High-throughput quantification of carboxymethyl lysine in serum and plasma using high-resolution accurate mass Orbitrap mass spectrometry

Abstract

Background Carboxymethyl lysine is an advanced glycation end product of interest as a potential biomarker of cardiovascular and other diseases. Available methods involve ELISA, with potential interference, or isotope dilution mass spectrometry (IDMS), with low-throughput sample preparation. Methods A high-throughput sample preparation method based on 96-well plates was developed. Protein-bound carboxymethyl lysine and lysine were quantified by IDMS using reversed phase chromatography coupled to a high-resolution accurate mass Orbitrap Exactive mass spectrometer. The carboxymethyl lysine concentration (normalized to lysine concentration) was measured in 1714 plasma samples from the British Regional Heart Study (BRHS). Results For carboxymethyl lysine, the lower limit of quantification (LLOQ) was estimated at 0.16 μM and the assay was linear between 0.25 and 10 μM. For lysine, the LLOQ was estimated at 3.79 mM, and the assay was linear between 2.5 and 100 mM. The intra-assay coefficient of variation was 17.2% for carboxymethyl lysine, 9.3% for lysine and 10.5% for normalized carboxymethyl lysine. The inter-assay coefficient of variation was 18.1% for carboxymethyl lysine, 14.8 for lysine and 16.2% for normalized carboxymethyl lysine. The median and inter-quartile range of all study samples in each batch were monitored. A mean carboxymethyl lysine concentration of 2.7 μM (IQR 2.0–3.2 μM, range 0.2–17.4 μM) and a mean normalized carboxymethyl lysine concentration of 69 μM/M lysine (IQR 54–76 μM/M, range 19–453 μM/M) were measured in the BRHS. Conclusion This high-throughput sample preparation method makes it possible to analyse large cohorts required to determine the potential of carboxymethyl lysine as a biomarker.

UPLC-MS/MS method for quantitative determination of the advanced glycation endproducts Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine

“…31–33 There are several methods that report CML, CEL, and/or lysine detection for AGEs studies utilizing various analytical techniques. 34–36…”

“…[31][32][33] There are several methods that report CML, CEL, and/or lysine detection for AGEs studies utilizing various analytical techniques. [34][35][36] The bioanalytical method validation by the European Medicines Agency (EMA) provides guidance and recommendations for bioanalytical assays, which can be seen as the "gold standard" in other types of method development. 37 By using the FDA approved guideline M10 by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), this study used sample analysis recommended chemical, biological, and metabolite drug guidelines to explore various analytical parameters, such as LOD, LOQ, matrix effects, intra-and inter-assay accuracy and precision, and matrix interferences.…”

Comprehensive Quantification of Carboxymethyllysine-Modified Peptides in Human Plasma

“…Second by glucose oxidation resulted glyoxal reaction with lysine and third by glyoxal derived from Schiff base decomposition reaction with lysine . Endogenously formed CML is more clinically relevant and has been studied mainly by GC–MS, enzyme-linked immunosorbent assay, fluorescence, multiple reaction monitoring (MRM), and isotope dilution methods . However, all of the above-mentioned studies measure free CML in enzymatic hydrolysates of either plasma, tissues, or cellular and extracellular proteins but fail in providing information about modified protein and the modification site, which are critical to claim CML as endogenous and understand the pathological roles of these proteins.…”

“…15 Endogenously formed CML is more clinically relevant and has been studied mainly by GC−MS, 16 enzyme-linked immunosorbent assay, 17 fluorescence, 18 multiple reaction monitoring (MRM), 19 and isotope dilution methods. 20 However, all of the abovementioned studies measure free CML in enzymatic hydrolysates of either plasma, tissues, or cellular and extracellular proteins but fail in providing information about modified protein and the modification site, which are critical to claim CML as endogenous and understand the pathological roles of these proteins. Previously, CML and CEL modifications are reported in isolated lens protein Crystallin and collagen from connective tissue.…”