Determination of Girisopam (2,3-Benzodiazepine Compound) and Its Four Metabolites in Human and Rat Plasma by Gradient RP-HPLC Method

Abstract

A gradient RP-HPLC bioanalytical method has been developed for the human pharmacokinetic studies of girisopam, the new 2,3-benzodiazepine compound with anxiolytic effect that has no myorelaxant and anticonvulsive side effects. The compound is an analogue of tofizopam (Grandaxin®, EGIS Pharmaceuticals Ltd., Budapest, Hungary). The method was found to be appropriate for the purposes of human pharmacokinetic studies performed at 25, 50, 100, 200, 325, and 525 mg dose levels. The method allowed the simultaneous determination of girisopam (G) and its four metabolites (4′-hydroxy-G, 7-demethyl-G, 4-hydroxymethyl-G and 4-demethyl-4-oxo-G) identified in previous studies in human plasma. The solutes were separated on Hypersil BDS C18 column and quantified by UV detection at 238 nm. A solid phase extraction (SPE) method using reversed-phase 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-demethyl-4-oxo-G and 4′-hydroxy-G. In the case of 7-demethyl-G, LLOQ amounted to 2 ng/mL. The calibration curves showed good linearity; r = 0.9959, 0.9928, 0.9954, and 0.9974 in the concentration range of 1–500 ng/mL and r = 0.9959 in the range of 2–500 ng/mL respectively. The validation results obtained for all the five solutes indicated that the present method complied with internationally accepted criteria and ensured quantitative determinations of appropriate accuracy and reproducibility. After small modification and validation, the developed method was applied for determination of girisopam and its metabolites in rat plasma in a toxicological study (in vivo rat liver micronucleus test) at 600 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 was good for all components (r ≥ 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 absorption of girisopam was verified by the measuring of girisopam and its metabolite (7-demethyl-G) in the plasma samples of toxicological study (micronucleus test).