Análise quantitativa de aminoácidos por HPLC em sangue e urina coletados em papel-filtro no período neonatal: validação de método, estabelecimento de valores de referência e aplicação prática em erros inatos do metabolismo

Abstract

INTRODUCTION: Inborn errors of metabolism constitute a diverse group of individually rare diseases, but collectively correspond to 10% of the genetic diseases already described. They occur by a specific enzymatic defect that leads to the interruption of a metabolic pathway. Among them, we have the aminoacidopathies. Quantitative analysis of amino acids in blood and urine are especially indicated for the diagnosis and follow-up of patients with amino acid disorders and the highperformance liquid chromatography (HPLC) technique is the most used for this detection. Sample collection on filter paper has been increasing exponentially and there are many advantages attributed to biological samples processed in this way. OBJECTIVES: To validate a method of quantitative analysis of amino acids by HPLC in blood and urine collected on filter paper and to establish reference values in the neonatal period. METHODS: Samples of blood collected by venipuncture and applied on filter paper (n = 58) and urine collected on filter paper (n = 45) from a group of healthy newborns aged 2 to 9 days. Pre-treatment and extraction of samples according to Yamaguchi (1997). Separation of amino acids by HPLC, Alliance HT Waters model e2795, C18 reverse phase column. RESULTS: Chromatographic separation of amino acids was adequate. The method demonstrated satisfactory linearity, limits of detection and quantification, repetibility and recovery. Matrix effect was detected for all amino acids. The reference values for the amino acids aspartate, serine, glutamate, glycine, histidine, arginine, threonine, alanine, proline, cystine, tyrosine, valine, methionine, lysine, isoleucine, leucine and phenylalanine were defined. CONCLUSIONS: This work described a simple, fast and effective method for simultaneous analysis of 17 amino acids in blood and urine collected on filter paper in a single run and the reference values were established and validated. This method can be used in routine laboratories and it is an ideal tool for the pediatric area, where obtaining a large sample volume is not always possible.