| Objective:To explore the best method of extraction and separation of 4 benzodiazepines including clozapinein,lorazepam,oxazepam and temazepam in human blood and urine samples,and establish an ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method to detect 4 benzodiazepines in human blood and urine samples.Methods:Biological samples were determined by UPLC-MS/MS method after processed with dilution method,protein deposition method and solid phase extraction method,respectively.The mobile phase was acetonitrile-0.1%ammonia water solution(lmmol ammonium bicarbonate)and running in gradient elution.The mass spectrometric acquisition was carried out by electrospray ionization in positive mode(ESI+)with multiple reaction monitoring(MRM)method.The multiple characteristic ion pairs were adopted to 4 benzodiazepines for qualitative analysis and the external standard calibration curve method was adopted to 4 benzodiazepines for quantitative analysis.Results:The LODs of 4 benzodiazepines in human urine sample were 0.13ng/mIL,0.08ng/mL,0.03ng/mL and 0.02ng/mL;the LOQs were 0.43ng/mL,0.28ng/mL,0.10ng/mL and 0.07ng/mL after they were diluted.The LODs of 4 benzodiazepines in human urine sample were 0.04ng/mL,0.05ng/mL,0.04ng/mL and 0.008ng/mL;the LOQs were 0.14ng/mL,0.16ng/mL,0.15ng/mL and 0.028ng/mL after they were pretreated by methanol precipitation.The LODs of 4 benzodiazepines in human blood sample were 0.04ng/mL,0.02ng/mL,0.06ng/mL and 0.005ng/mL;the LOQs were 0.15ng/mL,0.16ng/mL,0.12ng/mL and 0.017ng/mL after they were pretreated by methanol precipitation.The LODs of 4 benzodiazepines in human urine sample were 0.01ng/mL,0.045ng/mL,0.05ng/mL and 0.002ng/mL;the LOQs were 0.13ng/mL,0.15ng/mL,0.19ng/mL and 0.008ng/mL after they were pretreated with Waters Oasis MCX column.The LODs of 4 benzodiazepines in human blood sample were O.Olng/mL,0.02ng/mL,0.016ng/mL and 0.01ng/mL;the LOQs were 0.20ng/mL,0.07ng/mL,0.054ng/mL and 0.034ng/mL after they were pretreated with Waters Oasis MCX column.The extraction recoveries of 4 benzodiazepines in human urine sample were 53.2%-82.2%,55.5%-82.2%,53.4%?72.5%and 53.4%?83.3%;the matrix effects were 103.4%?122.4%,104.3%?119.9%,99.9%?114.5%and 90.1%?120.5%after they were diluted.The extraction recoveries of 4 benzodiazepines in human urine sample were 70.0%?82.4%,71.1%?83.0%,72.6%?81.4%and 74.8%?88.0%;the matrix effects were 101.6%?18.8%,103.9%?119.0%,108.9%?118.0%and 97.2%?114.7%after they were pretreated by methanol precipitation.The extraction recoveries of 4 benzodiazepines in human blood sample were 70.8%?85.4%,72.9%?80.2%,76.9%?82.8%and 70.4%?79.1%;the matrix effects were 88.8%?113.9%,105.4%?116.6%,104.5%?118.2%and 104.5%-118.7%after they were pretreated by methanol precipitation.The extraction recoveries of 4 benzodiazepines in human urine sample were 78.8%?101.0%,80.4%-89.6%,81.1%?97.8%and 80.2%?97.8%;the matrix effects were 76.6%?101.2%,81.6%~111.2%,79.8%?103.7%and 81.6%?99.6%after they were pretreated with Waters Oasis MCX column.The extraction recoveries of 4 benzodiazepines in human blood sample were 75.4%?99.4%,81.3%?97.4%,76.1%?99.3%and 76.1%?97.8%;the matrix effects were 80.3%?102.9%,85.0%?106.4%,83.9%-99.2%and 69.3%?99.5%after they were pretreated with Waters Oasis MCX column.The accuracies of 4 benzodiazepines in human urine sample were 75.2%?93.6%,75.7%-89.0%,70.4%?87.4%and 76.1%?90.6%after they were pretreated with Waters Oasis MCX column;precisions during the day and daysof 4 benzodiazepines in human urine sample were 3.3?6.4,3.1?7.1,4.6?6.6,3.7?8.0 and 1.9?6.5,3.3?7.8,3.3?5.7,2.5-7.2,respectively.The accuracies of 4 benzodiazepines in human blood sample were 64.6%?84.2%,72.5%?92.0%,66.6%?91.4%and 70.4%?93.0%after they were pretreated with Waters Oasis MCX column;precisions during the day and days of 4 benzodiazepines in human blood sample were 2.6?8.4,3.1?5.2,2.3?6.9,4.4?8.4 and 4.9-5.9,3.5-8.7,3.2-6.4,3.0?5.4,respectively.Conclusions:This paper has established an UPLC-MS/MS method to detect 4 benzodiazepines including clozapinein,lorazepam,oxazepam and temazepam in human biological fluid samples.The extraction effect of the Waters Oasis MCX column is the best for 4 benzodiazepines after comparing with the extraction recoveries and the matrix effect of three sample processing methods for human blood and urine samples,which could be used for the pretreatments in court on a scientific laboratory analysis of 4 benzodiazepines in human biological fluid samples. |
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