Rapid and simple method for determination of Nɛ-(carboxymethyl)lysine and Nɛ-(carboxyethyl)lysine in urine using gas chromatography[sol ]mass spectrometry

Abstract

A new procedure was developed to determine in urine the concentrations of N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine (CEL), the major products of oxidative modification of glycated proteins, to assess levels of oxidative stress in physiological systems. The urine samples were acetonitrile-deproteinized, then derivatized by ethylchloroformate, and N(O,S)-ethoxycarbonyl ethyl esters of amino acids were analysed by isotope dilution gas chromatography/mass spectrometry. Recovery averaged 89%. Linearity was excellent (r = 0.998-0.999) in the 0.5-25 micromol/L range for CML and CEL. The limit of detection of this assay was 0.1 micromol/L (corresponding to 0.20 pmol of CML or CEL on column). Intra-day and inter-day precisions were likewise excellent, with relative standard deviations <4.63 and <6.15%, respectively. Accuracy of CML and CEL determination (15 micromol/L) was 2.9 and 5.9% of the estimated theoretical value. The time from obtaining the urine sample to determination of the concentration from the chromatographic peak was 80 min or less. This method is sensitive, reproducible, accurate, relatively cheap and very simple. It can be useful for laboratories involved in the diagnosis and monitoring of age-related chronic diseases.

Simultaneous analysis of lysine, Nɛ-carboxymethyllysine and lysinoalanine from proteins

“…The average recovery values were 84.45 ± 1.74 and 107.76 ± 10.24% for UHT milk and soy beverage, respectively. Data agreed with those described by others [47][48][49].…”

“…One-way analysis of variance of the response factor of CML demonstrated significant homogeneity of the variance between the samples over the range of concentration studied since a F-value of 1.010 with P = 0.412 (P > 0.05) was obtained. The precision of the method determined for the whole procedure including sample work-up ranged from 1.6 to 10.0% that can be considered adequate for quantitative analysis of CML according to the bibliography [17,26,27,[47][48][49]. Accuracy was estimated by carrying out recovery assays.…”

Molecular pathology and age estimation

“…In addition, MALDI-TOF and LC-MS-approaches have been described with the advantages of detecting minute amounts [144,145]. Recently, a fast, simple, precise and reliable GC-MS approach was described, where the entire detection procedure takes less than 1.5 h [142].…”

“…These methods include enzyme linked immunosorbent assays (ELISA) applying either polyclonal or monoclonal antibodies [135,1,136], immunohistochemistry [1,137] HPLC [138][139][140], GC-MS [141,142] or fluorimetry using the autofluorescent properties of AGEs [143]. In addition, MALDI-TOF and LC-MS-approaches have been described with the advantages of detecting minute amounts [144,145].…”

Dietary glycotoxins and infant formulas

“…The best method for measuring AGEs in tissues is biochemical examination of biopsies (30)(31)(32)(33).…”