title: Development of a validated HPLC method for the determination of four 1,4-benzodiazepines in human biological fluids.
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 C8 analytical (250 mm x 4 mm, 5 microm) column, after selective extraction of benzodiazepine drugs from biological matrices by means of SPE. Isocratic elution was performed with a mobile phase consisting of CH3COONH4, 0.05 M CH3OH, and CH3CN (33:57:10 by volume). Quantification was performed at 240 nm with mefenamic acid (6 ng/microL) 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 microL injection volume. The method was applied to spiked biological matrices. Moreover, the method was applied to real samples of urine after an oral administration.
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece.
authors.forename: Victoria F
authors.initials: VF
authors.lastname: Samanidou
authors.forename: Anastasia P
authors.initials: AP
authors.lastname: Pechlivanidou
authors.forename: Ioannis N
authors.initials: IN
authors.lastname: Papadoyannis
mesh_terms: D000284:Administration, Oral; D001569:Benzodiazepines / Q000097:blood* / Q000737:chemistry / Q000494:pharmacology / Q000652:urine*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D015394:Molecular Structure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D001569:Benzodiazepines