A simple and sensitive high-performance liquid chromatographic method to measure antipyrine and its metabolites in human urine

RESUMO

A method which permits the simultaneous HPLC analysis of antipyrine (A), 3-hydroxymethylantipyrine (HMA), 4-hydroxyantipyrine (HOA) and norantiopyrine (NORA) using a single extraction step in a five minute chromatographic run is described. Only 500 micraLitro of urine were necessary for the quantitation of all compounds investigated. An analog derivative, 4-aminoantipyrine, was used as internal standard (IS). Urine samples were hydrolyzed with Limpet acetone powder, 37 graus centigrados, pH 5.0 for 2 h. Sodium chloride was added and urine samples were extracted with diclhormethane-isopropyl alcohol (9010, v/v) in acidic medium. The residue was dissolved with mobile phase, washed with n-hexane and 20 micralitro of the lower phase were injected into a 4-micron Nova-Pak (R) 'C IND. 18' column. Peaks monitored at 254 nm were eluted with 0.75 M sodium acetate buffer, pH 5.0 methanol, (7030, v/v) as mobile phase. The method, validated on the basis of the confidence limits for antipyrine and its metabolites, presented good stability, sensitivity, linearity, and intra or interassay precisions lower than 5 percente for all commpounds were investigated. This assay was applied for drug metabolism study carried out in ten healthy volunteers 23 about 5 yr and 63 about 10 kg(mean about SD) after p.o. single dose of antipyrine, 500 mg/capsule. Diuresis of 24 h up to 72 h of drug administration was preserved with sodium metabisulfite, 4 mg/mL. Biological fluids were stored at -20 centigrade degree until assay. The main hydroxy-metabolites (HMA + HOA) and NORA, minor N-demethylated metabolite of antipyrine, were excreted 60 percent and 20 percente as percentage of the given dose, respectively. Clearances for production of metabolites expressed as mL/min, were 8.61 about 4.28 (7.24) for HMA, 13.91 about 6.20(12.67) for HOA, and 8.08 about 4.01(5.93) for NORA, mean about SD (median).