Separation and quantification of fourteen chemical forms of fat soluble vitamins in food matrices using solid phase extraction coupled with liquid chromatography – mass spectrometry

A solid-phase extraction (SPE) method was developed for separation and quantification of vitamin A (retinol, beta-carotene, retinyl-palmitate, retinyl-acetate), vitamin D (D2, D3), vitamin E (alpha,beta,gamma,delta tocopherols, tocopheryl-acetate), and vitamin K (K1, K2, K3) from different food matrices. The polarities of all fourteen fat-soluble vitamins (FSVs) were determined and chemical properties of many different organic solvents were studied. Quantification was conducted using liquid chromatography-mass spectrometry (LC-MS). All fourteen fat-soluble vitamins were found to be non-polar in nature; beta-carotene was found to be the most non-polar vitamin in the group, followed by vitamin esters. Tocopherols, ketones (vitamins K1, K2, K3), and alcohols (vitamins D2, D3, and retinol) were found to be relatively more polar. All fourteen forms were soluble in dimethyl sulfoxide (DMSO) and were thus extracted with DMSO and methanol from food matrices.

To determine the recoveries of known concentrations of vitamin standards methanol and water were found to be suitable polar solvents for conditioning of the SPE column. Hexane was used for elution. This methodology was further applied to separate fourteen forms of FSVs from food matrices. 80-100% recoveries of FSVs were observed after SPE of provitamin samples (containing 2-3 ingredients). Poor recoveries of FSVs were observed after SPE of complex food samples such as multivitamin capsules, vitamin fortified drink mixes, and chewable nutrition tablets, which consisted of many different ingredients with varying polarities. The presence of other ingredients interfered with the SPE process leading to the elution of other compounds in the hexane solution along with FSVs.

Repeated measures ANOVA compared means of total FSVs after SPE and total FSVs from food labels. No significant differences were observed in the means (p < 0.05). A strong reliability (alpha>0.9) was observed between the three SPE extractions from each food sample. This SPE methodology can therefore be used for consistent and efficient separation of FSVs from products which do not have compounds that interfere with the extraction process.