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Development of a validated HPLC method for the determination of four 1,4‐benzodiazepines in human biological fluids

2007; Wiley; Volume: 30; Issue: 5 Linguagem: Inglês

10.1002/jssc.200600365

1615-9314

Victoria Samanidou, Anastasia P. Pechlivanidou, Ioannis N. Papadoyannis,

Analytical chemistry methods development

Abstract A simple and sensitive HPLC method was developed and validated for the determination of four frequently prescribed 1,4‐benzodiazepines: alprazolam (ALP), bromazepam (BRZ), diazepam (DZP), and flunitrazepam (FNZ). Separation was achieved on an Inertsil C 8 analytical (250 mm×4 mm, 5 μm) column, after selective extraction of benzodiazepine drugs from biological matrices by means of SPE. Isocratic elution was performed with a mobile phase consisting of CH 3 COONH 4 , 0.05 M CH 3 OH, and CH 3 CN (33:57:10 by volume). Quantification was performed at 240 nm with mefenamic acid (6 ng/μL) as the internal standard. DSC‐18 Supelco cartridges provided high absolute recoveries (81–115%). The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, and sensitivity. Repeatability ( n = 8) and between‐day precision ( n = 8) revealed RSD <12%. Recoveries from biological samples ranged from 81.2 to 115%. The detection limit of the method was calculated as 3.3–10.2 ng in blood plasma and 2.6–12.6 ng in urine for 20 μL injection volume. The method was applied to spiked biological matrices. Moreover, the method was applied to real samples of urine after an oral administration.