Simple and sensitive liquid chromatography/tandem mass spectrometry method for the determination of diazepam and its major metabolites in rat cerebrospinal fluid

Abstract

Diazepam (DZP) is one of the most commonly prescribed drugs for treating status epilepticus (SE). A simple, sensitive and selective LC/MS/MS method with a wide linear calibration range was developed to quantify DZP and its major metabolites, N-desmethyldiazepam (DMDZP), temazepam (TZP), and oxazepam (OZP), in rat cerebrospinal fluid (CSF). The method was used to simultaneously determine the concentrations of all analytes in a small sample volume (as little as 25 microL) of rat CSF. The lower limits of quantification (LLOQ) of the method are 0.04 ng/mL for DZP and 0.1 ng/mL for its metabolites. The calibration range is 0.04-200 ng/mL for DZP and 0.1-200 ng/ml for the metabolites. All intra- and inter-assay coefficients of variation (%CV) and mean percent errors of the method are less than 12%. This method successfully addresses the need to determine low therapeutic drug concentrations in small physiological samples, namely rat CSF. Moreover, it can be used to investigate the distribution of the drug and its metabolites among blood plasma, brain tissue, and CSF in pharmacokinetic and pharmacodynamic studies in a variety of laboratory animals. With respect to animal experiments involving assays in CSF, this method addresses two of the three criteria of Russell and Bruch (Principles of Humane Experimental Techniques, 1959, Methuen and Co., London) for minimizing animal use, namely refinement and reduction.

Novel positive allosteric modulators of GABAA receptors: Do subtle differences in activity at α1 plus α5 versus α2 plus α3 subunits account for dissimilarities in behavioral effects in rats?

“…At 100 nM concentration, which lies well in the range of typical brain levels of BZ site ligands achievable in vivo (cf. Pike et al, 2007; Wang et al, 2003), this potentiation was slightly above (SH-053-2’N and JY-XHe-053) or slightly below (SH-053-S-CH3-2’F) that one elicited through the α 5 -containing subtypes.…”

LC–UV and LC–MS evaluation of stress degradation behaviour of avizafone

“…Some methods were developed for these studies [11][12][13], but none have been associated with mass spectrometry. Numerous LC-MS methods have been reported previously concerning diazepam and its metabolite (desmethyldiazepam) in biological samples [14][15][16].…”

Determination of 14 benzodiazepines and hydroxy metabolites, zaleplon and zolpidem as tert-butyldimethylsilyl derivatives compared with other common silylating reagents in whole blood by gas chromatography–mass spectrometry

“…Giroud et al used the same analytical technique for the simultaneous quantitative analysis of zolpidem and zaleplon, but did not include benzodiazepines [12]. Recent quantitative methodology for the analysis of benzodiazepines in serum, plasma or whole blood includes dual-column GC [13], GC-MS [14][15][16][17], GC-tandem mass spectrometry (GC-MS/MS) [18,19], liquid chromatography (LC) [20,21], liquid chromatography-mass spectrometry (LC-MS) [22][23][24][25][26] and liquid chromatography-tandem mass spectrometry (LC-MS/MS) [27][28][29][30]. However, GC-MS based techniques remain a method of choice for many routine laboratories due to the separation efficiency, versatility, ease of operation and maintenance, as well as lower costs of the analyses and investment expenses of an analytical system compared to LC-MS/MS.…”