Simultaneous determination of fifteen low-dosed benzodiazepines in human urine by solid-phase extraction and gas chromatography–mass spectrometry

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Abstract

A gas chromatographic–mass spectrometric method was developed for the simultaneous analysis of 15 low-dosed benzodiazepines, both parent compounds and their corresponding metabolites, in human urine. The target compounds are alprazolam, α-hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylflurazepam, nitrogen-desalkylflurazepam, ketazolam, oxazepam, lormetazepam, lorazepam, triazolam and α-hydroxytriazolam. Nitrogen-methylclonazepam is used as the internal standard. The urine sample preparation involves enzymatic hydrolysis of the conjugated metabolites with Helix pomatia β-glucuronidase for 1 h at 56°C followed by solid-phase extraction on a phenyl-type column. The extracted benzodiazepines are subsequently analyzed on a polydimethylsiloxane column using on-column injection to enhance sensitivity. The extraction efficiency exceeded 80% for all compounds except for oxazepam, lorazepam and 4-hydroxyalprazolam which had recoveries of about 60%. The LODs ranged from 13 to 30 ng/ml in the scan mode and from 1.0 to 1.7 ng/ml in the selected ion monitoring (SIM) mode. Linear calibration curves were obtained in the concentration ranges from 50 to 1000 ng/ml in the scan mode and from 5 to 100 ng/ml in the SIM mode. The within-day and day-to-day relative standard deviations at three different concentrations never exceeded 15%.

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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