Adriele Rebeca Vieira da Silva¹, Ester Miranda Pereira¹, Semiramis Jamil Hadad do Monte¹, José Ribeiro dos Santos Júnior¹, Cícero Alves Lopes Júnior¹

Rare diseases are a group of illnesses that affect a small number of people when compared to the general population. Acute porphyria is a disease that falls into this group, characterized by an enzymatic deficiency in the heme biosynthesis pathway, which causes the accumulation of toxic precursors such as 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), metabolites that are excreted in the urine. Diagnosis in Brazil, made available by SUS, is carried out through clinical analysis and a qualitative test using the Ehrlich reagent, which indicates the high presence of PBG. For more accurate results, a quantitative test is required. However, this is not affordable and its availability is limited to study and reference centers. In view of this, the aim of this study was to develop a method for quantifying ALA in urine using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to a UV-VIS detector. To this end, the method optimization tests were based on the pre-column derivatization protocol, in which urine samples were obtained and analyzed following the guidelines approved by the ethics committee with opinion no. 7.305.492 of the Federal University of Piauí. Previously, the samples were filtered using 0.22 μm syringe filters with hydrophilic PTFE membranes and centrifuged at 10,000 rpm for 5 min. Pre-column derivatization was carried out with the reagent o-Phthalaldehyde (OPA) to increase the absorption of the compounds through the formation of isoindol compounds. The chromatographic conditions used to separate the analytes were as follows: Luna column (5μm, C18, 250 x 4.6 mm), with a mobile phase composed of methanol/phosphate buffer (0.5 M, 50:50 v/v, pH 5.0), at a flow rate of 0.7 mL/min, injection volume of 3 μL and 10 μL for the standard and sample, respectively, and a detection wavelength of 330 nm. The chromatographic analysis effectively separated ALA, with a retention time of 12.50 min. Twelve samples from patients with different clinical conditions were analyzed, and the values obtained for ALA ranged from 6.7 - 59.7 mg/L. The values found are within the range expected for the clinical condition of patients with suspected porphyria, with concentrations of less than 7 mg/L being considered normal. Thus, the results highlight the potential of the method for quantifying ALA, contributing to the early diagnosis and treatment of acute porphyria. 

Agradecimentos: To the Postgraduate Program in Chemistry of the Federal University of Piauí.