Bromazepam determination in human plasma by high‐performance liquid chromatography coupled to tandem mass spectrometry: a highly sensitive and specific tool for bioequivalence studies

Abstract

A rapid, sensitive and specific method to quantify bromazepam in human plasma using diazepam as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using diethyl ether-hexane (80 : 20, v/v). The extracts were analyzed by high-performance liquid chromatography (HPLC) coupled to electrospray tandem mass spectrometry (MS/MS). Chromatography was performed isocratically on a Genesis C(18) analytical column (100 x 2.1 mm i.d., film thickness 4 microm). The method had a chromatographic run time of 5.0 min and a linear calibration curve over the range 5.0-150 ng ml(-1) (r(2) > 0.9952). The limit of quantification was 5 ng ml(-1). This HPLC/MS/MS procedure was used to assess the bioequivalence of two bromazepam 6 mg tablet formulations (bromazepam from Medley SA Indústria Farmacêutica as the test formulation and Lexotan from Produtos Roche Químico e Farmacêutico SA as the reference formulation). A single 6 mg dose of each formulation was administered to 24 healthy volunteers (12 males and 12 females). The study was conducted using an open, randomized, two-period crossover design with a 3 week washout interval. Since the 90% CI for C(max), AUC(last), AUC(0-240 h) (linear) and AUC((0- infinity )) ratios were all inside the 80-125% interval proposed by the US Food and Drug Administration, it was concluded that the bromazepam formulation from Medley is bioequivalent to the Lexotan formulation for both the rate and the extent of absorption.

Validated method for determination of bromopride in human plasma by liquid chromatography–electrospray tandem mass spectrometry: application to the bioequivalence study

“…Bromopride is chemically related to metoclopramide and is administered as a single oral dose over a range of 10 to 30 mg. Bromopride has a volume of distribution (a measure of the apparent space in the body available to contain the drug) 1 of 3.07 l/kg, and it is only bound 40% to plasma protein 2,3 and reaches a dose-related maximum peak concentration (C max D 20 to 64 ng/ml) in 2.8 h. The systemic bioavailability (related to the concentration of the drug in the plasma) ranges from 54 to 78% and its elimination half-life is around 4.9 h. Quantification of drugs in biological matrices by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is becoming more common, because of the improved sensitivity and specificity of this technique. 4,5,6 Some other techniques have been used to determine bromopride in a variety of matrices. Such methods included high-performance liquid chromatography (HPLC) with UV detection, 2,3,7 thin-layer chromatography (TLC) and gas chromatography (GC).…”

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the quantitation of prazepam and its main metabolites in human plasma

“…6,7 A gas chromatographic (GC) procedure coupled to mass spectrometric (MS) detection has recently been reported. 8 An enzyme-linked immunosorbent assay (ELISA) has been also proposed for screening benzodiazepines and opiates in post mortem blood samples.…”

Experimental design for optimization of microwave-assisted extraction of benzodiazepines in human plasma

“…Liquid chromatography has become an important tool for routine determination of BZD owing to its specificity, rapidity and sensitivity [4][5][6][7][8]. Although high-performance liquid chromatography (HPLC) with mass spectrometry detection [5][6][7] is highly selective, the cost of the instrumentation makes it less suitable for clinical or toxicology laboratories.…”

“…Although high-performance liquid chromatography (HPLC) with mass spectrometry detection [5][6][7] is highly selective, the cost of the instrumentation makes it less suitable for clinical or toxicology laboratories. In contrast, HPLC-UV detection or diode array detection (DAD) instrumentation is widely available in most analytical laboratories [8][9][10][11][12].…”