Preparation and quantification of methylglyoxal in human plasma using reverse-phase high-performance liquid chromatography

Abstract

Objectives: To detect methylglyoxal (MG), a highly reactive α-oxoaldehyde found widespread throughout biological life, in human plasma using reverse-phase high-performance liquid chromatography method (RP HPLC) with UV detection. Design and methods: The processing of human plasma required protein precipitation with trifluoroacetic acid (TFA), incubation of the supernatant (2 h) with 1,2-diamino-4,5-dimethoxybenzene (DDB) to convert MG to 6,7-dimethoxy-2-methylquinoxaline (DMQ), freeze-drying, and RP HPLC analysis using 6,7-dimethoxy-2,3-dimethylquinoxaline (DMDQ) as an internal standard (IS). Simplified methods for the synthesis of MG and DDB are also described. Results: Calibration curves were linear in the range of 200–1000 nM. The limit of detection was 30.6 and 45.9 pmol, at 215 and 352 nm, respectively. The intraday coefficients of variation were 6.9–12.6% for 215 nm and 3.5–12.6% for 352 nm. The interday coefficients of variation were 9.6–12.8% for 215 nm and 7.2–14.7% for 352 nm. Sample storage conditions together with statistical evaluation are also described. Conclusions: Here we present a rapid and inexpensive method for the determination of methylglyoxal in human plasma using RP HPLC with UV detection. The simplicity of the reported RP HPLC method makes it suitable for the detection of methylglyoxal in many human plasma samples.