Rapid determination of benzodiazepines, zolpidem and their metabolites in urine using direct injection liquid chromatography–tandem mass spectrometry
Graphical abstract
Introduction
Benzodiazepines (BZD) are a group of central nervous system depressant drugs used as hypnotics, anxiolytics, anti-depressants, muscle relaxants and anticonvulsants [1]. There are four main types of BZD, 1,4-benzodiazepines, triazolobenzodiazepines, diazolobenzodiazepines and 7-nitrobenzodiazepines [2]. BDZ are metabolized primarily in the liver via several different microsomal enzymes. The pathways of the BDZ biotransformation involve hepatic microsomal oxidation, aliphatic hydroxylation or N-dealkylation, and glucuronide conjugation [3]. Many hydroxylated metabolites of BDZ are pharmacologically active. As many of these active metabolites have been circulating in the market, it may be not easy to confirm which substance was ingested simply based on the results of analysis. Therefore, caution must be taken in the interpretation of the positive findings. BDZ often had unexpected side effects including a high potential for abuse [4]. They were frequently involved with forensic cases, irrespective of their therapeutic applications.
Zolpidem (ZP) is a non-benzodiazepine hypnotic of the imidazopyridine type for the treatment of insomnia [5]. It binds to the BDZ site on the gamma amino butyric acid-1 receptor [6]. It was considered as a safer medicine than BDZ as there was no evidence of abuse potential in its early period. But over the past decade, numerous cases of zolpidem abuse have been reported in the United States and many other countries. It was revealed that zolpidem had a higher potential for abuse than previously reported [7].
Drug-facilitated crime (DFC) has become a significant problem. Alcohol is the drug most commonly connected with DFC, but recently the use of other drugs is on the rise. Aside from alcohol, BDZ and ZP are the most frequently observed compounds in DFC cases in Korea. According to a previous study, most common substances detected in postmortem samples were ZP, diazepam (DIZ), nordiazepam (NDZ), midzolam (MIZ), alprazolam (APZ), triazolam (TRZ), 7-aminoclonazepam (7-ACLZ), 7-aminoflunitrazepam (7-AFNZ), lorazepam (LRZ), flurazepam (FRZ), temazepam (TMZ), dealkylflurazepam (DA-FRZ), and oxazepam (OXZ) [8].
The steady increase in the nonmedical uses of BZDs and ZP requires convenient and rapid methods in biological samples for forensic purposes. To date, several hyphenated mass spectrometric methods have been reported including gas chromatography–mass spectrometry (GC–MS) [9], [10], [11], [12], [13], capillary electrophoresis–mass spectrometry [14], [15] and liquid chromatography–tandem mass spectrometry (LC–MS/MS) [16], [17], [18], [19], [20]. LC–MS/MS based technique is considered a powerful alternative to GC–MS. The main advantage of this technique is simplified sample preparation of direct measurement of hydrophilic analytes without hydrolysis and derivatization procedures. LC–MS/MS technique provides higher selectivity with less interference from co-elutes and matrices than single MS detection, resulting in less time consuming method development and faster analysis time [21]. Due to its usefulness, direct injection LC–MS/MS methods have been reported for measuring opiates, phenylethylamines, benzylpiperazine, and non-benzodiazepine hypnotics [22], [23], [24], while direct injection LC–MS method was reported for 10 benzodiazepines [25].
There have been continual demands for rapid drug tests for a large number of urine samples and multi-component analysis. LC–MS/MS-based technique is useful as a multi-component screening and is also thought to save the time of sample preparation and speed up the analysis [26], [16]. The aim of the study was to develop a convenient and rapid method for simultaneous determination of 18 benzodiazepines, zolpidem and their metabolites using direct injection LC–MS/MS. The method was validated and its applicability was confirmed by analysis of authentic urine samples.
Section snippets
Chemicals
The reference compounds of zolipem phenyl-4-carboxylic acid (ZPCA), 7-ACLZ, ZP, α-hydroxymidazolam (MIZ-OH), MIZ, FRZ, α-hydroxytriazolam (TRZ-OH), α-hydroxyalprazolam (APR-OH), OXZ, N-desmethylflunitrazepam (N-DFNZ), LRZ, DA-FRZ, NDZ, TMZ, flunirazepam (FNZ), APZ, TRZ, and DIZ were obtained from Cerilliant (Austin, TX, USA). The deuterated internal standards (IS) ZP-d6, MIZ-OH-d4, TRZ-OH-d4, APZ-OH-d5, OXZ-d5, LRZ-d4, NDZ-d5, TMZ-d5, TRZ-d4, and DIZ-d5 were also obtained from Cerilliant.
Optimization of sample preparation and HPLC condition
Urine is easily accessible, can be collected noninvasively and has numerous potential biomarkers including metabolites, proteins, nucleic acids and so on. However, matrix components not removed during sample preparation may cause ion suppression or enhancement. To minimize these difficulties, high speed centrifugation at 50,000 g was applied for fast and efficient clean-up.
In this work, the concentration effects of three acidic modifiers (formic acid, acetic acid and trifluoroacetic acid) and
Conclusions
In the study, a rapid and simple LC–MS/MS method for the determination of 18 drugs widely used for drug facilitated crimes in urine was developed and validated. High speed centrifugation at 50,000 g produced clean supernatants and reduced the interference from the chemical background noise. The direct injection was a crucial step to decreasing sample preparation time and sample loss. The direct injection analysis was also successfully applied to LC–MS/MS with simplicity and higher sensitivity
Acknowledgements
This study was supported in part by a grant (2012-0009835) from the National R&D program of Ministry of Science, ICT and Future planning (MSIP) and National Research Foundation (NRF) of Korea.
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