Analysis of N7-(2-carbamoyl-2-hydroxyethyl)guanine in Dried Blood Spot after Food Exposure by Ultra High Perfomance Liquid Chromatography – Tandem Mass Spectrometry

Abstract N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GAG) is a DNA adduct formed by glycidamide, which is the metabolite of acrylamide. Acrylamide can be found in foods containing reducing sugars and asparagine that are heated at high temperatures. Analysis of N7-(2-carbamoyl-2-hydroxyethyl)guanine was perfomed in Dried Blood Spot (DBS) samples from 25 subjects of group test who consumed a lot of acrylamide-containing foods and 25 subjects of negative control group. This study aimed to determine whether there is a significant difference in the levels of N7-(2-carbamoyl-2-hydroxyethyl)guanine between the two groups. DBS samples were extracted using the QIAamp DNA Mini Blood Kit and analyzed using Ultra High Performance Liquid Chromatography – Tandem Mass Spectrometry (UHPLC-MS/MS). Separation was performed using an Acquity UPLC BEH C18 column (2.1 mm x 100 mm; 1.7 µm), eluted a flow rate of 0.1 ml/minute under an isocratic of mobile phase of 0.1% formic acid and acetonitrile. The bioanalytical method of N7-(2-carbamoyl-2-hydroxyethyl)guanine in DBS with allopurinol as the internal standard by using UHPLC-MS/MS has been validated. The calibration curve range of N7-(2-carbamoyl-2-hydroxyethyl)guanine obtained was 10–300 ng/ml with a coefficient of correlation of 0.997. The results of the analysis on 25 test group subjects showed that the concentration of N7-(2-carbamoyl-2-hydroxyethyl)guanine ranged from 1.87 to 23.71 ng/ml, while the 25 subjects in the negative group ranged from 1.18 to 8.47 ng/ml. The results of the Mann Whitney test showed that there was a significant difference in the levels of N7-(2-carbamoyl-2-hydroxyethyl)guanine between the test group and the negative control group with p value less than 0.001.