Development and validation of two chromatographic methods for the quantification of E-6087 and one of its metabolites, E-6132, in rat plasma

E-6087 is a nonsteroidal anti-inflammatory compound under development that selectively inhibits cyclooxygenase 2. In vitro studies have shown that one of its metabolites, E-6132, also inhibits this enzyme. Due to chromatograp hic reasons, two reverse phase HPLC methods were developed and validated in order to elucidate which compound is responsible for the pharmacological a ctivity in vivo. Chromatographic separation of E-6087 was achieved using ac etonitrile-phosphate buffer (pH 2.5; 25 mM) (60:40, v/v) as mobile phase an d two 4.6 x 150 mm x 5 mum Inertsil ODS-2 columns. For E-6132, two Inertsil ODS-3 columns and 52% of acetonitrile were used instead. Internal standard s and fluorescence detection differed between both methods. The same on-lin e solid-phase extraction method was used. Mean retention times for E-6087 a nd E-6132 were 15.2 (+/- 1.3) and 36.1 (+/- 0.6) min, respectively. The met hods were selective and linear over the concentration range of 10-500 ng ml (-1) (r(2) > 0.996) for E-6087 and 5-200 ng ml(-1) (r(2) > 0.997) for E-613 2. The limits of quantitation were 10 ng ml(-1) (E-6087) and 5 ng ml(-1) (E -6132) with a precision and accuracy < 16% (E-6087) and < 11% (E-6132). Mea n recoveries from plasma were 43.2-61.9% (E-6087) and 60.4-65.2% (E-6132). For both compounds, both inter-assay and intra-assay precision and accuracy were within acceptable limits (< 15%). As an example of the suitability of these methods, the results from a pharmacokinetic study are reported. Afte r single oral administration of 5 mg kg(-1) of E-6087 to rats, plasma conce ntrations of E-6087 at peak time were higher than those of E-6132, suggesti ng that activity is mainly due to E-6087. (C) 2001 Elsevier Science B.V. Al l rights reserved.