Development and Validation of Liquid Chromatography-Tandem Mass Spectrometric Method for the Quantification of Diclofenac in Human Plasma

According to Food and Drug Administration requirements, a simple, sensitive and specific liquid chromatography-tandem mass spectrophotometry method for quantifying diclofenac in human plasma was developed and fully validated. Electrospray ionisation in the positive mode produced protonated ions, which were utilised to detect analyte and fluconazole (internal standard). Protein precipitation generated by acetonitrile was used to extract the analytes, followed by liquid-liquid extraction with ethyl acetate. The reversed phase separation was performed on a Chromolith speed ROD RP-18e (4.6 mmx50 mm) column with a simple isocratic mobile phase of 10 mM ammonium acetate in water, pH adjusted to 4.5 using acetic acid:acetonitrile in the ratio of (20:80, v/v) at a flow rate of 0.5 ml min-1. On a triple quadrupole mass spectrometer, fragmentation of Diclofenac m/z 296.10- (parent) and 250 (product) and Fluconazole m/z 307.20- (parent) and 220 (product) for internal standard was monitored. The devised method was verified in human plasma spanning a concentration range of 18.75 to 2000.25 ng ml-1, with a correlation coefficient (r2) of 0.9948. The detection was carried out using an electrospray ionisation approach on a triple quadrupole mass spectrometer that was used for multiple reaction monitoring. The intraday precision and accuracy values obtained from six different sets of quality control samples evaluated on different occasions ranged from 96.22-113.46 % and 2.66-9.95 %, respectively. For the analyte and internal standard, the overall recoveries were 61.98 3.97 and 55.01 1.18, respectively. The described approach was used to analyse diclofenac in human plasma samples with great performance.