Borges KB, Freire EF, Martins I, de Siqueira ME
Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma. [Journal Article]
Talanta 2009 Apr 15; 78(1):233-41.
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 (250mmx4.6mm, 5mum) 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 0.8mLmin(-1). UV detection was carried out at 245nm. Employing LLE, the best conditions were achieved with double extraction of 0.5mL plasma using ethyl acetate and Na(2)HPO(4) pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5mL plasma plus 3mL 0.1M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3mL 0.1M borate buffer pH 9.5, 4mL Milli-Q water and 1mL 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 50-1200ngmL(-1) plasma (r>/=0.9927), limits of quantification of 50ngmL(-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 30-1200ngmL(-1) plasma (r>/=0.9900), limits of quantification of 30ngmL(-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.
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