The 1,4-benzodiazepines constitute an important class of psychotherapeutic agents known for their hypnotic, tranquilizing and anti-convulsant properties. Comprehensive research has been performed to study their clinical performance.¹ Diazepam is one of the most frequently prescribed benzodiazepines for the treatment of anxiety, sleep disturbance and alcohol withdrawal.² However, the use of diazepam for alcohol withdrawal deserves clinical attention and monitoring because hepatic dysfunction usually accompanies alcohol dependence syndrome that depends on the metabolism rate in the liver.³

For the clinical, toxicological and biopharmaceutical study of benzodiazepines various biological fluids have been analysed. Clinically effective doses of benzodiazepines undergo extensive metabolic reactions and many of these metabolites are pharmacologically active.⁴

Identification of those pharmacologically active benzodiazepines is best performed on blood following a positive urine screening. It is, therefore, essential that assay methods are sensitive and specific (i.e., capable of separating and determining the parent drug, as well as its major metabolites).⁵

However, these immunoassay methods are not capable of differentiating the primal molecule from minor metabolites, so they are often applied together with other chromatographic techniques.

In Brief

The extraction procedures are relatively simple, derivatization is not necessary and operation at ambient temperature allows the determination of thermally stable benzodiazepines. Strong absorption in the 230–260 nm wavelength region gives sensitivity in the nanogramme range and linearity over a wide concentration range. Moreover, because the technique is non-destructive, the eluted drugs can be recovered for further examination.¹⁸ In recent years, the coupling of LC to MS (LC–MS) has provided a useful and rugged technique for the analysis of drug compounds and an alternative to GC–MS in which some compounds thermally decompose to give metabolites common to many of benzodiazepines.^19–21 Now, LC–MS is a fairly established technique for benzodiazepine analysis.^22,23 Compared with LC–UV systems, LC–MS is much more selective and sensitive. In applications where plasma concentrations are high, the use of the LC–UV is more than adequate. Whereas, for clinical purposes LC–MS is quite expensive where low costs are mandatory for routine clinical analysis. Because HPLC-UV assays are inexpensive and widely used, it appeared to be the best option to perform simultaneous separation, quantification and clinical monitoring of diazepam as a primary concern of this paper.

As well as clinical research applications, some other applications involving the determination and quantification of benzodiazepines with HPLC-UV analysis technique have also been reported recently.^24,25

Differences in the metabolism rates of benzodiazepines in humans can be expressed by monitoring drug levels.⁴ When considering the management of alcohol withdrawal, as well as the clinical assessments, the dose of diazepam and free diazepam levels in biological fluid is required to display the variations in half-life of benzodiazepines according to each patient.²⁶

The scope of this study is to construct and validate a rapid and simple method to determine and monitor free diazepam in human plasma and urine; and also to emphasize the correct dosage of diazepam by simultaneously monitoring its levels in plasma and urine. For this purpose two groups of patients being treated for alcohol-withdrawal syndrome in Ankara University Hospital Psychiatry Clinic were gathered. Free diazepam levels in the plasma and urine samples from patients in the first group who had single oral dose of diazepam, were investigated to display the personal variations of drug metabolism. From the same point of view, the findings from the second group who had repeated daily constant doses were investigated, to denote drug accumulation in individuals because of metabolism variations.

Experimental

Chemicals and reagents: Sodium carbonate, sodium dihydrogen phosphate dehydrate, disodium hydrogen phosphate, sodium hydroxide, ethyl acetate, diethyl ether and HPLC-grade methanol were purchased from Merck (Darmstadt, Germany). The standards of pharmaceuticals, diazepam and nitrazepam (Internal standard) were obtained from Sigma-Aldrich (St Louis, Missouri, USA). Prescribed diazepam tablets for patients were obtained from pharmaceutical firm Deva Medical Company (Istanbul, Turkey). All chemicals were of analytical-grade in the highest purity available.