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.

Development and validation of an HPLC method for the determination of benzodiazepines and tricyclic antidepressants in biological fluids after sequential SPE

“…Common HPLC procedures need sample pretreatment based on protein precipitation, liquid -liquid extraction [44], and SPE [5,33,45]. Some of these drugs have been determined in serum by direct on-column injection [46], automated online SPE [30], SPME [47,48], online dialysis [30], direct LC analysis using restricted access extraction materials [49], supported liquid membrane technique [50], column switching techniques [51,52] as well as liquid phase microextraction [53,54].…”

Mixed‐mode SPE for a multi‐residue analysis of benzodiazepines in whole blood using rapid GC with negative‐ion chemical ionization MS

“…The developed method has been compared with previously described methods suitable for the determination of benzodiazepines in plasma or whole blood after SPE. The extraction efficiency of QC by our method is higher in comparison to other published methods [6,12,21,22]. A review of these four studies with respect to the accuracy of benzodiazepine determination shows accuracies of 81-114% [6], 85-115% [12], 88-114% [21], and 83-93% [22].…”

Boron‐Doped Diamond Electrode Coupled to Liquid Chromatography: Application to Simultaneous Determination of Benzodiazepines

“…(vs. Ag/AgCl). In the case of NZP, CZP and FZP, the presence of the nitro-phenyl group, yields the possibility to detect those substances at lower potential, but in a general screening of benzodiazepines a working potential of À 1.4 V (Ag/AgCl) was shown to be appropriate [31]. In a study performed by Honeychurch and Hart [32] both nitrazepam and flunitrazepam have been determined by HPLC dual electrode detection in the reductive-reductive mode, using a carbon fiber veil electrode in conjugation with a glassy carbon electrode.…”