Determination of vitamin D in oily drops using a column-switching system with an on-line clean-up by supercritical fluid chromatography

Abstract

A column-switching system, composed of supercritical fluid chromatography (SFC) and reverse phase liquid chromatography/mass spectrometry (RPLC/MS) was developed for the analysis of vitamin D in oily and fatty matrices. The SFC with the similar retention behavior as normal-phase liquid chromatography (NPLC), was applied for an on-line clean-up of oily and fatty samples, then followed by separation and detection using a reverse-phase LC-MS/MS. Three SFC columns packed with materials of different functional groups (Silica, NH2, Diol) were compared and the column with diol groups, on which the retention time of vitamin D was the longest, was finally selected for purification of the samples. 100% methanol was chosen to carry vitamin D from the clean-up column to the pre-treatment column. It was also used as the mobile phase for the separation of vitamin D on a reverse phase C18 column. Vitamin D2 and D3 were baseline separated by using this system. The linearity was calculated with a value of coefficient of determination (r2) ≥ 0.998. The linear range is from 20 ng/mL to 200 ng/mL. Two kinds of liquid vitamin D3 supplements (Baby Ddrops and Vitamin AD drops) were directly analyzed using this system without any fussy preparation procedure. The limit of detection (LOD) for vitamin D3 in the two oily samples was estimated to be 10 ng/mL. The relative standard deviations (RSD) of intra- and inter-day precision, repeatability were 1.47%, 2.43% and 1.59% for Baby Ddrops and 5.76%, 8.24% and 5.86% for Vitamin AD drops. The recoveries vary between 84.3% and 102.8% with 7.1% RSD for Baby Ddrops and 90.8–109.6% with 5.83% RSD for Vitamin AD drops, respectively. These results suggest that the method based on the SFC-RPLC/MS column-switching system is simple and suitable for analysis of vitamin D in oily and fatty samples.