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

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 24; no. 5; pp. 897 - 911
• Main Authors: Reinoso, R.F., Farrán, R., Moragon, T., Garcı́a-Soret, A., Martı́nez, L.
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 01.03.2001; Elsevier Science
• Subjects: Analysis; Animals; Anti-inflammatory agents; Anti-Inflammatory Agents, Non-Steroidal - blood; Anti-Inflammatory Agents, Non-Steroidal - pharmacokinetics; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; Cyclooxygenase Inhibitors - blood; Cyclooxygenase Inhibitors - pharmacokinetics; E-6087; Female; General pharmacology; HPLC; Male; Medical sciences; On-line solid-phase extraction; Pharmacology. Drug treatments; Prospekt; Pyrazoles - blood; Pyrazoles - pharmacokinetics; Quality Control; Rats; Rats, Wistar; Sensitivity and Specificity; Spectrometry, Fluorescence; Sulfonamides - blood; Sulfonamides - pharmacokinetics; Validation; E-6087; Validation; Prospekt; Anti-inflammatory agents; HPLC; On-line solid-phase extraction; Solid phase extraction; Biological fluid; Fluorescence detector; Rat; Metabolite; Rodentia; Single dose; Oral administration; HPLC chromatography; Metabolism; Blood plasma; Non steroidal antiinflammatory agent; Separation method; Vertebrata; Mammalia; Animal; Pharmacokinetics; Quantitative analysis
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/S0731-7085(00)00558-6

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 chromatographic reasons, two reverse phase HPLC methods were developed and validated in order to elucidate which compound is responsible for the pharmacological activity in vivo. Chromatographic separation of E-6087 was achieved using acetonitrile–phosphate buffer (pH 2.5; 25 mM) (60:40, v/v) as mobile phase and two 4.6×150 mm×5 μm Inertsil ODS-2 columns. For E-6132, two Inertsil ODS-3 columns and 52% of acetonitrile were used instead. Internal standards and fluorescence detection differed between both methods. The same on-line solid-phase extraction method was used. Mean retention times for E-6087 and E-6132 were 15.2 (±1.3) and 36.1 (±0.6) min, respectively. The methods 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-6132. 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). Mean 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. After single oral administration of 5 mg kg −1 of E-6087 to rats, plasma concentrations of E-6087 at peak time were higher than those of E-6132, suggesting that activity is mainly due to E-6087.