Determination of Allopurinol and Oxypurinol in Dogs Plasma by High- Performance Liquid Chromatography with an Ultraviolet Detector: Application for Pharmacokinetic Studies

Abstract

High performance liquid chromatography with ultraviolet detection (HPLC-UV) was developed and validated for quantification of allopurinol and its active metabolite oxypurinol; in dog plasma with naturally acquired zoonotic visceral leishmaniasis. Allopurinol is a potent inhibitor of the xanthine oxidase enzyme; an enzyme that catalyzes the conversion of hypoxanthine to xanthine and from xanthine to uric acid. In veterinary medicine allopurinol is indicated in the treatment of canine visceral leishmaniasis. Allopurinol is utilized to inhibit the synthesis of Leishmania RNA. The conditions defined for development of the chromatographic analysis of dog plasma samples by utilizing the mobile phase of the HPLC-UV consist of a mixture of 0.1% water, 88% formic acid and 0.25% acetonitrile. Allopurinol and oxypurinol were separated on a LiChroCART® 125-4 LiChrospher® 100 RP-8 (5 μm) column utilizing a flow rate of 0.7 mL min-1 and detector operation was at a wavelength of 254 nm. Acyclovir was utilized as an internal standard. The validation of the HPLC-UV method was determined by limits of detection and quantification, linearity, reproducibility and repeatability. The method had a lower quantification limit of 0.1 μg.mL-1 for both allopurinol and oxypurinol. The analysis was linear at concentrations of 0.1-20.0 μg.mL-1 for both allopurinol and its oxypurinol. The precision value intracurrent and intercurrent for all concentrations presented had a coefficient variation lower than 15%. The confidence limits of HPLC-UV for analysis of allopurinol and oxypurinol in dog plasma indicates that the method is applicable to the multiple dose pharmacokinetic study of allopurinol in dogs. It is efficient, accurate and sensitive. This study of Pharmacokinetic research of allopurinol and oxypurinol in dogs with Stage I and II visceral leishmaniasis resulted in similar research outcomes that correlated with the healthy dog investigation.