I. Urmos et al., DETERMINATION OF GIRISOPAM (2,3-BENZODIAZEPINE COMPOUND) AND ITS 4 METABOLITES IN HUMAN AND RAT PLASMA BY GRADIENT RP-HPLC METHOD, Journal of liquid chromatography & related technologies, 21(6), 1998, pp. 803-818
Citations number
8
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
A gradient RP-HPLC bioanalytical method has been developed for the hum
an pharmacokinetic studies of girisopam, the new 2,3-benzodiazepine co
mpound with anxiolytic effect that has no myorelaxant and anticonvulsi
ve side effects. The compound is an analogue of tofizopam (Grandaxin(R
), EGIS Pharmaceuticals Ltd., Budapest, Hungary). The method was found
to be appropriate for the purposes of human pharmacokinetic studies p
erformed at 25, 50, 100, 200, 325, and 525 mg dose levels. The method
allowed the simultaneous determination of girisopam (G) and its four m
etabolites (4'-hydroxy-G, 7-demethyl-G, 4-hydroxymethyl-G and 4-demeth
yl-4-oxo-G) identified in previous studies in human plasma. The solute
s were separated on Hypersil BDS C18 column and quantified by UV detec
tion at 238 nm. A solid phase extraction (SPE) method using reversed-p
hase cartridges was developed for sample processing, whereby girisopam
and the much more polar metabolites, as well as the internal standard
could be extracted in a single step. The limit of quantitation (LLOQ)
was: 1 ng/mL in the case of Girisopam (G), 4-hydroxymethyl-G, 4-demet
hyl-4-oxo-G and 4'-hydroxy-G. In the case of 7-demethyl-G, LLOQ amount
ed to 2 ng/mL. The calibration curves showed good linearity; r = 0.995
9, 0.9928, 0.9954, and 0.9974 in the concentration range of 1-500 ng/m
L and r = 0.9959 in the range of 2-500 ng/mL respectively. The validat
ion results obtained for all the five solutes indicated that the prese
nt method complied with internationally accepted criteria and ensured
quantitative determinations of appropriate accuracy and reproducibilit
y. After small modification and validation, the developed method was a
pplied for determination of girisopam and its metabolites in rat plasm
a in a toxicological study (in vivo rat liver micronucleus test) at 60
0 and 1200 mg/kg dose levels. The LLOQ was 10 ng/mL for girisopam and
50 ng/mL for metabolites in rat plasma. The validation parameters for
determination of solutes in rat plasma were internationally acceptable
. The linearity tvas good for all components (r greater than or equal
to 0.992) in the wide calibration range of 10-18000 ng/mL and 50-6000
ng/mL in the case of girisopam and its metabolites respectively. The a
bsorption of girisopam was verified by the measuring of girisopam and
its metabolite (7-demethyl-G) in the plasma samples of toxicological s
tudy (micronucleus test).