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Diazepam is frequently used as an adjuvant during antidepressant therapy. Recently, some studies have suggested that the treatment with benzodiazepines could have different efficacy in depressed patients as opposed to non-depressed ones. To clarify the matter, a study is currently underway, regarding the drug metabolism in rats. In order to obtain a more complete and significant set of data, the main diazepam metabolites have also been considered, namely: nordiazepam, temazepam and oxazepam. A feasible and reliable HPLC method has been developed for the simultaneous determination of these compounds in plasma and brain tissue of rats. The method has been applied to "normal" rats and to genetic rat models of depression in order to estimate drug metabolism in different breeds. Analyte separation was achieved on a C8 reversed phase column using an acidic phosphate buffer/acetonitrile mixture as the mobile phase. The detection wavelength was 238 nm. An original sample pre-treatment, based on solid-phase extraction (SPE) was developed in order to eliminate endogenous interference, using only 250 microL of matrix (brain homogenate or plasma) for a complete analysis. The method has been validated with good results in terms of precision, extraction yield, sensitivity, selectivity and accuracy on both matrices and has been successfully applied to samples from some rats subjected to the preliminary study. The obtained data will hopefully contribute to the clarification of possible differences between depressed and non-depressed subjects with respect to benzodiazepine biotransformation.

The enantioselective high-performance liquid chromatography (HPLC) of three racemic 3-hydroxybenzodiazepines, oxazepam (Oxa), lorazepam (Lor), and temazepam (Tem), is a difficult operation because of the spontaneous chiral inversion in polar solvent. To solve this problem, we have developed an HPLC method based on a chiral Cyclobond I-2000 RSP column, maintained at 12 degrees C, and a reversed mobile phase (acetonitrile in 1% triethylamine acetate buffer, TEAA) at a flow rate of 0.4 ml/min. Peaks were detected by a photodiode-array detector at 230 nm for quantification and by an optical rotation detector for identification of (+) and (-) enantiomers. The results showed that peak resolutions of Oxa, Lor, and Tem enantiomers, analyzed under the same conditions, were 3.2, 2.0, and 1.8, respectively. For the determination of Oxa enantiomers in plasma of rabbits, extraction with diethyl ether at pH 1.5, a polar organic mobile phase, and a Cyclobond I-2000 SP column were used. Other analytical conditions were the same as previously described. Blood samples were immediately cooled at 4 degrees C and centrifuged at 0 degrees C for the collection of plasma. The results showed a difference in plasma S(+)- and R(-)-oxazepam concentrations in rabbits. No racemization of S(+)- or R(-)-Oxa enantiomers, added alone to blank plasma, was observed after extraction and enantioselective HPLC analysis., (Copyright 2002 Elsevier Science B.V.)

Enantiomers of 3-O-methyloxazepam and 3-O-ethyloxazepam were resolved by chiral stationary phase high-performance liquid chromatography (CSP-HPLC). Temperature-dependent and acid-catalysed racemization of 3-O-methyloxazepam enantiomers in methanol and 3-O-ethyloxazepam enantiomers in ethanol were studied by quenching reaction products at various times by neutralization. Enantiomeric contents of reaction product were determined by CSP-HPLC. Thermodynamic parameters in the formation of the activated complex (Eact, delta H++, delta S++ and delta G++) were consistent with those determined by a spectropolarimetric method. A nucleophilically solvated and transient C3 carbocation intermediate resulting from an N4-protonated enantiomer is proposed to be an intermediate and responsible for the acid-catalysed stereoselective homonucleophilic substitution and the resulting racemization.

The resolution of four racemic 3-hydroxy-1,4-benzodiazepin-2-ones, widely used in therapeutics, by means of a chiral stationary phase is described. The chiral selector used is (S)-N-(3,5-dinitrobenzoyl)phenylalanine. This chiral stationary phase showed both good enantioselectivity and efficiency for the compounds. Elution times were in all cases shorter than those previously reported for such compounds on different stationary phases. Racemic oxazepam was used to evaluate the loading capacity of the chiral stationary phase.

For studies on the excretion of drugs into milk a sensitive high-performance liquid chromatographic assay was developed to quantitate diazepam and nordazepam in the milk and plasma of humans and rabbits in the presence of their major metabolites, oxazepam and temazepam. Flurazepam was used as an internal standard. The assay involves extractions with diethyl ether and an additional acid clean-up step. Chromatographic separation was achieved by a LiChrospher 60 RP-select B (5 microns) column and KH2PO4- acetonitrile (69:31, v/v) adjusted to pH 2.80 as a mobile phase. The same extraction and chromatographic conditions were suited to both types of samples, milk and plasma. The limits of determination using ultraviolet detection at 241 nm was for diazepam 20 ng/ml and for nordazepam 15 ng/ml. The absolute recoveries of diazepam, nordazepam and flurazepam in human milk were 84, 86 and 92% and in human plasma 97, 89 and 94%, respectively. The within- and between-day accuracy and precision for diazepam and nordazepam in milk and plasma at all concentrations tested (20-1500 ng/ml) were better than 8%. The high fat content which occurs in rabbit milk presented no limitation for the extraction of lipophilic diazepam: the method was successfully used to monitor milk and plasma concentrations of diazepam and nordazepam in lactating New Zealand White rabbits during 26-h infusions of diazepam (1.4 mg/h).

The advent of several new column materials for the resolution of chiral compounds in high-performance liquid chromatography has opened up new possibilities for the analysis of drug enantiomers both in the dosage form and in bioanalytical studies. The utility of simplex optimisation, modified simplex and response surface mapping are considered with reference to the antischistosomal drug, oxamniquine, separated on an alpha 1-acid glycoprotein column. The resolution of the enantiomers of three closely related benzodiazepines, temazepam, oxazepam and lorazepam, is attempted on three new column systems: cellulose triacetate, beta-cyclodextrin and the reversed-phase column porous graphitic carbon with beta-cyclodextrin as a mobile phase additive.