Validation of an HPLC Method for Pretreatment of Steviol Glycosides in Fermented Milk

Abstract

Steviol glycosides are used in food and beverages worldwide as natural sweeteners, serving as a low-calorie sugar substitute. The acceptable daily intake of steviol is 0–4 mg/kg body weight. The rising demand for dairy products has led to a corresponding increase in the use of steviol glycosides in such products. Therefore, it is important to analyze the levels of steviol glycosides in dairy products. Dairy products have high fat contents and unique emulsion characteristics, conferred by a mixture of fat globules, casein micelles, whey proteins, and numerous other small molecules. These characteristics may interfere with the estimation of steviol glycoside levels; therefore, dairy samples require pretreatment. We aimed to develop an objective test for measuring the levels of steviol glycosides through the development of an efficient pretreatment method. In this study, the steviol glycoside content in dairy products was evaluated by using various methods, and an optimal pretreatment method was determined. We used high-performance liquid chromatography to assess the selectivity, linearity, limit of detection, limit of quantification, accuracy, precision, and recovery rate. Calibration curves were linear in the range of 1–50 mg/kg, with a coefficient of determination of ≥0.999. The limit of detection and limit of quantification were in the ranges of 0.11–0.56 and 0.33–1.69 mg/kg, respectively. The relative standard deviation (%) represents the precision of a measurement. The RSD relative standard deviationof recovery varied between 0.16% and 2.83%, and recovery of the analysis varied between 83.57% and 104.84%. These results demonstrate the reliability of the method for measuring the steviol glycoside content. This method can be used for the simple pretreatment of steviol glycosides and can provide an accurate determination of steviol glycoside content in emulsified food matrices, such as dairy products.

Validation of UHPLC-ESI-MS/MS Method for Determining Steviol Glycoside and Its Derivatives in Foods and Beverages

“…In this study, the ionization suppression or enhancement effect was calculated as the percentage of an absolute matrix effect (%ME A ) by comparing the peak area of the standard with the peak area of the standard when added to a sample matrix. The equation for the calculation is shown below [12,47].…”

“…Some studies used an acidic solution and extraction buffer, such as formic acid and N,N-diisopropylethylamine (DIPEA), for sample pretreatment [64], but this process was not applied in our study. We adopted the use of Carrez solutions to remove proteins and fat from dairy samples, which was found in several previous studies [12,19,70].…”

“…An important step in risk assessment is exposure assessment, which is calculated based on the consumption amount, concentration of the chemicals and average body weight in the population. There are many techniques used to determine the concentration of steviol glycoside and its derivatives [12], including high-performance thin-layer chromatography (HPTLC) [13], enzymatic hydrolysis [14,15], capillary electrophoresis [16], near-infrared spectroscopy, ionexchange resin chromatography [17,18], high-performance liquid chromatography (HPLC) with UV detection [19][20][21][22], LC/MS [23] and UHPLC-MS [2]. Nowadays, mass spectrometry is a powerful analytical technique based on ion separation; therefore, ionization is of importance for high sensitivity and selectivity.…”