Journal of Chromatography B: Biomedical Sciences and Applications
Simultaneous determination of fifteen low-dosed benzodiazepines in human urine by solid-phase extraction and gas chromatography–mass spectrometry
Introduction
Benzodiazepines are among the most widely prescribed drugs, being used in the treatment of stress, anxiety, sleep disorders, muscle spasms and seizures. Many patients develop a dependence on these drugs which are often involved in intoxications. Consequently, benzodiazepines are frequently encountered both in clinical and forensic toxicological analyses.
For identification purposes, urine is the preferred matrix as the concentrations of benzodiazepines and their metabolites are higher in urine than in plasma. This is especially relevant for the low-dosed compounds. Benzodiazepines are extensively metabolized and many metabolites are excreted in urine as glucuronide conjugates. Cleavage of these conjugates by acid hydrolysis is fast and therefore often applied but decomposes the benzodiazepine molecules to benzophenones [1], [2], [3]. In this way unequivocal identification is impaired as some compounds yield common benzophenones. During enzymatic hydrolysis, which is a more gentle procedure, benzodiazepines remain intact. The commonly used β-glucuronidases are produced from different sources like snail intestinal juice (Helix pomatia), bovine liver and bacteria (Escherichia coli), and the reported hydrolysis conditions also vary widely [4], [5], [6], [7].
A large number of analytical methods have been published for the determination of benzodiazepines. Most of these methods can only be applied to the analysis of either parent compounds alone or one parent benzodiazepine and its corresponding metabolites. Traditional liquid–liquid extraction (LLE) techniques are still very popular and solvents used to extract benzodiazepines include chloroform [8], [9], diethylether [10], [11], n-butyl acetate [12], [13] or mixtures of different solvents [14], [15]. Extractions are mostly performed under slightly alkaline conditions (pH 9–10) obtained with dilute sodium hydroxide or sodium carbonate, –phosphate and –borate buffers.
Existing solid-phase extraction procedures for benzodiazepines cover a broad range of apolar bonded-phase cartridges: C18 [16], [17], C8 [18] or C2 [19], [20]. Mixed-phase Bond Elut Certify columns are also commonly used, as well for gas chromatography (GC) [21], [22] as for high-performance liquid chromatography (HPLC) applications [23], [24].
As none of the sample preparation methods described in the literature proved satisfactory, our aim was to develop a new, sensitive and simple extraction procedure using phenyl-type solid-phase extraction columns for the simultaneous determination of the following low-dosed benzodiazepines and their corresponding metabolites in human urine: ketazolam (Solatran, Unakalm), oxazepam; flunitrazepam (Rohypnol, Hypnocalm), 7-aminoflunitrazepam, desmethyl-flunitrazepam; flurazepam (Staurodorm), hydroxyethylflurazepam, N-desalkylflurazepam; lormetazepam (Loramet), lorazepam (Serenase, Temesta); alprazolam (Xanax), 4-hydroxyalprazolam, α-hydroxyalprazolam; triazolam (Halcion) and α-hydroxytriazolam. Unequivocal identification of each benzodiazepine is guaranteed by the combination of enzymatic hydrolysis and mass spectrometric detection.
Section snippets
Solvents and reagents
Standards of ketazolam, alprazolam, 4-hydroxyalprazolam, α-hydroxyalprazolam, triazolam and α-hydroxytriazolam were a gift from Upjohn (Kalamazoo, MI, USA). Flunitrazepam, desmethylflunitrazepam, 7-aminoflunitrazepam, 7-aminodesmethyl-flunitrazepam, and the internal standard N-methylclonazepam were a gift from Hoffman-La Roche (Basel, Switzerland) and flurazepam and hydroxyethylflurazepam were a gift from Madaus-Therabel (Brussels, Belgium). N-Desalkylflurazepam was obtained from Mikromol
Results and discussion
Enzymatic hydrolysis conditions were optimized using a urine sample from a patient on lormetazepam prescription. Absolute recoveries could not be determined because of the unavailability of the benzodiazepine–glucuronide conjugate standard. In four series of experiments the following parameters were optimized: enzyme activity, hydrolysis pH, temperature and incubation time. In the first series of experiments 1-ml aliquots of urine were adjusted to pH 5.0 and incubated at 56°C for 2 h with
Conclusion
The optimized enzymatic hydrolysis conditions, followed by the developed phenyl-phase extraction and acetylation procedures provided satisfying recoveries of all selected benzodiazepines. The obtained extracts were suitable for on-column injection. This injection technique in combination with GC–MS proved to be very sensitive and selective for the determination of 15 commonly used low-dosed benzodiazepines in human urine samples.
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