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

作　　者：Elisangela Jaqueline Magalhaes Clesia Cristina Nascentes Rodinei Augusti Maria Eliana Lopes Ribeiro de Queiroz Julio Cesar Cardoso da Silva Robson Jose de Cassia Franco Afonso 

机构地区：[1]Departamento de Quimica,Universidade Federal de Minas Gerais,Belo Horizonte,Brazil [2]Departamento de Quimica,Universidade Federal de Vicosa,Vicosa,Brazil [3]Departamento de Quimica,Universidade Federal de Ouro Preto,Ouro Preto,Brazil

出　　处：《American Journal of Analytical Chemistry》2012年第2期118-124,共7页美国分析化学（英文）

基　　金：Conselho Nacional de Desenvolvimento Cientifico e Tecnologico(CNPq)and Fundacao de Amparo a Pesquisa do Estado de Minas Gerais(FAPEMIG)for financial support and research fellowships.

摘　　要：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.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.

关 键 词：BENZODIAZEPINES URINE LC-HRMS Extraction Procedure Low-Temperature Partitioning 

分 类 号：R73[医药卫生—肿瘤]