The assay of methylglyoxal in biological systems by derivatization with 1,2-diamino-4,5-dimethoxybenzene.

A procedure for the assay of methylglyoxal in biological systems is described, together with sample storage, sample processing procedures, and statistical evaluation. Specimen data are presented. Methylglyoxal was assayed by derivatization with 1,2-diamino-4,5-dimethoxybenzene and high-performance liquid chromatography (HPLC) of the resulting quinoxaline, 6,7-dimethoxy-2-methylquinoxaline, with spectrophotometric or fluorescence detection. Derivatization, solid-phase extraction, and HPLC were performed under acid conditions to prevent the spontaneous formation of methylglyoxal from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate during the assay. The limits of detection in the biological matrix were 45 pmol (absorbance detection) and 10 pmol (fluorimetric detection), the recovery was 58%, and the intra- and interbatch coefficients of variance were 7.7 and 30.0%, respectively. The concentration of methylglyoxal in whole blood from normal healthy human individuals was (mean +/- SE, nM) 256 +/- 92 (n = 12) and that from diabetic patients was 479 +/- 49 (n = 55), showing a significant increase in diabetes mellitus (P < 0.01; Mann-Whitney U test). Sample processing under acidic conditions was essential to avoid interferences. Previous estimates of the concentration of methylglyoxal in biological samples require re-evaluation.