High-performance liquid chromatographic determination of glyoxal and methylglyoxal in urine by prederivatization to lumazinic rings using in serial fast scan fluorimetric and diode array detectors

Glyoxal and methylglyoxal are two important markers of oxidative stress and both are involved in the evaluation of several diseases. A new HPLC method for determining glyoxal and methylglyoxal in urine was developed. The method is based on the reaction of α-dialdehydes, glyoxal and methylglyoxal, with 5,6-diamino-2,4-hydroxypyrimidine sulfate in basic medium to form highly fluorescent lumazine derivatives. Creatinine was also included in the method even though it does not react with the reagent. The derivatives and creatinine are separated on a C18 reversed-phase column with a mobile phase consisting of acetonitrile:citrate buffer, pH 6.0 (3:97 v/v). The flow rate was 1.0 mL min−1 and the effluent was monitored photometrically at 250 nm for determination of creatinine and fluorimetrically at 500 nm (exciting at 330 nm) for determination of glyoxal and methylglyoxal derivatives. Recording time of the separation is less than 10 min. Determination of the analytes is performed in urine after incubation of the sample, with the reagent in alkaline medium, for 30 min at 60 °C. Urinary levels of glyoxal and methylglyoxal, expressed as glyoxal/creatinine and methylglyoxal/creatinine ratios, in healthy young women and men were determined. For women, values of 0.80 ± 0.37 and 0.60 ± 0.22 μg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. For men, values of 0.63 ± 0.15 and 0.49 ± 0.05 μg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. These results were also related to the body mass index of each individual.