Determination of glyoxal and methylglyoxal in serum by UHPLC coupled with fluorescence detection

  • Anal Biochem. 2019 May 15:573:51-66. doi: 10.1016/j.ab.2019.02.014.
Karthik Dhananjayan  1 ,  Felix Irrgang  1 ,  Ritesh Raju  1 ,  David G Harman  1 ,  Chris Moran  2 ,  Velandai Srikanth  3 ,  Gerald Münch  4
Affiliations
  • 1. Department of Pharmacology, School of Medicine, Western Sydney University, Locked Bag 1797, Penrith, NSW, 2751, Australia.
  • 2. Department of Medicine, Peninsula Health & Peninsula Clinical School, Monash University, Victoria, 3199, Australia.
  • 3. Department of Medicine, Peninsula Health & Peninsula Clinical School, Monash University, Victoria, 3199, Australia; Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania 7000, Australia.
  • 4. Department of Pharmacology, School of Medicine, Western Sydney University, Locked Bag 1797, Penrith, NSW, 2751, Australia; NICM Health Research Institute, Western Sydney University, Locked Bag 1797, Penrith, NSW, 2751, Australia. Electronic address: [email protected].
PMID: 30796906 DOI: 10.1016/j.ab.2019.02.014
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 ± 27 and 192 ± 10 nmol/L, respectively. In human serum, GO and MGO levels in non-diabetic subjects (n = 14) were determined to be 154 ± 88 and 98 ± 27 nmol/L, and in serum samples from subjects with diabetes (n = 14) 244 ± 137 and 190 ± 68 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.

Keywords
Blood samples; Fluorescence; Glyoxal; Methylglyoxal; UHPLC; Validation.
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