Optimization of assay method of trans-10-hydroxy-2-decenoic acid (10-hda) in royal jelly and food supplements by high performance liquid chromatography

Abstract

Introduction Royal jelly (RJ) is a worker bee gelatinous secretion from the hypofaringeal and mandibular glands. It is composed by proteins, carbohydrates, lipids, amino acids, sugars, minerals and small amount of vitamins. Nowadays RJ is widely used in producing of food supplements, in pure state or lyophilized, also in cosmetic products. The major and the most important of RJ is (2E)-10-hydroxydec-2-enoic acid (10-HDA). 10-HDA is unsaturated fatty acid, no one other bee products contain such substance. Since it is specific, the substance can be used as biomarker of RJ. Thus an amount of 10-HDA shows the quality of those products. Materials and Methods HPLC analysis has been carried out using Waters 2695 chromatography system. Separation was done with usage of ACE 5 C18 column (250 x 4.6 mm, particle size 5μm. The two elution solvents were exchanged: the solvent A (0.1% TFA) and the solvent B (acetonitrile). The following linear gradient elution profile was used: 95% A/5% B–0 min, 25% A/75% B–20 min, 5% A/95% B–21 min, and 5% A/95% B–28 min. The flow rate was 1 mL/min and injection volume was 10. The effluent was determined at a wavelength of 240 nm. Results and discussion For extraction of 10-HDA from raw material and products, such as tablets and capsules, which contains RJ, we used 3 different solvents (methanol, chloroform and ethyl acetate). Standard was dissolved in each of these solvents, the best peak shape was obtained from methanol solvent. For preparation test solutions were used the same method, weights of samples were placed into measure flask added methanol and sonicated for 15 minutes. This method was suitable for tablets, but not for capsules and raw material, we could not receive separation of 10-HDA and other excipients. Because of that, the content of capsules was extracted with a portion of diethyl ether (15 ml), then non-soluble material was extract