A sensitive and selective method for the detection of diazepam and its main metabolites in urine by gas chromatography–tandem mass spectrometry
Introduction
Benzodiazepines constitute a class of versatile and widely prescribed CNS-depressant. They are prescribed as anxiolytics, sedative hypnotics, anticonvulsants and muscle relaxants [1], [2], [3], [4]. These drugs are very frequently encountered in clinical and forensic toxicology, having been featured in an increasing number of misuses and abuses over the past years [5], [6], [7]. Benzodiazepines may affect performances such as driving a vehicle or flying an aircraft [8], [9]. Their detection and quantification at the low levels encountered in body fluids require reliable and sensitive methods [10].
An important number of studies have been reported about the analysis of benzodiazepines and their metabolites. Because of cross reactivity, immunological techniques need to be confirmed by chromatography–mass spectrometry techniques that are known to be more specific [11], [12]. Even though the recent developments of liquid chromatography–mass spectrometry (LC–MS) have shown very promising results, most of the literature devoted to benzodiazepines analysis involves gas chromatography–mass spectrometry (GC–MS) techniques. For a long time, most of these studies have only been performed on urine [13], [14], [15], blood, serum or plasma extracts [16], [17], [18], [19], [20]. However, in the last decade, particular attention has been focused on hair analysis [21]. Electron capture ionization appeared to be the most powerful ionization mode for the analysis of benzodiazepines, in terms of sensitivity and selectivity; it has been widely used, especially for hair analysis [22], [23], [24].
In a general way, selectivity and specificity of MS techniques are greatly enhanced when MS/MS experiments can be performed. Concerning specificity, a recent European recommendation about validation of GC–MS methods attributes four points to MRM methods involving two transitions from one precursor ion and “n” points to SIM methods monitoring “n” ions [25]. We did not find in the literature any method associating electron capture ionization and MS/MS detection for the analysis of benzodiazepines. The aim of this study was to develop and test such a method. Diazepam, nordazepam and oxazepam were chosen for this study because diazepam (Valium®) is undoubtedly one of the most consumed benzodiazepines worldwide. Nordazepam and oxazepam are the metabolites of diazepam as well as being active drugs (Nordaz® and Seresta®, respectively). Diazepam is metabolized in the liver by P450 mediated reactions [26]. About 60% of a given dose is N-demethylated to yield nordazepam, the major metabolite found in plasma (Fig. 1). C3-hydroxylation of diazepam yields temazepam. Both metabolites can be further converted to oxazepam. The chemical structures of the studied benzodiazepines are displayed in Fig. 1. The applicability and reliability of the method have been tested on urine extracts.
Section snippets
Chemicals
Manufacturers kindly provided the three benzodiazepines: diazepam from Roche (Neuilly-sur-Seine, France), nordazepam and oxazepam from Parke-Davis (Courbevoie, France). All benzodiazepines were more than 99% pure. The deuterated standard [2H5]-Diazepam (hydrogen atoms of the C6H5 phenyl group were replaced at 99.9% by deuterium atoms), was purchased from Promochem (Molsheim, France). Methanol HPLC grade was obtained from Prolabo (Fontenay-sous-Bois, France).
Extraction protocol
TOXI-TUBES A liquid/liquid extraction
MS–MS method development
As previously reported by Joice et al., oxazepam undergoes thermal decomposition (water elimination via a Frigerio-type rearrangement) during the injection step [26]. We confirmed in this work that H2O elimination from oxazepam is complete when performing injection at 280 °C, so we decided to quantify oxazepam on the chromatographic peak of its degradation product (molecular weight of 268) to avoid a derivatization step during sample preparation. The three analytes, diazepam, nordazepam and
Conclusions
The method presented associates electron capture ionization and multiple reactions monitoring performing CID with xenon as collision gas. The sample preparation part involves liquid/liquid extraction with TOXI-TUBES A; it takes only a few minutes and provides recovery yields between 68 and 95% with CV below 6% (n = 10). From 1 mL of urine, the method provides quantitation limits of 0.15 ng/mL for diazepam, 1.0 ng/mL for nordazepam and 1.5 ng/mL for oxazepam. The applicability of the method was
Acknowledgements
We greatly acknowledge VARIAN S.A. (France) for its technical support, especially Michel Lesieur and John Ogden for their helpful advices.
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