[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.
Dordević
Snezana
S
Vojnomedicinska akademija, Nacionalni centar za kontrolu trovanja, Odeljenje za toksikolosku hemiju, Beograd, Srbija.
Kilibarda
Vesna
V
D000818:Animals; D002853:Chromatography, Liquid; D003975:Diazepam / Q000031:analogs & derivatives / Q000493:pharmacokinetics*; D005260:Female; D013058:Mass Spectrometry; D051381:Rats; D017208:Rats, Wistar
D003975:Diazepam