Godoy et al., J Chromatogr Sep Tech 2017, 8:4 DOI: 10.4172/2157-7064.1000373 Research Article Open Access Journal of Chromatography Separation Techniques J o u r n a l o f C h r o m a t o g r a p h y & S e p a r a t i o n T e c h n i q u es ISSN: 2157-7064 Volume 8 • Issue 4 • 1000373 J Chromatogr Sep Tech, an open access journal ISSN: 2157-7064 *Corresponding authors: Ana Leonor Pardo Campos Godoy, Laboratory of Toxicology - School of Pharmacy, Federal University of Bahia (UFBA) , Rua Barão de Jeremoabo, 147, Salvador, BA, Brazil, Tel: +5571992463177; E-mail: leonor.godoy@ufba.br Stella Maria Barrouin Melo, Laboratory of Veterinary Infectology, Hospital- School of Veterinary Medicine Federal University of Bahia (UFBA), Av. Adhemar de Barros500, Salvador, BA, 40170- 110, Brazil, Tel: +557132836756;E-mail: barrouin@ufba.br Received June 15, 2017; Accepted June 22, 2017; Published June 30, 2017 Citation: Godoy ALPC, De Jesus C, De Oliveira ML, Rocha A, Pereira MPM, et al. (2017) Determination of Allopurinol and Oxypurinol in Dogs Plasma by High- Performance Liquid Chromatography with an Ultraviolet Detector: Application for Pharmacokinetic Studies. J Chromatogr Sep Tech 8: 373. doi: 10.4172/2157- 7064.1000373 Copyright: © 2017 Godoy ALPC, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Determination of Allopurinol and Oxypurinol in Dogs Plasma by High- Performance Liquid Chromatography with an Ultraviolet Detector: Application for Pharmacokinetic Studies Godoy ALPC 1,2 * , De Jesus C 2 , De Oliveira ML 3 , Rocha A 3 , Pereira MPM 3 , Larangeira DF 2,4 , Lanchote VL 3 , Barrouin-Melo SM 2,4 * 1 Department of Clinical and Toxicological Analyzes, School of Pharmacy, Federal University of Bahia (UFBA) , Rua Barão de Jeremoabo, 147, Salvador, BA, Brazil 2 Laboratory of Veterinary Infectology, Hospital-School of Veterinary Medicine, Federal University of Bahia, Av. Adhemar de Barros, 500, Salvador, BA, Brazil 3 Department of Clinical, Toxicological and Bromatological Analyzes, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café, S / N, Ribeirão Preto, SP, Brazil 4 Department of Anatomy, Pathology and Clinics, School of Veterinary Medicine and Animal Science, UFBA, Salvador, BA, Brazil Abstract High performance liquid chromatography with ultraviolet detection (HPLC-UV) was developed and validated for quantifcation 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 defned 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 fow 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 quantifcation, linearity, reproducibility and repeatability. The method had a lower quantifcation 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 coeffcient variation lower than 15%. The confdence 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 effcient, 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. Keywords: HPLC-UV; Allopurinol; Oxypurinol; Pharmacokinetic; Canine visceral leishmaniasis Introduction Allopurinol (1H-pyrazolo (3,4-d) pyrimidin-4-ol) is an oxypurine base. It was frst synthesized in the 1950s by George Hitchings and Gertrude Elion in New York with the aim to increase the efcacy of antineoplastic drugs [1,2]. However, it was later discovered that allopurinol was a strong inhibitor of the enzyme xanthine oxidase [1]. Currently allopurinol is utilized in the treatment of humans diagnosed with hyperuricemia [2,3]. In veterinary medicine, this drug has been used to dissolve uroliths formed by ammonia uric acid calcifcations in dogs. Dalmatian genetics elicit a predisposition to developing high concentrations of uric acid [4,5]. Its use also extends to the treatment of canine visceral leishmaniasis (LV) therapy. In leishmaniostatic treatment is utilized either as monotherapy [6,7] or associated with leishmanicidal drugs [8-10]. Allopurinol is an analog of hypoxanthine and is converted by xanthine oxidase into its active metabolite oxypurinol (alloxanthin) (Figure 1). Both allopurinol and oxypurinol bind competitively to xanthine oxidase promoting its inhibition. Tey inhibit xanthine oxidase which is the enzyme that catalyzes the conversion of hypoxanthine to xanthine and xanthine into uric acid in both humans and dogs [3,11,12]. Peak plasma concentrations of allopurinol are obtained within 30 minutes to 1 hour afer ingestion and about 60- 70% of the drug is converted rapidly by xanthine oxidase to the active metabolite oxypurinol [3,13]. In humans, allopurinol and oxypurinol are excreted primarily via the kidneys. A dose of 100 mg allopurinol orally generates 90 mg of oxypurinol [11]. Allopurinol is excreted in the urine in its unchanged form at a rate of 10%; in the feces at a rate of 20%. Te metabolite oxypurinol is eliminated in the urine at a concentration of 70% [12- 14]. Limited research has been conducted on the pharmacokinetics of allopurinol in dogs [5,15,16]. Te pharmacokinetics of allopurinol has been demonstrated in healthy Dalmatians where maximum concentration (C max ) was reached at 6.43 ± 0.18 μg.mL -1 at a time of (T max ) 1.9 hours and its apparent volume of distribution was (Vd / F) 1.17 ± 0.07 L.kg -1 . Tis illustrates it was well distributed within the tissues [5].