High-throughput ultra-performance LC-MS-MS method for analysis of diclofenac sodium in rabbit plasma

A new UPLC-MS-MS method was developed and validated for quantification of diclofenac sodium in rabbit plasma. Acetonitrile-based protein precipitation method was used to extract the drug from plasma samples. Chromatographic separation was carried out on Acquity UPLC(®)BEH phenyl C18 1.7 µm, 2.1 × 50 mm column. Drug elution was facilitated by using mobile phase containing acetonitrile (0.1% glacial acetic) and water (pH 3.5), in a ratio of 75 : 25, flowing at 0.2 mL/min. Molecular ions were generated by using the positive electrospray ionization mode (ESI(+)) and analyzed on a triple-quadrupole mass spectrometer. The ionic transitions of diclofenac (m/z 296 > 214 and 249.9) and flufenamic acid (internal standard) (m/z 282.1 > 166.9 and 244) were measured in multiple reaction modes. Developed method is simple, quick, precise and accurate over a linearity range of 80-4,000 ng/mL. The lower limit of quantification (LLOQ) for diclofenac was 80 ng/mL. The percentage recoveries of diclofenac at three quality control samples were 54 ± 6.1, 67.1 ± 5.4 and 62.3 ± 1.4%. Precision and accuracy of the assay at LLOQ, middle limit of quantitation and higher limit of quantitation were 100 ± 7.0, 100 ± 1.0 and 100 ± 2.0% and 81.8 ± 4.6, 106.6 ± 3.1 and 103.3 ± 4.0%, respectively.