Validation of a chromatographic method to determine E-6006 and its metabolite E-6332 in rat and dog plasma by solid-phase extraction and capillary gas chromatography. Application in pharmacokinetics.

Abstract

E-6006, 5-(alpha-[2-(dimethylamino)ethoxy]-2-thienylmethyl)-1-methyl-1H-pyrazole is a new antidepressive compound and E-6332, 5-(alpha-[2-(methylamino)ethoxy]-2-thienylmethyl)-1-methyl-1H-pyrazole is its desmethylate metabolite. With the aim of quantifying E-6006 and E-6332, simultaneously in rat or dog plasma, a method of analysis based on solid-phase extraction coupled with capillary gas chromatographic system with N-P detection was developed and validated. E-6006, E-6332 and its internal standard (E-4018) were isolated from plasma using an off-line semiautomatic solid-phase extraction method. Gas chromatography separations were carried out by means of 12 m length, 0 2 mm (i.d.) and 0.33 microm (f.t.) ULTRA 1 type capillary column in splitless mode of injection at 190 degrees C, with a TSD or specific nitrogen--phosphorus detector. No peaks interfering with the quantification of E-6332 and E-6006 were observed. The limit of quantification was 5 ng/ml with a precision and accuracy <17%. The peak height ratios were proportional to E-6332 and E-6006 concentration over the range from 5 to 600 ng/ml (r(2)>0.998). Mean recoveries of E-6332, E-6006 and internal standard from rat plasma were between 57.1 and 82.6. Intra-assay precision coefficients were <8.0 and <11.8%, respectively, for E-6332 and E-6006, with an accuracy <12.6 and <9.7%. Both inter-assay precision and accuracy were within acceptable limits (<15%). In dog, the results were very similar to those obtained in rat. To show an example of the suitability of the method to determine E-6332 and E-6006, plasma profiles obtained after single oral and intravenous administration of 20 mg/kg to rats and 25 mg/kg to dogs are reported.