Borges Keyller Bastos, Freire Ellen Figueiredo, Martins Isarita, de Siqueira Maria Elisa Pereira Bastos
Departament of Physics and Chemistry, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil.
Talanta. 2009 Apr 15;78(1):233-41. doi: 10.1016/j.talanta.2008.11.003. Epub 2008 Nov 12.
A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm x 4.6 mm, 5 microm) at 35 degrees C under isocratic conditions using 5mM KH(2)PO(4) buffer solution pH 6.0:methanol:diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min(-1). UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL plasma using ethyl acetate and Na(2)HPO(4) pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1M borate buffer pH 9.5, 4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether:n-hexane:methanol (50:30:20). In both methods the solvent was evaporated at 40 degrees C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL(-1) plasma (r>or=0.9927), limits of quantification of 50 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL(-1) plasma (r>or=0.9900), limits of quantification of 30 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis.
描述了一种使用液-液萃取和固相萃取，随后进行高效液相色谱（HPLC）分析，以N-去烷基氟西泮为内标同时测定人血浆中七种苯二氮䓬类药物（BZPs）（氟硝西泮、氯硝西泮、奥沙西泮、劳拉西泮、氯氮卓、去甲西泮和地西泮）的方法。使用LC-18 DB柱（250 mm×4.6 mm，5 µm）在35℃等度条件下，以5mM KH(2)PO(4)缓冲溶液pH 6.0:甲醇:乙醚（55:40:5，v/v/v）作为流动相，流速为0.8 mL min(-1)分离分析物。在245 nm处进行紫外检测。采用液-液萃取时，最佳条件是用乙酸乙酯和pH 9.5的Na(2)HPO(4)对0.5 mL血浆进行两次萃取以调节pH值。采用固相萃取时，最佳条件是0.5 mL血浆加3 mL 0.1M pH 9.5的硼酸盐缓冲液，然后通过预先用这些溶剂平衡过的C18柱：3 mL 0.1M pH 9.5的硼酸盐缓冲液、4 mL超纯水和1 mL 5%乙腈，最后用乙醚:正己烷:甲醇（50:30:20）洗脱BZPs。两种方法中，溶剂均在40℃氮气流下蒸发。液-液萃取得到的验证参数为：血浆线性范围50 - 1200 ng mL(-1)（r≥0.9927），定量限为50 ng mL(-1)血浆，日内和日间CV%以及用于精密度和准确度的E%低于15%，所有BZPs的回收率高于65%。在固相萃取中，得到的参数为：血浆线性范围30 - 1200 ng mL(-1)（r≥0.9900），定量限为30 ng mL(-1)血浆，日内和日间CV%以及用于精密度和准确度的E%低于15%，所有BZPs的回收率高于55%。这些萃取程序随后进行HPLC分析显示它们在检测人血浆中的一种或多种BZPs方面具有合适的适用性。此外，可以认为这些程序可用于各种分析应用，特别是毒理学/法医分析。
