Simultaneous determination of piroxicam and norfloxacin in biological fluids by high-performance liquid chromatography with fluorescence detection at zero-order emission mode.

A new highly sensitive high-performance liquid chromatographic method with fluorescence detection (HPLC-FLD) in zero-order emission mode was developed for the first time for the simultaneous determination of piroxicam (PRX) and norfloxacin (NRF) in biological fluids. The fluorescence detector wavelengths were set at 278 nm for excitation and zero-order mode for emission. The zero-order emission mode produced greater sensitivity for the measurement of both drugs than a fixed emission wavelength (446 nm). The new developed method was validated according to International Conference of Harmonization (ICH) guidelines. Linearity was found to be over concentration ranges 0.001-20 μg/ml and 0.00003-0.035 μg/ml for PRX and NRF, respectively. The limits of detection were 4.87 × 10-4 and 1.32 × 10-5 μg/ml for PRX and NRF, and the limits of quantitation were 1.47 × 10-3 and 4.01 × 10-5 μg/ml, respectively. The current fluorescence method was found to be more sensitive than most commonly used analytical methods and was successfully applied for simultaneous determination of PRX and NRF in biological fluids (serum and urine) with recoveries ranging from 91.67% to 100.36% for PRX and from 96.00% to 101.43% for NRF.