EXTRACTIONLESS DETERMINATION OF DICLOFENAC SODIUM IN SERUM USING REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH FLUOROMETRIC DETECTION

The author describes a method of using reversed-phase high-performance liquid chromatography with fluorimetric detection for the assay of diclofenac sodium in serum. The method is sensitive down to 20 ng/ml (250-mu-l loop). Elution is at pH 6.2 with methanol in 0.05 M phosphate buffer (43:57, v/v) on a 25-cm Spherisorb S5 ODS2 column. Detection is at an excitation wavelength of 282 nm and an emission wavelength of 365 nm. Serum sample size is 100-mu-l. Sample protein, to which diclofenac is highly bound, is first denatured by heat and then with methanol to release the diclofenac prior to centrifugation and injection of 100-mu-l (or 250-mu-l) of the clear supernatant. Harmol, with similar fluorescence and polarity characteristics to diclofenac, is a satisfactory internal standard. At the 1-mu-g/ml level intra-sample reproducibility is better than 2%, whilst inter-sample reproducibility is 4.6%. Detector response is linear from 40 ng/ml to 20-mu-g/ml (100-mu-l loop).