Validation of Enzytec™ Liquid D-Glucose/D-Fructose for Enzymatic Determination of D-Glucose and D-Fructose in Selected Foods and Beverages: Official Method 2024.04 First Action.

Abstract

Background: D-Glucose and D-fructose are present in honey, wine and beer, and in a range of other foodstuffs such as bread, pastries, or chocolate. Both sugars can occur as a monosaccharide or in di-, oligo-, and polysaccharides.

Objective: To validate the performance of the Enzytec™ Liquid D-Glucose/D-Fructose test kit for the determination of D-glucose and D-fructose in food and beverages such as fruit and vegetable juices, soft drinks, wines, and beer.

Methods: The method is based on enzymes that are part of a prepackaged kit that contains three ready-to-use components. Both sugars are phosphorylated by a hexokinase. Glucose-6-phosphate (G-6-P) and nicotinamide adenine dinucleotide (NAD) are converted to gluconate-6-phosphate and reduced nicotinamide adenine dinucleotide (NADH). NADH is measured at 340 nm. Phosphoglucose isomerase converts fructose-6-phosphate to G-6-P, which in turn is converted to gluconate-6-phosphate and NADH.

Results: The test is specific to D-glucose and D-fructose and shows no side-activities or interferences with the exception of mannose and sulphite that interfere at 5.1 and 1.25 g/L or more, respectively. The measurement range is from 6.1 to 2000 mg/L for D-glucose and 5.6 to 1000 mg/L for D-fructose (100 µL test volume). Trueness was evaluated using NIST SRM 3282 (cranberry juice) and one reference wine. The recoveries ranged from 101 to 102%. Spiking of wine, beer, soft drinks, and juices resulted in recoveries between 93 and 105%. Intermediate precision is below 6% for concentrations at 25 mg/L and below 4% for higher concentrations. For automation, three applications with different test volumes and different measurement ranges were validated. Linearity is given from 5.0 to 10 000 mg/L for the sum of both sugars.

Conclusion: The method is robust and accurate for manual and automated applications and was approved as an AOAC Official Method of Analysis℠.

Highlights: The ready-to-use components of the test kit have a shelf life of at least 29 months.