Desenvolvimento de metodologia para monitorização terapêutica da azatioprina por cromatografia líquida de alta eficiência-UV (HPLC-UV) em transplantados renais / Development of a methodology for therapeutic drug monitoring of azathioprine by high performance liquid chromatography-UV (HPLC-UV) in renal transplant recipients

Maurilio Pacheco Neto

Azathioprine (AZA) is an immunosuppressant used in autoimmune pathologies like lupus erythematosus, Chrons disease, inflammatory bowel disease and against rejection in solid organs transplant. After more than 40 years of use, AZA continues exerting a central role in immunomodulatory regimens, due to the fact that it combines effectiveness, safety and low cost. It is well known that thiopurine methyltransferase activity may determine, at least in part, the clinical efficacy of AZA therapy. This enzyme exhibits codominant genetic polymorphism and the distribution of these variant alleles differs significantly among populations. The considerable pharmacokinetic variability in AZA metabolism justify the therapeutic drug monitoring of this drug. In this work a methodology was improved to quantify the metabolites of AZA, 6-TGN and 6-MMP, by high performance liquid chromatography (HPLC/UV-Vis) with an ultraviolet-visible detector, using a single wavelength reading, following a simple acid deproteinization and heating to convert the metabolites into their respective free bases. The values of these metabolites were determined in a population of 124 renal transplant recipients. To adequate the process to international and local legislation, Anvisa, FDA and CLSI guidelines were followed. Separation was achieved on a reversed-phase column; mobile phase A potassium phosphate and mobile phase B methanol. Detection of 6-TGN and 6-MMP was performed at 342 m (UV-Vis). Assay linearity for 6-TGN ranged from 0.30 to 89.71 mol/L and from 0.30 to 93.86 mol/L for 6-MMP. The recoveries were 95.08, 97.76 and 100.80% for 6-TGN and 104.79, 95.38 and 105.06% for 6-MMP. Repeatability CV were 3.50, 5.06, 1.09 and 0.04, 0.35, 1.58%, while reproducibility CV were 8.65, 7.18, 8.44 and 12.73, 6.40, 4.88% for 6-TGN and 6-MMP, respectively. LOQ and LOD of 6-TNG and 6-MMP were respectively 0.30 mol/L and 0.13 mol/L for both metabolites. The washed erythrocytes and the samples treated and ready for injection into the HPLC system were stored below -5 °C until analysis, at this temperature the samples were stable for 8 weeks and for 1 day, respectively. 6-TGN and 6-MMP patient analysis values ranged from non detectable to 1569 mol/8 x 108 RBC (median of 200.50) and non detectable to 113057 mol/8 x 108 RBC (median of 5166), respectively. The correlations between 6-TGN or 6-MMP levels and variables sex, time post-transplant, number of transplants and AZA dosage (mg/kg) were examined in different groups. The proposed HPLC method has a good cost-benefit ratio, is straightforward, precise, accurate and fast at the determining 6-TGN and 6-MMP concentrations in red blood cells of patients under AZA therapy. The validated method is good enough to enable the laboratory to routinely provide useful and accurate pharmacokinetic data in time to adjust patient regimens. It can be easily adopted for routine analysis of these drug metabolites. The results of patient samples are in agreement with others studies, thus certifying the usefulness of this analytical tool in monitoring of patients

AUTOR(ES)

DATA DE PUBLICAÇÃO

RESUMO

ASSUNTO(S)

ACESSO AO ARTIGO

Documentos Relacionados