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Rapid analysis of piroxicam level in microsample of human plasma by fully validated HPLC assay

Rajaa Farhan Hussein, Muhammad M.Hammami

A rapid, simple HPLC assay for piroxicam measurement in human plasma was developed and validated. 50 l of 24%perchloric acid and 0.2ml acetonitrile were mixed with 0.1 ml plasma sample, and the supernatant was injected directly into 4.6 ï‚´ 150 mm, XTerra® RP18, 5msteel column at room temperature (RT). Themobile phase, 0.2%triflouroacetic acid, and acetonitrile (70:30, v:v), was delivered at 1.2ml/minwith a run time of 6min.Doxycycline (internal standard, IS) and piroxicam were detected using Waters 996 photodiode array detector set at 339 nm IS at 3.7 and 4.8 min, respectively. The response was linear over the range of 0.2-20g/ml, and the intraand inter-run coefficient of variationswere ï‚£ 5.2%and ï‚£ 6.7%, respectively. Extraction recovery and intra- and inter-run biaswere  86%(mean 93%), ï‚£ 9%, and ï‚£ 11%, respectively. Piroxicamwas stable in plasma for 24 hours at RT ( 96%), 8weeks at -20 0C ( 100%), and after 3 cycles of freeze at -200C and thawat RT ( 94%). In processed samples, piroxicamwas stable for 24 hours at RT (100%) and 48 hours at -200C (100%). Stock solution of piroxicam(1mg/ml inmethanol) was stable for 48 hours at RT and 8 weeks at -200C(100%).