Fast Determination of Benzodiazepines in Human Urine via Liquid-Liquid Extraction with Low Temperature Partitioning and LC-HRMS

Elisângela Jaqueline Magalhães, Clésia Cristina Nascentes, Rodinei Augusti, Maria Eliana Lopes Ribeiro de Queiroz, Júlio César Cardoso da Silva, Robson José de Cássia Franco Afonso

Departamento de Química, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Departamento de Química, Universidade Federal de Ouro Preto, Ouro Preto, Brazil.
Departamento de Química, Universidade Federal de Vi?osa, Vi?osa, Brazil.

DOI: 10.4236/ajac.2012.32017                

Abstract

A simple and high-throughput method to simultaneously determine selected benzodiazepines (i.e., diazepam, lorazepam, clonazepam, and bromazepam) in urine was developed and validated. The entire methodology consisted of the application of an innovative extraction/cleanup procedure, namely liquid-liquid extraction with low-temperature partitioning (LLE-LTP), and analysis by liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS). The LLE-LTP procedure was optimized via factorial design and by evaluating crucial variables, specifically the freezing mode (either slow or fast), the urine/acetonitrile volume ratio, and the sample ionic strength. The benzodiazepines were quantified using matrix-matched calibration curves where the following parameters were assessed by validation protocol: in general, linearity range of 17 - 200 μg?L–1 (r > 0.9957); limits of detection lower than 5 μg?L–1; relative standard deviations (RSD) lower than 12.5%; and accuracy ranging from 72.3 to 117%. To test this procedure’s performance, the method was applied to determine the content of diazepam in actual urine samples. The validation results obtained for the method demonstrated that the present methodology could be potentially applied in proficient laboratories as a routine approach for determining benzodiazepines compounds content in urine.

Keywords

Benzodiazepines; Urine; LC-HRMS; Extraction Procedure; Low-Temperature Partitioning

Conflicts of Interest

The authors declare no conflicts of interest.

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