APPLICATION WITH VARIATION OF EXPERIMENTAL PARAMETERS OF THE HPLC METHOD IN THE PHARMACEUTICAL ANALYSIS OF THE ASCORBIC ACID SUBSTANCE

Abstract

The article is devoted to the identification and investigation of the purity of the ascorbic acid substance, namely the detection of accompanying and unacceptable impurities, by the method of high-performance liquid chromatography (HPLC) with the adaptation of chromatography conditions and modification of research methods. The purpose of the work. To introduce an alternative chromatography method - the method of high-performance liquid chromatography (HPLC) as a method with a higher identification capacity in the analysis of the tested substance of ascorbic acid in order to detect impurities of various origins in its composition; adapt the chromatography conditions and modify the research methods with optimal conditions for the protection of ascorbic acid molecules – active substance (API) of medicines from chemical degradation. Methodology. Ascorbic acid (Vitamin C) is a necessary nutrient for the human body, found in natural sources - leafy vegetables, fruits and berries. Ascorbic acid exists in the form of two enantiomers (L-, D-), among which the L-isomer is more common in nature. The D-isomer can be synthesized by organic synthesis, but has no significant biological value. In case of vitamin C deficiency, scurvy develops. Vitamin C has antioxidant properties and is a powerful regenerator. Therapeutic use of ascorbic acid includes prevention of ascorbic acid deficiency, as well as treatment of scurvy and anemia. Vitamin C lowers blood pressure and cholesterol levels in the body. During the synthesis of ascorbic acid substances, the formation of by-products, semi-products, accompanying impurities is possible, the presence of most of which is not regulated by the State Pharmacopoeia of Ukraine (SPU), European Pharmacopoeia (Eur.Ph.) and British Pharmacopoeia. In addition, the pharmacopoeial analysis of ascorbic acid involves the use of the liquid chromatography (LC) method. The highly selective method of HPLC chromatography provides a thorough analysis of the substance and a high degree of separation of components, which allows you to detect unregulated pharmacopoeias and unacceptable accompanying impurities in the composition of the tested substance and to draw a conclusion about the degree of its purity. Scientific novelty. Implementation of the modern highly selective HPLC method into the practice of pharmaceutical analysis of the ascorbic acid substance by adapting the chromatographic conditions and modifying the research methods with optimal conditions for the protection of API molecules – ascorbic acid from chemical degradation. Materials and methods. Samples of the substance of ascorbic acid, pharmacopoeial standard samples of SPU of ascorbic acid impurity C, ascorbic acid impurity D; HPLC, Agilent 1260 Infinity II chromatograph with UV detector, Zorbax NH2 column with a temperature of 25˚С; flow – 0.6 ml/min; injection volume – 20 μl; chromatography time – 60 min; UV detection at 210 nm; reagents were used to determine impurities by the HPLC method: acetonitrile (pure for HPLC), water for chromatography P (pure for HPLC), potassium dihydrogen phosphate; computer analysis by the OpenLab CDS program. Conclusions. The conditions of HPLC chromatography of the ascorbic acid substance were adapted in order to determine its purity. The proposed system of mobile phases: mobile phase A - potassium dihydrogen phosphate - water for chromatography P (filtrated), mobile phase B - acetonitrile P, mobile phase - phosphate buffer solution (phase A) - acetonitrile (phase B), 25:75 (V/V). Mobile phase A is proposed to dissolve the test substance of ascorbic acid. In order to adapt the chromatography conditions and protect the molecular structure of the substance from chemical and thermal degradation, it is proposed to vary some procedure parameters: flow rate, column temperature, which are milder and do not affect the quality of the analysis.4 unidentified impurities were found in the tested sample of the ascorbic acid substance: imp1 (Rt=2.984 min), imp2 (Rt=3.641 min), imp3 (Rt=7.744 min), imp4 (Rt=14.523 min), the presence of which is not regulated by pharmacopoeias. During the study, no specified impurity С.