Quantitation of meloxicam in the plasma of koalas (Phascolarctos cinereus) by improved high performance liquid chromatography

An improved method to determine meloxicam (MEL) concentrations in koala plasma using reversed phase high performance liquid chromatography equipped with a photo diode array detector was developed and validated. A plasma sample clean-up step was carried out with hydrophilic-lipophilic copolymer solid phase extraction cartridges. MEL was separated from an endogenous interference using an isocratic mobile phase [acetonitrile and 50 mM potassium phosphate buffer (pH 2.15), 45:55 (v:v)] on a Nova-Pak C₁₈ 4-µm (300 × 3.9 mm) column. Retention times for MEL and piroxicam were 8.03 and 5.56 min, respectively. Peak area ratios of MEL to the internal standard (IS) were used for regression analysis of the calibration curve, which was linear from 10 to 1,000 ng/mL (r² > 0.9998). Average absolute recovery rates were 91% and 96% for MEL and the IS, respectively. This method had sufficient sensitivity (lower quantitation limit of 10 ng/mL), precision, accuracy, and selectivity for routine analysis of MEL in koala plasma using 250-µL sample volumes. Our technique clearly resolved the MEL peak from the complex koala plasma matrix and accurately measured MEL concentrations in small plasma volumes.