Development and Validation of a RP-HPLC Method for the Simultaneous Determination of LevamisoleHCl and Oxyclozanideand its Application in the Assay of Veterinary Bolus Formulations
Introduction: A reverse-phase high performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous analysis of two drugs, levamisole hydrochloride(LH) and oxyclozanide(OX), in co-formulations for veterinary use. Materials and Methods: The new HPLC method was validated per ICH and other guidelines. A C18 column was used with agradient program; eluent A was an equal mixture of methanol and acetonitrile, and eluent B a 25 mM phosphate buffer at pH 7.0 containing 30 mM sodium decanesulfonate andtriethylamine(50:50:1 v/v)then pH adjusted to 7.0 with H3PO4 [51:49 v/v] .The detection wavelength was set at 220 nm.For the final gradient program, the retention times were 8.2(for LH)and 13.6(for OX) minutes respectively at flow rate of 1 ml/min over 20 minute run time. Results: The method wasprecise, specific and robust.The correlation coefficients, R2 were 0.9998 and 0.9999 for LH and OX respectively in the ranges of 5 – 280 µg / mL.The percent y-intercepts and percent residual standard deviations were 1.6%/0.4% and 1.4%/1.0% for LH and OX, respectively. The LOD and LOQ of the method were 0.21 µg / mL and 0.62 µg / mL for LHand 0.06 µg / mL and 0.18 µg / mL for OX. The method has average accuracy of 100.5% for LH and 101.1% for OX when tested on veterinary bolus formulations, and the samples couldbe stored under typical lab conditions for about 7 days without significant degradation. Conclusion: This HPLC method is suitable forassayinglevamisole hydrochloride and oxyclozanide simultaneously from veterinary formulations.