ABSTRACT
Glyoxal (GO) and methylglyoxal (MGO) are two important biomarkers in diabetes. Analytical methods for determination of GO and MGO in serum samples are either HPLC with UV-Vis (low sensitivity) or MS/MS (expensive) detection. These disadvantages have hampered the introduction of these biomarkers as a routine analyte for diabetes diagnostics into the clinical laboratory. In this study, we introduce a UHPLC method with fluorescence detection for the measurement of GO and MGO in serum samples by pre-column derivatization at neutral pH with 5, 6-diamino-2,4-dihydroxypyrimidine sulfate (DDP) to form lumazines. The method was validated as per FDA guidelines. Using this method, we have determined GO and MGO in a variety of animal serum samples, and for example, determined the GO and MGO concentration in adult bovine serum to be 852⯱â¯27 and 192⯱â¯10â¯nmol/L, respectively. In human serum, GO and MGO levels in non-diabetic subjects (nâ¯=â¯14) were determined to be 154⯱â¯88 and 98⯱â¯27â¯nmol/L, and in serum samples from subjects with diabetes (nâ¯=â¯14) 244⯱â¯137 and 190⯱â¯68â¯nmol/L, respectively. In addition, interference studies showed that physiological serum components did not lead to an artificial increase in the levels of GO and MGO.
Subject(s)
Chromatography, High Pressure Liquid/methods , Fluorescent Dyes/chemistry , Glyoxal/blood , Pyruvaldehyde/blood , Aged , Aged, 80 and over , Animals , Calibration , Chromatography, High Pressure Liquid/standards , Diabetes Mellitus/pathology , Female , Glyoxal/chemistry , Glyoxal/standards , Humans , Hydrogen-Ion Concentration , Limit of Detection , Male , Mass Spectrometry , Middle Aged , Pteridines/chemistry , Pyruvaldehyde/chemistry , Pyruvaldehyde/standards , Reproducibility of ResultsABSTRACT
BACKGROUND: The reactive α-oxoaldehydes glyoxal (GO), methylglyoxal (MGO) and 3-deoxyglucosone (3-DG) have been linked to diabetic complications and other age-related diseases. Numerous techniques have been described for the quantification of α-oxoaldehydes in blood or plasma, although with several shortcomings such as the need of large sample volume, elaborate extraction steps or long run-times during analysis. Therefore, we developed and evaluated an improved method including sample preparation, for the quantification of these α-oxoaldehydes in blood and plasma with ultra performance liquid chromatography tandem mass spectrometry (UPLC MS/MS). METHODS: EDTA plasma and whole blood samples were deproteinized using perchloric acid (PCA) and subsequently derivatized with o-phenylenediamine (oPD). GO, MGO and 3-DG concentrations were determined using stable isotope dilution UPLC MS/MS with a run-to-run time of 8 min. Stability of α-oxoaldehyde concentrations in plasma and whole blood during storage was tested. The concentration of GO, MGO and 3-DG was measured in EDTA plasma of non-diabetic controls and patients with type 2 diabetes (T2DM). RESULTS: Calibration curves of GO, MGO and 3-DG were linear throughout selected ranges. Recoveries of these α-oxoaldehydes were between 95% and 104%. Intra- and inter-assay CVs were between 2% and 14%. CONCLUSIONS: To obtain stable and reliable α-oxoaldehyde concentrations, immediate centrifugation of blood after blood sampling is essential and the use of EDTA as anticoagulant is preferable. Moreover, immediate precipitation of plasma protein with PCA stabilized α-oxoaldehyde concentrations for at least 120 min. With the use of the developed method, we found increased plasma concentrations of GO, MGO and 3-DG in T2DM as compared with non-diabetic controls.