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HPLC method for determination of DRF‐4367 in rat plasma: validation and its application to pharmacokinetics in Wistar rats
Author(s) -
Mullangi Ramesh,
Kallem Raja Reddy,
Bhamidipati Ravi Kanth,
Mamidi Rao N. V. S.,
Srinivas Nuggehally R.
Publication year - 2004
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.359
Subject(s) - chemistry , chromatography , high performance liquid chromatography , analyte , detection limit , standard curve , elution , calibration curve
A specic, accurate, precise and reproducible high‐performance liquid chromatography (HPLC) method was developed for the estimation of DRF‐4367, a novel cyclooxygenase‐2 inhibitor in rat plasma. The assay procedure involved simple liquid/liquid extraction of DRF‐4367 and internal standard (IS, celecoxib) from plasma into dichloromethane. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40°C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100‐5C 18 column (4.6 × 250 mm, 5 µm). The mobile phase consisting of 0.01 m potassium dihydrogen ortho ‐phosphate (pH 3.2) and acetonitrile (40:60, v/v) was used at a ow rate of 1.0 mL/min. The eluate was monitored using an UV detector set at 247 nm. The ratio of peak area of analyte to IS was used for quantication of plasma samples. Nominal retention times of DRF‐4367 and IS were 6.6 and 11.2 min, respectively. The standard curve for DRF‐4367 was linear ( r 2 > 0.999) in the concentration range 0.1–20 µg/mL. Absolute recovery was >86% from rat plasma for both analyte and IS. The lower limit of quantication of DRF‐4367 was 0.1 µg/mL. The inter‐ and intra‐day precisions in the measurement of quality control samples, 0.1, 0.3, 8.0 and 15.0 µg/mL, were in the range 6.93–9.34% relative standard deviation (RSD) and 0.48–6.59% RSD, respectively. Accuracy in the measurement of QC samples was in the range 91.24–109.36% of the nominal values. Analyte and IS were stable in the battery of stability studies, viz. benchtop, autosampler and freeze–thaw cycles. Stability of DRF‐4367 was established for 1 month at −80°C. The application of the assay to a pharmacokinetic study in rats is described. Copyright © 2004 John Wiley & Sons, Ltd.