Robust HPLC-Refractive Index Analysis of Simple Sugars in Bev erages Using Silica Hydride Columns

Background: Chromatographic analysis of sugars is impaired by several factors: polarity of the analytes, lack of chromophores in the structures, and chemical reactivity/degradation of HPLC columns typically used. As a result, existing routine sugars assays tend to be difficult and lack robustness. Objective: The unique characteristics of silica hydride-based columns are investigated for separation of two common sugars, fructose and glucose. Methods: Two stationary phase types are studied, and their chromatographic properties are compared in terms of analyte retention, selectivity, and peak shape. As a proof of concept, the developed chromatographic approach is used to obtain estimates of the concentration of fructose and glucose in three selected beverage samples. Robustness in analyte retention is also addressed in both intra- and inter-day timescales. Results: The silica hydride based phases can overcome the commonly reported issue of peak shape distortion from mutorotation using carefully chosen mobile phase conditions. Concentrations in the samples were obtained using calibration curve data (0.5-10.0 mg/mL range) and, accounting for sample dilutions, were 42.3 and 25.0 mg/mL for fructose and glucose respectively in cola, 1.22 and 1.46 mg/mL in red wine, and 13.7 and 13.1 mg/mL in grape juice. For robustness, percent relative standard deviation of analyte retention was not more than 0.010% for intra-day (ten injections) and not more than 1.0% for inter-day (five days). Conclusion: Silica hydride columns can be used in routine sugar analysis in a robust manner, which may be of great benefit to QC laboratories that need reliable assay methods.