Simultaneous Quantitation of 2-Acetyl-4-tetrahydroxybutylimidazole, 2- and 4-Methylimidazoles, and 5-Hydroxymethylfurfural in Beverages by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

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Abstract
An ultrahigh-performance liquid chromatography (UHPLC) tandem mass spectrometric (MS/MS) method was developed for the simultaneous quantification of 2-acetyl-4-tetrahydroxybutylimidazole (THI), 2- and 4-methylimidazoles (2-MI and 4-MI), and 5-hydroxymethylfurfural (HMF) in beverage samples. A C30 reversed-phase column was used in this method, providing sufficient retention and total resolution for all targeted analytes, with an MS/MS instrument operated in selected reaction monitoring (SRM) mode for sensitive and selective detection using isotope-labeled 4-methyl-d3-imidazole (4-MI-d3) as the internal standard (IS). This method demonstrates lower limit of quantification (LLOQ) at 1 ng/mL and coefficient of determination (r2) >0.999 for each analyte with a calibration range established from 1 to 500 ng/mL. This method also demonstrates excellent quantification accuracy (84.6–105% at 5 ng/mL, n = 7), precision (RSD < 7% at 5 ng/mL, n = 7), and recovery (88.8–99.5% at 10, 100, and 200 ng/mL, n = 3). Seventeen carbonated beverage samples were tested (n = 2) in this study including 13 dark-colored beverage samples with different flavors and varieties and 4 light-colored beverage samples. Three target analytes were quantified in these samples with concentrations in the range from 284 to 644 ng/mL for 4-MI and from 706 to 4940 ng/mL for HMF. THI was detected in only one sample at 6.35 ng/mL.

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