Determination of fat-soluble vitamins A, D2, D3, E and K3 by isotope dilution and LC-MS/MS instrument assembly

Abstract

The purpose of our publication is the determination of the total amount (of natural origin and added) of fat-soluble vitamins A, D2, D3 and E in low amounts in foods (wheat flour, soft drinks, effervescent tablets) and dietary supplements using a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The samples were diluted with isotope-labeled derivatives of the target components (vitamin A-d6, vitamin D2-d3, vitamin D3-d3, vitamin E-d6), and after extraction and saponification, they were purified by liquid-liquid extraction. After a solvent exchange, the concentration of the vitamins was determined on a C8 HPLC column using acidic mobile phases (0.1% formic acid in water/methanol) and LC-MS/MS technique. In dietary supplements, the analysis of the fat-soluble vitamin K3 may also be important, because the use of vitamin K3 is currently not approved in human formulations. During the determination of vitamin K3, saponification is not necessary, due to its structure, alkaline hydrolysis would lead to the decomposition of vitamin K3, so this component was analyzed by a method different from the one used for the other vitamins. LC-MS/MS analysis of small amounts of vitamin K3 is more complicated than that of other vitamins due to the low sensitivity of the MS instrument to vitamin K3. The determination of vitamin K3 was therefore carried out after chemical derivatization with L-cysteine as a derivatizing reagent, also with isotope dilution and LC-MS/MS technique. After intralaboratory validation, the methods were successfully used in domestic and international proficiency tests in infant formulas and liquid vitamin preparations.