Author: Yan Jing Chao 1, level 2 soup, Chan Peng 1,2, any country spectra 1, *
Abstract: Objective: To develop a reversed-phase high performance liquid chromatography method for rapid determination of vitamin A esters in infant formula. Method: use directly
The sample was extracted with a mixed solvent of dichloromethane and methanol in a volume ratio of 2.5:1, and isocratic elution was carried out at 30 ° C. The mobile phase was used in a volume ratio of
80:20 methanol-trichloromethane solvent, mobile phase flow rate 1.0 mL/min; column temperature 30 ° C, detection wavelength 325 nm. Result: Vitamin A vinegar
The recoveries of acid esters and vitamin A palmitate were 100.1% and 102.8%, respectively, and the relative deviations of multiple determinations were 1.18% and 0.96%, respectively.
The limits are 3.1 ng and 4.3 ng, respectively. Conclusion: This method is simple, precise, and has high recovery and sensitivity. It is suitable for infant formula milk powder.
Detection of vitamin A esters.
Key words: vitamin A ester; infant formula; reversed-phase high performance liquid chromatography
Rapid Determination of Retinyl Esters in Infant Formula MilkPowder by Reversed-Phase
High-performance Liquid Chromatography
YAN Jing-chao1, TANG Shui-ping2, PENG Can1, 2, REN Guo-pu1,*
(1. College of Food Science and Engineering, Central South University of Forestry and Technology, Changsha 410004, China;
2. Hunan Avadairy Co. Ltd., Changsha 410200, China)
Abstract :Objective: To develop a rapid reversed-phase highperformance liquid chromatographic (RP-HPLC) method for
Measuring retinyl esters in infant formula milk powder. Methods:Samples were directly extracted with a mixture of
And methanol (2.5:1, V/V). The chromatographic separation wasperformed at 30 °C in the isocratic elution mode using a
Phase composed of a mixture of methanol and chloroform (80:20,V/V). Results: Retinyl acetate and retinyl palmitate presented
Average spike recoveries of 6 replicates at 2 levels of 100.1% and 102.8%, with relative standard deviations of 1.18% and 0.96%,
The detection limits for retinyl acetate andretinyl palmitate were 3.1 ng and 4.3 ng, respectively.
Method demonstrates simplicity and high precision, recovery andsensitivity, and thus has promising potential for practical
Applications.
Key words:retinyl esters;infant formula milkpowder;reversed-phase high performance liquid chromatography
CLC number: TS252.1 Document code: A Article ID: 1002-6630(2010)08-0154-04
VA is an important fat-soluble vitamin whose main physiological functions
It is to maintain vision and bone health, participate in cell division and cell recognition, participate in growth, reproduction and maintain the integrity of the immune system [1]. GB 10766-1997 "Infant formula milk powder II, III" and GB 10765 - 1997
The content of VA in infant formula is specified in Infant Formula I
It is 1250-2500 IU/100g, which allows the addition of vitamin A acetate and vitamin A palmitate [2]. Currently measuring infants
The VA in the milk powder is mostly subjected to high performance liquid chromatography [3-9]. Before the analysis, the sample is generally saponified [3-7]. The process consumes a large amount of solvent, the operation is cumbersome and the vitamin A ester cannot be detected. The content of the class. In this experiment, the vitamin A ester in the reverse-phase chromatography legal infant formula was used. The pretreatment was not saponified. The protein was precipitated directly by using a mixture of dichloromethane and methanol, and the vitamin A ester was extracted. The vitamin A acetate and the vitamin A were quickly detected. Vitamin A palmitate content, control and wind for vitamins in the production process
※Analytical Testing and Food Science 2010, Vol. 31, No. 08 155
The establishment of the risk assessment system provides theoretical basis and practical guidance.
1 Materials and methods
1.1 Materials, reagents and instruments
Infant formula 1 (for infants from 0 to 6 months) Commercially available. Methanol, chloroform, dichloromethane (all chromatographically pure) American edia company; NaCl (analytical pure) Nanjing Chemical Reagent Co., Ltd.; vitamin A acetate, vitamin A palmitate standard American Sigma company.
Waters alliance 2695 High Performance Liquid Chromatography System (with Model 1200, Agilent UV Detector, Empower Control Software); UV-1800 UV-Spectrophotometer Japan Shimadzu Corporation; BF2000 Nitrogen Blowing Instrument; 800B Low Speed ​​Table
Centrifuge Shanghai Anting Scientific Instrument Factory; TC-C18 column (4.6mm × 25cm, 5μm) Agilent, USA.
1.2 Methods
1.2.1 Preparation of standard solutions
Vitamin A acetate and vitamin A palmitate standard stock solution: accurately weigh 10 mg of vitamin A acetate or vitamin A palmitate, and dilute to 100 mL volumetric flask with methanol to prepare a mass concentration of 100 μg/mL. Standard stock solution. Vitamin A acetate and vitamin A palmitate standard working solution: Take 1mL of vitamin A acetate or vitamin A palmitate standard stock solution in a 50mL volumetric flask and dissolve it with methanol to prepare a standard concentration of 2μg/mL. Working fluid.
1.2.2 Sample preparation
Accurately weigh 0.25g sample in a 10mL centrifuge tube, add 1mL warm water to dissolve completely, add 7mL volume ratio of 2.5:1 dichloromethane and methanol mixed solvent to extract the sample, vortex for 5min, after 5min, add 1mL saturation The NaCl solution was inverted and shaken 3 times, and centrifuged at 2500 r/min for 5 min. The methylene chloride layer was dried under nitrogen and dissolved in methanol.
solution. Filter through a 0.22 μm filter and collect in a brown vial.
1.2.3 Determination of UV Spectra of Standards
The standard stock solution of vitamin A acetate and vitamin A palmitate and standard working solution are scanned at a wavelength of 200-400 nm with a wavelength interval of 1 nm.
1.2.4 Orthogonal test design of chromatographic conditions
The factors are mobile phase volume ratio, column temperature, and mobile phase flow rate. This experiment does not consider the interaction, using L9 (34) orthogonal test for analysis, there is an empty column, can be used as experimental error to measure the reliability of the test, the factor level is shown in Table 1.
1.2.5 Quantitative analysis
Quantification by external standard method.
2 Results and analysis
2.1 Standard curve and linear relationship
Standard solutions with mass concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 μg/mL were prepared separately for injection and the corresponding peak areas were obtained. Taking the standard mass concentration as the abscissa and the peak area as the ordinate, the standard curve is shown in Figure 1.
factor
Level A Mobile phase volume ratio B Column C Mobile phase flow (methanol: chloroform) Temperature / ° C speed / (mL / min)
1 100:1 30 1.0
2 90:10 40 0.8
3 80:20 50 0.6
Table 1 Orthogonal test factor level
Table 1 Factors and levels in orthogonal array designfor optimizing instrumental parameters for HPLC analysis showed that the measured standard curve has a good linear relationship in the range of standard concentration of 1~5μg/mL. Vitamin A acetate and vitamin A palmitate standard curve The regression equations are y = 1 6 2 0 0 4 x -6012.6 and y=99332x - 2628.1, and the correlation coefficients R2 are 0.9992 and 0.9991, respectively.
2.2 zui good choice of chromatographic conditions
2.2.1 Selection of zui good detection wavelength
It can be seen from Fig. 2 that vitamin A acetate and vitamin A palmitate have two characteristic absorption peaks in this wavelength range, which are 263, 326 nm and 260 and 324 nm, respectively, and the absorption peaks of the two substances are large. At a wavelength of about 325 nm, so Zui Jiatu Figure 1 Vitamin A Acetate and Vitamin A Palmitate Standard Curve
Fig.1 Standard curves of retinyl acetate and palmitate
900
800
700
600
500
400
300
200
100
0
y=162004x - 6012.6
R2=0.9992
Peak area / (mV · s)
Standard mass concentration / (μg / mL)
0 1 2 3 4 5 6
y=99332x - 2628.1
R2=0.9991
Vitamin A Acetate Vitamin A Palmitate
Figure 2 Ultraviolet absorption spectrum of vitamin A acetate and vitamin A palmitate
Fig.2 UV absorption spectra of retinyl acetate and retinyl
Palmitate standards
2.0
1.5
1.0
0.5
0
260nm
Absorbance
Wavelength / nm
200 250 300 350 400
- 0.2
263nm
2
2
1
326nm
324nm vitamin A acetate
Vitamin A palmitate
1
The main chromatogram affecting the detection of vitamin A esters in infant formula 156 2010, Vol. 31, No. 08 Food Science ※ Analytical detection and determination wavelength is 325nm.
2.2.2 Influence of chromatographic conditions
2.2.2.1 Influence of mobile phase ratio
In the experiment, it was found that using methanol as the mobile phase, the retention time of vitamin A palmitate exceeded 40 min, which was not suitable for daily rapid analysis. The volume ratio of methanol to chloroform was measured. When the volume ratio of methanol to chloroform was 80:20, the separation of vitamin A esters was better, the retention time was shorter, and the analysis was completed within 15 minutes. The excessive mobility of chloroform is too small, and the retention time is too short to be suitable as a reverse phase mobile phase. Therefore, according to the liquid phase analysis experience, the volume ratio of methanol to chloroform is selected in the orthogonal test to be 100:1. 90:10, 80:20.
2.2.2.2 Effect of mobile phase flow rate
When the mobile phase flow rate is reduced, the chromatographic peak separation effect is slightly better, but the retention time of the vitamin A ester is prolonged, and the peak width is increased, the peak height is lowered, and the sensitivity of the vitamin A ester detection is lowered. In the liquid phase determination process, the flow rate is generally set to about 1 mL / min, the flow rate is too large, resulting in excessive system pressure; the flow rate is too small, resulting in too long retention time, and the peak broadens, the sensitivity is reduced, so flow in the orthogonal test The phase flow rate was selected to be 1.0, 0.8, and 0.6 mL/min.
2.2.2.3 Effect of column temperature
The temperature of the three columns at 30, 40, and 50 °C showed that the temperature of the column had little effect on the separation of the sample, but had a slight effect on the retention time. The low temperature of 30 °C was slightly longer than the high temperature of 50 °C, and the peak height was not affected. Or peak area, but the high temperature mobile phase is prone to bubbles. Therefore, the temperature of the column is selected in the orthogonal test to be in the range of 30 to 50 °C.
2.2.3 Chromatographic conditions orthogonal test results
Figure 3 Chromatogram of standard samples and samples
Fig.3 HPLC chromatograms of mixed retinyl acetate and retinylplamitate standards and a sample (both at a mixing ratio of 1:1)
Test number
A mobile phase volume with B column empty column C mobile phase flow
overall ratings
Ratio (methanol: chloroform) column temperature / ° C speed / (mL / min)
1 1(100:1) 1(50) 1 1(1) 67
2 1 2(40) 2 2(0.8) 65
3 1 3(30) 3 3(0.6) 61
4 2(90:10) 1 2 3 77
5 2 2 3 1 85
6 2 3 1 2 84
7 3(80:20) 1 3 2 92
8 3 2 1 3 87
9 3 3 2 1 95
K1 64.3 78.7 79.3 82.3 Total score 713
K2 82 79 79 77
K3 91 80 77.7 75
R 26.7 1.3 1.6 7.3
Table 2 Orthogonal test results
Table 2 Arrangement of experimental results of orthogonal array
Design for optimizing instrumental parameters for HPLC analysis
The effect of mobile phase volume ratio, mobile phase flow rate and column temperature on the determination of vitamin A esters was investigated by L9(34) orthogonal test. The method was judged by comprehensive scoring from three aspects of retention time, peak shape and system pressure. Good or bad. Out of 100 points, where the retention time is 50 points (50 points for 3min, 0 points for 53min), and the peak type is 40 points (25 points for each chromatographic condition, 3 points for the peak, 3 points for no tailing) Then, add 9 to 1 point according to the peak type. The system pressure is 10 points (10 points for pressure 1000-1500 psi, 6 points for 1500-2000 psi, and 2 points for 2000 psi). The results are shown in Table 2.
The data were analyzed by the range analysis method. The results showed that the effect of the mobile phase ratio on the determination of vitamin A esters was large, and the order of influence was A mobile phase volume ratio > C mobile phase flow rate > B column temperature. Combined with the UV absorption spectrum of the standard solution, the chromatographic conditions were determined as follows: methanol-trichloromethane solution with a mobile phase volume ratio of 80:20, column temperature 30 ° C, mobile phase flow rate 1.0 mL/min, measurement wavelength 325 nm .
Based on the experimental parameters identified above for vitamin A ester standards and
The sample was analyzed and the results are shown in Figure 3.
It can be seen from Fig. 3 that the vitamin A ester retention time is short, the vitamin A acetate is about 5 min, the vitamin A palmitate is about 10 min, there is no interfering substance near the chromatographic peak, and the separation effect with other components is good. The sample is consistent with the standard peak shape and peak time.
2.3 Repeatability experiment
0.032
0.024
0.016
0.008
0
4.793
AU
Time/min
0 3 6 9 12 15 9.893
a. Standard sample (the two standard solutions are mixed after 1:1 mixing)
0.0100
0.0075
0.0050
0.0025
0
4.668
AU
Time/min
0 3 6 9 12 15
9.893
b. Sample (the two samples of Zui final extract are mixed after 1:1 injection)
Experiment number
Vitamin A Acetate Vitamin A Palmitate
Measured value / (IU / 100g) Relative standard deviation / % Measured value / (IU / 100g) Relative standard deviation / %
1 1942.43 1773.19
2 1937.19 1765.00
3 1987.82 1.18 1731.69 0.96
4 1963.38 1757.90
5 1931.95 1771.55
Table 3 Repeatability experiment results (n=5)
Table 3 Repeatability test for the method (n=5) ※Analytical Testing and Food Science 2010, Vol. 31, No. 08 157
Five samples of the commercially available infant formula 1 milk powder sample were repeatedly measured in the same day as above, and the results were in terms of retinol equivalent. The results are shown in Table 3. Statistical analysis showed that the method was reproducible, and the relative standard deviations of vitamin A acetate and vitamin A palmitate were 1.18% and 0.96%, respectively.
2.4 spike recovery experiment
The standard sample is added to the milk powder of known vitamin A ester content, and the sample is treated as described above, and the result is determined by retinol equivalent.
See Table 4.
Substrate background value / (IU / 100g) Addition amount / (IU / 100g) Measured value / (IU / 100g) Recovery rate / % average recovery rate /%393299.5
2000 3794 96.0
Vitamin A acetate 1952 3972 100.5 100.1
6012 101.0
4000 6190 104.0
5910 99.3
3835 102.0
2000 3733 99.3
Vitamin A palmitate 1760 3812 101.4 102.8
6100 105.9
4000 5979 103.8
6002 104.2
Table 4 Recovery rate experiment results (n=6)
Table 4 Spike recovery test for the method (n=6)
Two levels of addition were chosen and assayed as described above. The results showed that the recovery rate of the method was high, and the average recoveries of vitamin A acetate and vitamin A palmitate were determined to be 100.1% and 102.8%, respectively.
2.5 zui low detection limit
According to the experimentally selected chromatographic conditions, the low detection limits of vitamin A acetate and vitamin A palmitate were calculated to be 3.1 ng and 4.3 ng, respectively, with a signal-to-noise ratio (RSN) of 3 times.
3 Conclusion
The chromatographic conditions for the determination of vitamin A esters were determined by orthogonal test: the mobile phase was methanol-trichloromethane solvent with a volume ratio of 80:20, the mobile phase flow rate was 1.0 mL/min, the column temperature was 30 ° C, and the detection wavelength was 325nm. The results of spiked recovery experiments showed that the average recoveries were 100.1% and 102.8% (n=6). Repeatability experiments showed that the relative deviations were 1.18% and 0.96% (n=5), respectively. The detection limits were 3.1 ng and 4.3 ng, respectively. The method of this method is simple in pretreatment, high in accuracy and precision, and is suitable for quantitative analysis of vitamin A esters in infant formula milk powder, and is also suitable for determination of vitamin A esters of other similar products.
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