[Determination of diazepam and its metabolites in serum by the use of liquid chromatography-mass spectrometry method].
OBJECTIVE
Diazepam is a benzodiazepine anxyolitic. Metabolism of diazepam takes place in liver which generates pharmacologically active metabolites N-desmethyldiazepam, temazepam and oxazepam. The aim of this study was to develop and validate the method of liquid chromatographymass spectrometry (LC-MS) for separation and determination of diazepam and its active metabolites in the serum of rats samples after i.p. application of diazepam in a dose of 10 mg/kg.


METHODS
The serum samples taken from Wistar rats, were used in LC-MS analysis after the application of 10 mg/kg of diazepam i.p.


RESULTS
After alkaline extraction from the serum samples with diethylether and separation on a C18 reversed-phase column by using mobile phase methanolglacial acetic acid-water (50:1:49 v/v), diazepam and its metabolites were quantified. Determination was performed in a selective ion monitoring (SIM) mode, thereby the other exogenous and endogenous compounds did not interfere with this assay. Diazepam, N-desmethyldiazepam, oxazepam and temazepam were eluted in 14 minutes. The standard curve was linear in the range from 10-2 000 ng/ml. The limits of detection for diazepam, N-desmethyldiazepam, oxazepam and temazepam were 4.37, 3.13, 4.38 and 7.31 ng/ml, respectively. The limits of quantitation for diazepam, N-desmethyldiazepam, oxazepam and temazepam were 14.58, 10.41, 14.59 and 24.36 ng/ml, respectively.


CONCLUSIONS
The described LC-MS is a simple, sensitive, specific and accurate method and could be used for routine identification and quantification of small concentrations of diazepam and its metabolites in biological fluids.
Vojnomedicinska akademija, Nacionalni centar za kontrolu trovanja, Odeljenje za toksikolosku hemiju, Beograd, Srbija.
Snezana
S
Dordević
Vesna
V
Kilibarda
D000818:Animals; D002853:Chromatography, Liquid; D003975:Diazepam / Q000031:analogs & derivatives / Q000493:pharmacokinetics*; D005260:Female; D013058:Mass Spectrometry; D051381:Rats; D017208:Rats, Wistar
D003975:Diazepam