻¹ range. The detection limit of this method was 35 ng mL⁻¹ for k = 3 and the throughput was 25 samples h⁻¹. The method was successfully applied for the screening of oxazepam in urine samples collected at different times after the single oral administration of Valium and Tranxilium by two healthy volunteers.

Keywords:

• Benzodiazepines screening
• Flow injection
• Fluorimetry
• Oxazepam
• Urine

Acknowledgments

This article is part of a Special Issue on Automated Flow Injection Techniques organized by Dr. Paraskevas Tzanavaras of Aristotelian University of Thessaloniki, Greece.

This work has been funded by Spain's Ministry of Science and Innovation, within the framework of Project AGL2009-12589. Authors thank Adrian Burton for reviewing the manuscript.

Notes

^a Linear range for chlordiazepoxide and diazepam.

^b Dimethylsulfoxide.

^c Time span between sample injection and the maximum fluorescence intensity of the signal.