Foreword

Vitamins A, D, and E are fat soluble vitamins necessary for maintaining normal metabolism and function in the body, but human and animal bodies cannot synthesize vitamins and must rely on food sources to provide sufficient vitamins. Health food, special medical formula food, and infant formula food are important ways to supplement vitamins. Accurately measuring the vitamin content in food is of great significance for scientific dietary guidance and ensuring food safety.

At present, the determination of vitamin A and E in food refers to the "GB 5009.82-2016 National Food Safety Standard for the Determination of Vitamin A, D, and E in Food (Partially Effective)", and the determination of vitamin D refers to the "GB 5009.296-2023 National Food Safety Standard for the Determination of Vitamin D in Food". GB 5009.296-2023 will be officially implemented on March 6, 2024, and the new standard mainly adds the second method of reverse phase liquid chromatography with online column switching. This scheme refers to the second method of the standard, using the BIKAI-UVTech’s two-dimensional liquid chromatography system to achieve first dimensional analysis of VA and VE (containing four isomers), and cutting the VD center on one dimension to the two-dimensional chromatographic column to eliminate matrix interference in the sample and achieve quantitative detection of low content VD.

1. Experimental conditions

1.1 System construction and valve switching process

· Instrument system: BIKAI-UVTech’s X-1511 two-dimensional liquid chromatography system

· System configuration: P60 dual ternary gradient pump

· A50C automatic sampler

· C10V2 column temperature box (with 2 built-in two position six port valves)

· D10 UV detector 

This scheme uses two pieces of six ports valves, with alternating use of a UV detector for one-dimensional and two-dimensional detection, to display the separation of the two dimensions in one chromatogram. By switching the valve, the VD on the one-dimensional chromatographic column is cut to the top of the trapping column, and then reverse eluted to the two-dimensional reverse phase chromatographic column through a two-dimensional pump, achieving purification and separation of VD. The specific flow path connection method and experimental steps are shown in the following figure.

1.2 Method conditions

2. Experimental Results and Discussion

2.1 Investigation of retention effect of trapping column

Due to the high proportion of methanol in the one-dimensional mobile phase during central cutting, there is a risk of "flow through" of the target on the trapping column. Therefore, this experiment investigated the retention effect of the capture column on VD during the cutting process. The specific operation is as follows: After 13.9 minutes, keep the valve status unchanged (left valve and right valve: 1-2), connect another UV detector to port 3 of the right valve, inject VD2/VD3 mixed standard solution, and the spectrum is shown below. From the results, it can be seen that the peak interval time between the two detectors is 2.5 minutes. Therefore, within the 1-minute cutting window (13.9-14.9 minutes), VD still remains on the trapping column, and the central cutting effect is better.

2.2 Chromatographic separation effect

According to the above method, the mixed standard solution was analyzed, and the resulting chromatogram is shown in the following figure. From the results, VA VD2、VD3、α-VE、β-VE、γ-VE、

The δ-VE was well separated. The separation degree of β-VE and γ-VE in the first dimensional chromatography is 1.66, and the separation degree of VD2 and VD3 is 2.75, which meets the quantitative requirements of the method. Based on the valve configuration of this scheme, the one-dimensional visible peak signal of VD can assist in determining the impact of one-dimensional retention time drift on the central cutting window, making this method more applicable in actual sample analysis.

2.3 Methodological investigation

2.3.1 Linear range, sensitivity, repeatability

Prepare ADE mixed standard working solution with methanol (VA: 100 μ g/mL; α, β, γ, δ - VE: 1000 μg/mL; VD2, VD3:1 μg/mL), and then dilute step by step to obtain a series of standard solutions. Due to the low amount of VD added in formula foods and the high and fixed content of VA and VE, this study investigated the linear relationship between VA in the concentration range of 0.1-10 μg/ml, VE in the range of 1-100 μg/ml, and VD in the range of 1-100 ng/mL, and focused on the instrument detection limit of VD. From the results, it can be seen that under the condition of a sample injection volume of 100 μL, all compounds have a good linear relationship, with a correlation coefficient>0.9999; When the concentration of VD2 and VD3 is 1 ng/mL, S/N>3, Based on a sample size of 5 g and a constant volume of 10 mL, the detection limit of this method is estimated to be 0.2 μg/100g, which is lower than the detection limit of the method in GB 5009.296-2023 (0.6 μg/100g)

Select ADE mixed standard solution (VA: 1 μg/mL; α, β, γ, δ - VE: 10 μg/mL; VD2, VD3:10 ng/mL) and repeat six needles (overlapping spectra shown below) to investigate the repeatability of compound peak area and retention time. From the results, it can be seen that the retention time RSD of all compounds is less than 0.1%, and the peak area RSD is less than 1%, indicating that this method has good stability.

2.3.2 Sample Testing

Accurately weigh 5g of a certain brand of infant formula milk powder, prepare the sample according to the first method of GB 5009.82-2016, inject the sample for analysis, and the sample spectrum is shown below. From the reults, it can be seen that this scheme can achieve simultaneous detection of vitamin ADE with one pre-treatment and one injection. Through reverse phase two-dimensional liquid chromatography center cutting for purification and separation of VD, matrix interference is reduced, and low content VD in milk powder can be accurately quantified.

3. Conclusion

This scheme uses the BIKAI-UVTech’s X-1511 two-dimensional liquid chromatography system to establish an analytical method for the content of vitamin ADE in food. This method uses reverse phase two-dimensional liquid chromatography center cutting to avoid the tedious steps of VD positive phase purification, and after one pre-treatment, one needle injection can achieve simultaneous detection of vitamin ADE; The solution of using BIKAI-UVTech’s P60 dual ternary gradient pump with single detector makes the instrument configuration more streamlined and the project operating cost lower. After preliminary methodological investigation, the linear range, repeatability, and sensitivity of this scheme meet the requirements of the national standard, and the results are accurate and reliable. It can be used as an effective means for rapid analysis of fat soluble vitamins in food matrices.