Разработка и валидация методов количественного определения витаминов В1 и В2 в пищевых продуктах с помощью высокоэффективной жидкостной хроматографии с диодно-матричным детектированием

Abstract

The main sources of vitamins, which are essential substances, are mainly aliment products, foods for special dietary uses and dietary supplements. Therefore, the study of the native content of vitamins in aliment foods has always been of interest. For the chromatographic separation of vitamins, rather versatile C18 columns are used as a stationary phase, which allow one to obtain reliable results using UV detection for vitamin-enriched foods, dietary supplements and vitamin premixes. However, for unfortified foods, this stationary phase in a UV detection system does not give acceptable results. The aim of the work was to develop a technique for the chromatographic separation of vitamins B1 and B2 in unfortified foods using a diode array detector. Material and methods. To prepare samples of foods, concentrated acid hydrolysis (1.0 g of sample and 4 ml of 0.1 N hydrochloric acid) was carried out in a water bath for 30 min at a temperature of 95 °C, followed by enzymatic hydrolysis and degreasing. Further studies of the samples were carried out on an Agilent Technologies 1100 chromatographic system with diode array detection. For the determination of vitamin B1, a Poroshell 120 Hilic column 4.6×150 mm, grain size 2.7 μm was used. As eluent A, a 10 mM aqueous solution of ammonium acetate with 0.5% acetic acid was used, eluent B was acetonitrile (gradient elution: 0-2 min - 90% B, 8-12 min - 50% B, 14-18 min - 90% B). To determine vitamin B2, a C18 Poroshell column 4.6×250 mm, grain size 5 μm was used. As eluent A, a classical phosphate buffer with pH 2.5 was used, eluent B - acetonitrile (gradient elution: 0-5 min - 0% B, 5-15 min - 90% B, 15-22 min - 90% B, 22-24 min - 0% B, 24-27 min - 0% B). Vitamin B1 was detected at a wavelength of 270 nm, vitamin B2 at 450 nm. Under selected conditions, good retention and efficient separation of vitamins B1 (over 16,000 theoretical plates) and B2 (over 20,000 theoretical plates) was observed. Results. It was demonstrated that the HPLC method with diode array detection can be used to quantify the native content of vitamins B1 and B2 in products with a complex food matrix. For the selective determination of these vitamins, a complex of chromatographic conditions is optimal: reverse-phase HPLC for vitamin B2 and hydrophilic interaction chromatography for vitamin B1. A suitable sample preparation of food products for the content of vitamins B1 and B2 under selected chromatographic conditions is concentrated acid-enzymatic hydrolysis. The limit of quantitation for vitamins B1 and B2 was 40 μg/100 g. Comparison of the enzymatic activity of amylorizin and thermostable α-amylase showed that during long-term hydrolysis for 16 hours (37 °С) with amylorizin, the degree of vitamin extraction was two fold higher than during hydrolysis (95 °С, 1 h) with α-amylase. Conclusion. The selected conditions for determining the native content of vitamins B1 and B2 in unfortified and low-fortified foods can be used in practice, which has been proven through their successful validation and practical application on real samples of cereals.