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Development and Validation of a Bioanalytical Method for Direct Extraction of Diclofenac Potassium from Spiked Plasma
Abstract
Purpose: To develop and validate a user-friendly spiked plasma method for the extraction of diclofenac potassium that reduces the number of treatments with plasma sample, in order to minimize human error.
Method: Instead of solvent evaporation technique, the spiked plasma sample was modified with H2SO4 and NaCl, respectively, and then the drug was extracted after vortexing the sample with acetonitrile as precipitating agent. The separation of diclofenac potassium and internal standard (ketoprofen) was achieved at preset conditions: 5 μm ODS Hypersil C-18 (4.0 mm x 250 mm) column, eluted with 50% acetonitrile in water (v/v) as mobile phase containing ammonium acetate and triethylamine (TEA), at a
flow rate of 1 mL min-1.
Results: The peaks of the drug and internal standard (I.S.) were resolved at 14 ± 1 min and 7 ± 1 min, respectively. The calibration curve and linearity were determined over the concentration range of 0.25 to 40 μg mL-1 and they were linear (r2 = 0.9991 and 0.9982, respectively). The accuracy was > 81.32 %. Limit of detection and limit of quantification were 0.05 and 0.25 μg mL-1, respectively, while the recovery range for diclofenac potassium and ketoprofen was more than 79 and 85 %, respectively. The absolute average difference of 0.18 between the observed concentrations for intra- and inter-day studies indicated that the sample was stable for over one month.
Conclusion: The proposed method may be applied to routine bioanalysis, particularly for NSAIDs, due to its high sensitivity, specificity, repeatability, reproducibility, robustness and ruggedness.
Keywords: Bioanalytical method, Diclofenac potassium, RP-HPLC method, NSAIDs, Plasma
