Bioanalytical Method Validation of Acrylamide and Glycidamide in Dried Blood Spot Using Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

Abstract

Acrylamide (AA) is a carcinogenic compound that can be found in food, coffee, and cigarette smoke. When it enters human body, acrylamide will be metabolized by CYP2E1 to glycidamide (GA) which can then react with DNA to form DNA adducts. To analyze acrylamide and glycidamide simultaneously in the blood, commonly used biosampling technique is venipuncture, which is invasive and requires special expertise. The biosampling technique that was used in this study is Dried Blood Spot (DBS) method as it is easy and non-invasive. Methods for analyzing acrylamide and glycidamide simultaneously using DBS have not been carried out in previous studies. Therefore, the aim of this study is to obtain an optimum and validated method of acrylamide and glycidamide simultaneous analysis with propanamide as an internal standard. Sample preparation was done by protein precipitation using a mixture of methanol and water (1:1). Separation of compounds used reversed phase chromatography with the Acquity® UPLC BEH C18 column (1.7 μm, 2.1 mm x 100 mm) and elution flow rate of 0.20 mL/min under gradient conditions with a mobile phase of 0.2% formic acid in water and acetonitrile for 5 minutes. Quantification was performed using triple quadrupole mass spectrometry with positive electrospray ionization and multiple reaction monitoring (MRM) mode set at m/z 72.0> 55.02 for acrylamide, 88.1> 44.0 for glycidamide, and 74.01> 57.1 for propanamide. The lower limit of quantification was obtained at 1 μg/ml for both acrylamide and glycidamide. The range of linear concentration was 1 - 40 µg/ml. The analysis method was valid according to FDA 2018 guidelines.