Diflunisal, naproxen, ketoprofen, etodolac, mefenamic acid, rofecoxib, and celecoxib are nonsteroidal anti-inflammatory drugs, which have analgesics, antipyretics, and anti-inflammatory activities. The aim of this work was to develop and validate a simple assay that could be implemented in most laboratories for the purpose of clinical and toxicological screening, pharmacokinetic studies, and in therapeutic drug monitoring.
A new and simple high-performance liquid chromatography assay was developed and validated for the simultaneous determination of the above-mentioned drugs in small samples of human plasma (0.25 mL). After protein precipitation with acetonitrile, satisfactory separation was achieved on a Hypersil BDS C18 column (250 × 4.6 mm, 5 m) using a mobile phase comprising 20 mmol/L ammonium phosphate buffer (pH = 3) and acetonitrile at a ratio of 35:65, vol/vol; the elution was isocratic at ambient temperature with a flow rate of 1 mL/min. The UV detector was set at 265 nm.
The method was validated according to the recommendations of the Food and Drug Administration, including assessment of linearity, selectivity, precision, accuracy, and stability in human plasma. The use of betamethasone dipropionate as internal standard improved accuracy and precision. Response was linear over the calibration ranges. The limits of quantification were 0.2 g/mL for diflunisal and naproxen, 0.05 g/mL for ketoprofen, 0.1 g/mL for etodolac and mefenamic acid, and 0.02 g/mL for celecoxib and rofecoxib. The percent coefficient of variation for the QCs and the limit of quantification were within 10%, and the accuracies ranged between 96% and 106% for all the analytes. Mean drug recovery values were in the range of 95%–98% and 90.0% for all analytes and internal standard, respectively. All the analytes were stable in frozen plasma over a period of 3 months at −80°C.
This assay method was valid within a wide range of plasma concentrations and may be proposed as a suitable method for pharmacokinetic studies, therapeutic drug monitoring implementation, and routine clinical applications and suitable for special populations of patients who receive a combination of these drugs.