Abstract

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-formulation for veterinary use.

Materials and Methods: The new HPLC method was validated as per the ICH and other guidelines. A C18 column was used with a gradient program; eluent A was an equal mixture of methanol and acetonitrile, and eluent B included a 25 mM phosphate buffer at pH 7.0 containing 30 mM sodium decanesulfonate and triethylamine (50:50:1 v/v) with pH adjusted to 7.0 using 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 a flow rate of 1 ml/min over 20 minutes run time.

Results: The method was precise, specific and robust. The correlation coefficient, R² was 0.9998 and 0.9999 for LH and OX, respectively in the range 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 LH and 0.06 μg / mL and 0.18 μg / mL for OX. The method has an average accuracy of 100.5% for LH and 101.1% for OX when tested on veterinary bolus formulations, and the samples could be stored under typical lab conditions for about 7 days without significant degradation.

Conclusion: This HPLC method is suitable for assaying levamisole hydrochloride and oxyclozanide simultaneously from veterinary formulations.

Keywords: Analytical method development, bolus formulations, RP-HPLC, levamisole, oxyclozanide, imidazothiazole.

Graphical Abstract