Vitamins A, D and E are fat-soluble vitamins necessary to maintain normal metabolism of the body, so the vitamin content in the blood is often concerned. However, the blood vitamin D content is low, and the matrix is complex, and the detection is relatively difficult. The multi-dimensional chromatography separation mode has the characteristics of expanding the separation space and improving the separation efficiency, among which the two-dimensional chromatography separation technology is the most common separation technology in the multi-dimensional chromatography separation means, which can enrich and purify the detected objects and improve the sensitivity.
Here we share an on-line two-dimensional liquid chromatographic method for the rapid determination of vitamins A, D, and E in serum.The first dimension analyzes vitamins A and E and realizes the enrichment and purification of vitamin D, and then the second dimension separates and analyzes vitamin D. The separation is based on the reversed-phase liquid chromatography (RPLC) mode. The first dimension and the second dimension are based on reversed-phase separation mode, and the system is stable and reproducible. This solution can quickly realize the quantitative analysis of vitamin A, D and E in the sample with one injection after the sample has been pre-treated, which improves the analytical efficiency.
Instrument: S6000 HPLC
Column
First dimension：Alphasil S-PFP, 4.6 × 150 mm, 3.5 μm
Second dimension：SunFire C18, 4.6 × 150 mm, 3.5 μm
Determination of actual samples
The on-line two-dimensional detection of vitamins A, D, and E in serum was performed using this protocol, and the results
are shown in the figure below.
Determination results of vitamin A, D and E standards chromatogram
Vitamin A, D, E actual sample determination results chromatogram
Linearity and detection limits
The linearity and detection limits of vitamins A, D and E were investigated in the experiment, and the results were shown in the table below.
Name | Concentration | R2 | Detection limit |
VA | 0.04~10µg/mL | R2=0.9997 | 0.01µg/mL |
α-VE | 0.1-5µg/mL | R2=0.9999 | 0.05µg/mL |
25-OH-VD2 | 1.0-100.0ng/mL | R2=0.9993 | 0.4ng/mL |
25-OH-VD3 | 1.0-100.0ng/mL | R2=0.9999 | 0.4ng/mL |
Conclusion
This paper presents an on-line two-dimensional liquid chromatography method for the rapid determination of vitamin A, D and E in serum. The experimental results show that this method has the characteristics of strong specificity and high sensitivity, and is a rapid and effective means for the analysis of serum vitamins.
