Elsevier

Talanta

Volume 78, Issue 1, 15 April 2009, Pages 233-241
Talanta

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid–liquid and solid-phase extractions in human plasma

https://doi.org/10.1016/j.talanta.2008.11.003Get rights and content

Abstract

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid–liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm × 4.6 mm, 5 μm) at 35 °C under isocratic conditions using 5 mM KH2PO4 buffer solution pH 6.0:methanol:diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min−1. UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL plasma using ethyl acetate and Na2HPO4 pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5, 4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether:n-hexane:methanol (50:30:20). In both methods the solvent was evaporated at 40 °C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50–1200 ng mL−1 plasma (r  0.9927), limits of quantification of 50 ng mL−1 plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30–1200 ng mL−1 plasma (r  0.9900), limits of quantification of 30 ng mL−1 plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis.

Introduction

Benzodiazepines (BZPs) are an important class of drugs commonly used as minor tranquillizers, hypnotics, muscle relaxant and anticonvulsive. The usefulness of BZPs varies considerably because there are large differences in selectivity among these drugs. They are among the most frequently prescribed drugs for the treatment of anxiety, sleep disturbance and status epilepticus. In addition, BZPs are used in treatment of alcohol withdrawal, to relieve tension in the preoperative period and to induce amnesia in surgical procedures. The abuse of these drugs is very common among its users and illegal drug users. Moreover, the continued abuse of these drugs leads to dependence. Therefore, these compounds may be involved in cases of sudden death and be linked to different crimes [1].

Laboratories, thus, frequently receive request for the determination of BZPs in plasma, serum, urine, or gastric lavage fluid. The analysis of BZPs in biological fluids is complicated by the diversity of these drugs commercially available and the fact that each product has a particular therapeutic and toxic range. The classical BZPs are chemically characterized by the presence of a benzene ring fused on the -10 and -11 positions of the 1,4-diazepine ring. All the important BZPs contain a 5-phenyl ring (in many cases, this ring suffers an additional substitution of halogen, F, Cl or Br, at RH-position), a halogen or nitro group in the 7-position (Fig. 1).

Several methods have been described in the literature for the clinical, toxicological and biopharmaceutical study of the BZPs. Besides spectrophotometric, spectrofluorimetric [2], [3], [4], potentiometric [5], [6] and immunoassay [7] methods, the majority of the papers describe methods employing HPLC (using UV or MS detector) [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], GC [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], and CE [14], [31].

The sample preparation techniques commonly used involve liquid–liquid extraction (LLE) [39], [40], [41], solid-phase extraction (SPE) [28], [33], [37], solid-phase microextraction (SPME) [17], [23], [34], liquid phase microextraction (LPME) [34], column switching techniques [42], [43] and direct LC analysis using restricted access materials (RAM) [44], [45].

Since HPLC/UV assays are inexpensive and widely utilized, it has been a good choice in the analysis of BZPs for simultaneous separation and quantification. Moreover, SPE and LLE are still the most employed techniques for sample preparation more used in routine analysis. The presence of different methods in the literature using these techniques is a great advantage for analysts from different areas. In this context, there are numerous detection methods, but no one is ideal due to the large number of chemically similar compounds, the wide variation in therapeutic and toxic range, and the intensive metabolism suffered by BZPs. In addition, exhaustive extraction techniques, such as LLE and SPE, are necessary because many analyses require a significant pre-concentration step. The purpose of this investigation was to develop a simple analytical HPLC method alongside a liquid–liquid extraction and an off-line solid-phase extraction procedures for the simultaneous analysis of seven benzodiazepines (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) (Fig. 2) in plasma samples.

Section snippets

Standard solutions and chemicals

BZPs, flunitrazepam, clonazepam, oxazepam, lorazepam, N-desalkylflurazepam (internal standard, I.S.), chlordiazepoxide, nordiazepam and diazepam were all obtained from Sigma–Aldrich® (St Louis, USA).

Stock standard solutions of flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam were prepared by dissolution of each drug in methanol to obtain a concentration of 1 mg mL−1. These standard solutions were used in dilution series to result in the following

Results and discussion

The methods of BZPs analysis in plasma must present high detectability to allow detection of low quantities of analytes (ng mL−1). The HPLC with UV detection employing LLE and SPE are still the most employed techniques used in routine analysis of BZPs. These drugs are amphoteric and relatively polar, moreover the biological sample preparation step using LLE is usually laborious and time consuming. SPE would, therefore, be a simple and reliable alternative, despite been expensive, since the

Conclusions

A suitable high-performance liquid chromatography method was developed and validated using LLE and SPE as extraction procedures for the determination of flunitrazepam, clonazepam, oxazepam, lorazepam, N-desalkylflurazepam (internal standard, I.S.), chlordiazepoxide, nordiazepam and diazepam simultaneously in human plasma. This HPLC method provides a good alternative to existing methods for analyzing BZPs employing LLE and SPE. The major advantage of this method over similar methods is that it

Acknowledgements

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), sub-program BIOTA/FAPESP (Rede BIOprospecTA), Financiadora de Estudos e Projetos - CT-INFRA (FINEP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support and for granting research fellowships.

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