CN108663462B - Method for measuring vitamin A, D and E in milk powder - Google Patents
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Abstract
The invention relates to a method for measuring vitamins A, D and E in milk powder, which comprises the following steps: uniformly mixing milk powder samples, weighing a proper amount, adding water, absolute ethyl alcohol, ascorbic acid, BHT (butylated hydroxytoluene) and KOH (potassium hydroxide) aqueous solution, heating for saponification, and cooling to a constant volume after saponification is finished; determining the content of vitamins A, D and E in the solution by adopting a high performance liquid chromatograph with a double-valve three-pump system; determining whether the sample contains vitamin A and E by comparing the retention time of the sample and the standard substance in the one-dimensional liquid chromatography; determining whether the sample contains vitamin D by comparing the retention time of the sample and the standard substance in the two-dimensional liquid chromatography; finally, calculating the contents of the vitamins A, D and E in the sample after conversion according to the obtained amounts of the vitamins A, D and E; the invention simplifies the operation steps, greatly shortens the pretreatment time, saves the labor and the reagent cost, realizes automatic online purification detection, improves the sensitivity and ensures the accuracy and the reproducibility of the test result.
Description
[ technical field ]
The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for determining vitamins A, D and E in milk powder.
[ background art ]
Vitamins are widely present in a variety of organisms, and there are currently found about tens of classes, which vary in chemical structure, among aliphatic, aromatic, alicyclic, heterocyclic and steroidal compounds. In physiological function, they are neither the main raw materials constituting various tissues nor the source of energy in the body, but they are small molecule organic compounds essential for participating in the regulation of metabolic processes of substances. The vitamins A, D and E are all nonpolar hydrophobic isoprene derivatives, are soluble in lipid and fat solvents and insoluble in water, and belong to fat-soluble vitamins. Excessive intake, unbalanced intake or lack of intake all cause corresponding symptoms, and influence the growth and development of the body and our life.
Fortified foods gradually enter our lives in order to meet the nutritional needs of different people and prevent diseases such as malnutrition. The vitamin enhancer is also the nutrient enhancer which is used earliest and most widely, and is added into various foods to supplement vitamins for human bodies. China issued ' standards for health use of food nutrition enhancers ' (trial runs) ' and food nutrition enhancer sanitation management methods in 1986. GB14880 'standards for use of food nutrition enhancers' was published in 1994, and is updated to 2012 at present, which plays a positive role in standardizing food nutrition enhancement in China, guiding enterprise production and ensuring the health and safety of consumers. Wherein the usage amount of vitamin A is 600 μ g/kg-17000 μ g/kg according to the requirement of food category, vitamin D is 2 μ g/kg-112 μ g/kg, and vitamin E is 5 μ g/kg-1450 μ g/kg.
The vitamins A, D and E are unstable in the external environment, are easily oxidized and damaged by the influence of illumination, air and the like, and meanwhile, the food matrix components are complex, the content of the vitamin D in the product is low, especially the content of the vitamin D in breast milk and milk is extremely low, so that the requirement on the sensitivity of the detection method is high. Based on the difficulties, the pretreatment method of the general sample comprises the steps of saponification wall breaking, organic solvent extraction phase inversion, concentration, purification and the like. The pretreatment process is complicated, time and labor are consumed, and the analysis efficiency of the sample is greatly influenced. At present, the national standard method for food safety is limited to simultaneous determination of vitamins A and E. The research on the simultaneous testing method of the vitamins A, D and E is also reported, and mainly comprises a liquid chromatography and a few liquid chromatography tandem mass spectrometry.
A method for measuring vitamin A, D3 and E in feed by using high performance liquid chromatography is reported in journal of environmental monitoring management and technology in 2013 of Wang' an swarm, and the method comprises the steps of soaking by using 90% of ethanol and then carrying out ultrasonic extraction by using 95% of ethanol, but the feed has complex matrix, serious damage to chromatographic columns, low VD content and incapability of being detected due to matrix interference, and is difficult to popularize. Subsequently, the Saimerfin issues a solution for simultaneously measuring the vitamins A, D and E in the feed by an online two-dimensional column switching method, and the application range of the method is expanded to a milk powder matrix in 2018. But the pretreatment inevitably needs to be carried out by the complicated processes of saponification, wall breaking, extraction, organic solvent phase inversion and concentration. Although the two-dimensional column switching method is adopted, the problem of matrix interference is solved, the situation that a chromatographic peak is widened and cannot be detected easily occurs due to the diffusion phenomenon of a target object in the chromatographic distribution process, and the influence is more obvious in a two-dimensional chromatographic column, so that the detection limit of VD (vacuum degassing) is difficult to reduce, and the test requirement of a low-content sample cannot be met. In 2015, the method for testing vitamins A, D and E in milk powder by using a high performance liquid chromatography tandem mass spectrometry reported by Yangzhou quality inspection improves the sensitivity of the method, but the steps of saponification extraction, organic solvent phase inversion concentration and the like cannot be avoided in the pretreatment process.
[ summary of the invention ]
The invention aims to solve the defects and provide a method for measuring vitamins A, D and E in milk powder, which simplifies the operation steps, greatly shortens the pretreatment time, saves the labor and reagent cost, realizes automatic online purification detection, improves the sensitivity and ensures the accuracy and the reproducibility of the test result.
In order to achieve the purpose, the method for measuring the vitamins A, D and E in the milk powder comprises the following steps:
1) sample treatment: uniformly mixing milk powder samples, weighing a proper amount, adding water, absolute ethyl alcohol, ascorbic acid, BHT (butylated hydroxytoluene) and KOH (potassium hydroxide) aqueous solution, heating for saponification, and cooling to a constant volume after saponification is finished;
2) determining whether the solution obtained in the step 1) contains vitamin A, D and E by adopting a high performance liquid chromatograph with a double-valve three-pump system; if the one-dimensional liquid chromatography result shows that the sample is suspected to contain vitamin A and vitamin E, comparing the retention time of the sample with that of the vitamin A and vitamin E standard substances to confirm whether the sample contains the vitamin A and the vitamin E; if the two-dimensional liquid chromatography result shows that the sample is suspected to contain vitamin D, comparing the retention time of the sample with that of a vitamin D standard substance to confirm whether the sample contains the vitamin D or not;
3) and if the vitamin A, D and E are confirmed to be contained in the sample, calculating the content of the vitamin A, D and E in the sample by a formula.
Further, in the step 1), weighing 2g of sample, adding 20mL of warm water, 30mL of absolute ethyl alcohol, 1g of ascorbic acid, 1g of BHT and 20mL of 50% KOH aqueous solution into the sample, heating in 80 ℃ water bath for 30min, cooling to room temperature after completion, diluting to 100mL with 50% ethanol aqueous solution, taking 2mL of the solution, filtering with a 0.45 μm filter membrane, and performing on-machine analysis.
Further, in step 1), if the sample contains starch, an appropriate amount of amylase is added before saponification.
Further, in the step 2), in the determination conditions of the high performance liquid chromatograph equipped with the double-valve three-pump system, the operation conditions of the on-line solid phase extraction part are as follows: (1) the enriching and purifying column for vitamin A, D and E adopts PLRP-S column with specification of 4.6 × 12.5mm and aperture of 15-20 um; (2) the mobile phase is 40% ethanol water solution, and the running time is 22 min; flow rate of mobile phase: 0-4min is 1 mL/min; 4-15min is 0.2 mL/min; 1mL/min for 15-22min, and 200 μ L of sample injection volume; (3) the flow path of the external six-way valve 3 is set as follows: 0-4min is 4 → 5 → 7 → 6; 8 → 9; 4-18min is 8 → 7 → 5 → 9; 4 → 6; 18-22min is 4 → 5 → 7 → 6; 8 → 9.
Further, in the step 2), in the determination conditions of the high performance liquid chromatograph equipped with the double-valve three-pump system, the operation conditions of the one-dimensional liquid chromatograph are as follows: (1) the liquid chromatographic column adopts a Poroshell 120EC-C8 column, the specification is 4.6 multiplied by 100mm, and the particle size is 4 mu m; (2) the mobile phase is water and acetonitrile, gradient elution is adopted, the running time is 22min, and the flow rate of the mobile phase is 1.5 mL/min; (3) adopting an ultraviolet detector, setting the wavelength to be 325nm for 0-11min and setting the wavelength to be 294nm for 11-22 min; (4) the flow path of the in-tank six-way valve 25 is set to: 0-12.4min is 17 → 16 → 19 → 18; 14 → 15; 12.4-13.1min is 14 → 19 → 16 → 15; 17 → 18; 13.1-22min is 17 → 16 → 19 → 18; 14 → 15.
Further, in the step 2), in the measuring conditions of the high performance liquid chromatograph equipped with the double-valve three-pump system, the operating conditions of the two-dimensional liquid chromatograph are as follows: (1) the vitamin D capturing column adopts a Poroshell 120EC-C18 column, the specification is 4.6 x 5mm, and the particle size is 4 mu m; (2) the liquid chromatographic column adopts an Eclipse PAH column, the specification is 2.1 x 100mm, and the particle size is 3.5 mu m; (3) the mobile phase is methanol and acetonitrile, gradient elution is adopted, the running time is 22min, and the flow rate is 0.4 mL/min; (4) an ultraviolet detector was used, with a wavelength set at 264 nm.
Further, in the step 2), the retention time of the sample peak and the retention time of the standard sample peak are compared to confirm whether the retention time of the chromatographic peak is consistent, so as to determine whether the vitamin A, D and E to be detected in the sample is detected, and if the vitamin A, D and E are contained, the standard curve external standard method is adopted for quantification.
Further, in the step 2), whether the sample contains vitamin A and vitamin E is confirmed by comparing the retention time of the sample and the standard substance in the one-dimensional liquid chromatography; and (3) comparing the retention time of the sample and the standard substance in the two-dimensional liquid chromatography to confirm whether the sample contains vitamin D.
Further, in the step 3), the contents of the vitamins A, D and E in the sample are calculated after conversion according to the contents of the vitamins A, D and E in the sample extracting solution obtained in the step 2).
Compared with the prior art, the invention has the following advantages:
(1) the invention establishes a multidimensional chromatographic mode which combines a plurality of separation mechanisms to directly purify the saponified extract of the milk powder on line, tests the high performance liquid chromatography of the vitamins A, D and E, simplifies the operation steps, greatly shortens the pretreatment time, saves the manpower and reagent cost, realizes the automatic on-line purification detection, improves the sensitivity, and ensures the accuracy and the reproducibility of the test result, thereby guiding the production regulation of food enterprises and preventing the adverse effect of inaccurate vitamin strengthening dosage on the health of consumers from the source.
(2) In the pretreatment of the existing method, the saponification liquid of a sample needs to be extracted and phase-inverted repeatedly by an organic solvent, the extracted organic solvent is washed by water for many times until the solution is neutral, and then the saponification liquid is concentrated to be nearly dry by rotary evaporation. This process is the most time and labor intensive step in the overall pretreatment process, limiting the efficiency and capacity of the overall process. The method adopts the saponification liquid to directly fix the volume and then carry out the on-machine analysis, skillfully avoids the steps of phase inversion, concentration and redissolution of the organic solvent, greatly simplifies the operation steps of the pretreatment of the sample, and obviously improves the analysis efficiency of the sample. Meanwhile, the pretreatment process is simplified, so that the risk of oxidative degradation of the vitamins A, D and E in the analysis process is reduced, and the accuracy and the reproducibility of a test result are ensured. The comparison graph is shown in the specification and figure 8.
(3) The method adopts the saponification liquid to directly fix the volume and then carry out the on-machine analysis, namely the saponification liquid does not need to adjust the pH value, thereby avoiding the loss of the target object caused by the solid precipitation phenomenon in the process of adjusting the pH value of the sample solution and expanding the application range of the method.
(4) The invention adopts the high performance liquid chromatography with a double-valve three-pump system to simultaneously detect the vitamins A, D and E in the sample, and the solid phase extraction is connected with the high performance liquid chromatography system on line, thereby realizing the automatic connection of the purification of the sample solution and the two-dimensional chromatographic analysis. Vitamin A, E is analyzed by the one-dimensional chromatography, vitamin D is analyzed by the two-dimensional chromatography, and meanwhile, a vitamin D capturing column is used in the two-dimensional chromatography for the first time, so that the problem that the sensitivity of a target substance is reduced due to too wide chromatographic peak caused by a diffusion phenomenon in the chromatographic distribution behavior process is effectively solved.
(5) The invention adopts special filler and purification column and chromatographic column with specification, and realizes the high performance liquid chromatography analysis of strong alkaline sample solution.
(6) After the target substance is enriched in the purifying column and the capturing column, the target substance is transferred to the analytical column in a way of eluting in the direction opposite to the flow path in the purifying process, and the obtained chromatographic peak has moderate peak width and good symmetry.
(7) The quantitative limits of vitamin A, alpha vitamin E, delta vitamin E and gamma vitamin E in the invention are all 80 mug/100 g, the quantitative limit of vitamin D2 is 2 mug/100 g, and the quantitative limit of vitamin D3 is 2 mug/100 g.
[ description of the drawings ]
FIG. 1 is a chromatogram of vitamin A, E in a one-dimensional chromatogram of example 1 according to the present invention;
FIG. 2 is a chromatogram of vitamin D in a two-dimensional chromatogram according to example 1 of the present invention;
FIG. 3 is a schematic view of an analysis solution flow path on SPE sample application equipped with a double-valve three-pump system in example 1 of the present invention;
FIG. 4 is a schematic view of an analytical solution flow path when VA/VD/VE is transferred to a one-dimensional chromatographic column during SPE elution with a double-valve three-pump system in example 1 of the present invention;
FIG. 5 is a schematic view of the analytical solution flow path when VA/VD/VE is eluted from a one-dimensional chromatographic column in SPE cleaning with a double-valve three-pump system in example 1 of the present invention;
FIG. 6 is a schematic view of an analytical solution flow path when VD is transferred from a one-dimensional chromatographic column to a capture column during SPE cleaning with a double-valve three-pump system according to example 1 of the present invention;
FIG. 7 is a schematic view of an analytical solution flow path when VD is transferred from a trap column to a two-dimensional analytical column during SPE equilibration with a double-valve three-pump system in example 1 of the present invention;
FIG. 8 is a comparison of a conventional process flow with a process flow of a direct saponification solution analysis method of the present invention;
in fig. 3 to 7: 1. the device comprises a solid-phase extraction sample pump 2, an automatic sample injector 3, an external six-way valve 4, an external first valve port 5, an external second valve port 6, an external third valve port 7, an external fourth valve port 8, an external fifth valve port 9, an external sixth valve port 10, a waste liquid collecting device 11, a solid-phase extraction column 12, a one-dimensional analysis pump 13, a one-dimensional analysis column 14, an in-tank first valve port 15, an in-tank second valve port 16, an in-tank third valve port 17, an in-tank fourth valve port 18, an in-tank fifth valve port 19, an in-tank sixth valve port 20, a variable wavelength detector 21, a two-dimensional analysis pump 22, a two-dimensional capture column 23, a two-dimensional analysis column 24, a diode array detector 25 and an in-tank six-.
[ detailed description of the invention ]
The invention belongs to the field of analytical chemistry for measuring vitamins A, D and E in milk powder, and the principle is as follows: heating and saponifying the vitamins A, D and E in the sample with water, absolute ethyl alcohol, ascorbic acid, BHT and KOH aqueous solution, cooling to constant volume after saponification, measuring the content by a high performance liquid chromatograph with a double-valve three-pump system, and determining the detection result by comparing the chromatographic peak retention time of the sample and a standard substance.
The method for measuring the vitamins A, D and E in the milk powder comprises the following steps: 1) sample treatment: uniformly mixing milk powder samples, weighing a proper amount, adding water, absolute ethyl alcohol, ascorbic acid, BHT (butylated hydroxytoluene) and KOH (potassium hydroxide) aqueous solution, heating for saponification, and cooling to a constant volume after saponification is finished; 2) determining whether the solution obtained in the step 1) contains vitamin A, D and E by adopting a high performance liquid chromatograph with a double-valve three-pump system; if the one-dimensional liquid chromatography result shows that the sample is suspected to contain vitamin A and vitamin E, comparing the retention time of the sample with that of the vitamin A and vitamin E standard substances to confirm whether the sample contains the vitamin A and the vitamin E; if the two-dimensional liquid chromatography result shows that the sample is suspected to contain vitamin D, comparing the retention time of the sample with that of a vitamin D standard substance to confirm whether the sample contains the vitamin D or not; 3) and if the vitamin A, D and E are confirmed to be contained in the sample, calculating the content of the vitamin A, D and E in the sample by a formula.
Wherein, in the step 1), if the sample contains starch, a proper amount of amylase needs to be added before saponification. And 2) comparing the retention time of the sample peak with that of the standard sample peak, and determining whether the retention time of chromatographic peaks is consistent, thereby determining whether the vitamins A, D and E to be detected in the sample are detected, and if so, quantifying by adopting a standard curve external standard method. Specifically, whether the sample contains vitamin A and vitamin E can be determined by comparing the retention time of the sample and the standard substance in the one-dimensional liquid chromatography; and (3) comparing the retention time of the sample and the standard substance in the two-dimensional liquid chromatography to confirm whether the sample contains vitamin D. In the step 3), calculating the contents of the vitamins A, D and E in the sample after conversion according to the contents of the vitamins A, D and E in the sample extracting solution obtained in the step 2).
The invention relates to a high performance liquid chromatograph with a double-valve three-pump system, namely, a double-valve three-pump high performance liquid chromatograph system, which comprises a solid phase extraction sample pump 1, an automatic sample injector 2, an external six-way valve 3, a waste liquid collecting device 10, a solid phase extraction column 11, a one-dimensional analysis pump 12, a one-dimensional analysis column 13, a variable wavelength detector 20, a two-dimensional analysis pump 21, a two-dimensional capture column 22, a two-dimensional analysis column 23, a diode array detector 24, an in-tank six-way valve 25 and a control system, wherein the external six-way valve 3 is provided with an external first valve port 4, an external second valve port 5, an external third valve port 6, an external fourth valve port 7, an external fifth valve port 8 and an external sixth valve, the in-tank six-way valve 25 is provided with an in-tank first valve port 14, an in-tank second valve port 15, an in-tank third valve port 16, an in-tank fourth valve port 17, an in-tank fifth valve port 18 and an in-tank sixth valve port 19; the solid-phase extraction sample injection pump 1 is connected with the automatic sample injector 2 and is used for realizing the absorption and sample injection of a sample analysis solution; the other end of the automatic sample injector 2 is connected with an external first valve port 4; two ends of the solid phase extraction column 11 are respectively connected with the external second valve port 5 and the external fourth valve port 7, a forward flow path of the solid phase extraction column is used for realizing the primary purification of a sample analysis solution, and a reverse flow path is used for realizing the elution sample introduction of an enriched analyte; the waste liquid collecting device 10 is connected with the external third valve port 6 and is used for collecting eluent; the one-dimensional analysis pump 12 is connected with an external fifth valve port 8 of the external six-way valve 3 and is used for transporting a mobile phase balance one-dimensional analysis column 13 and a target object which is reversely eluted and enriched in the solid phase extraction column 11; one end of the one-dimensional analytical column 13 is connected with the external sixth valve port 9, and the other end of the one-dimensional analytical column 13 is connected with the first valve port 14 in the tank and is used for analyzing part of the target object; the variable wavelength detector 20 is connected with the second valve port 15 in the tank and is used for detecting a target object; two ends of the two-dimensional trapping column 22 are respectively connected with a third valve port 16 in the tank and a sixth valve port 19 in the tank, a forward flow path of the two-dimensional trapping column is used for realizing elution sample injection of enriched analytes, and a reverse flow path of the two-dimensional trapping column is used for realizing secondary purification of a sample analysis solution; the two-dimensional analysis pump 21 is connected with a fourth valve port 17 in the box and is used for transporting a mobile phase equilibrium two-dimensional analysis column 23 and forward eluting the target substances enriched in the two-dimensional capture column 22; one end of the two-dimensional analytical column 23 is connected with the fifth valve port 18 in the tank, and the other end of the two-dimensional analytical column 23 is connected with the diode array detector 24 for detecting the target object.
The method adopts special filler, a purification column and a chromatographic column with specifications, realizes the direct volume fixing and then the on-machine analysis of the strong alkaline saponification solution for the first time, namely the saponification solution does not need to adjust the pH, avoids the loss of a target object caused by the solid precipitation phenomenon of a sample solution in the process of adjusting the pH, enlarges the application range of the method, skillfully avoids the steps of phase inversion, concentration and redissolution of an organic solvent, greatly simplifies the operation steps of the pretreatment of the sample, and obviously improves the analysis efficiency of the sample. Meanwhile, the pretreatment process is simplified, so that the risk of oxidative degradation of the vitamins A, D and E in the analysis process is reduced, and the accuracy and the reproducibility of a test result are ensured. The vitamin D capturing column is used in the two-dimensional chromatographic column, so that the problem of over-wide chromatographic peak and reduced sensitivity caused by diffusion phenomenon of the target in the chromatographic distribution behavior process is effectively solved.
The invention is further illustrated below with reference to specific examples:
example 1: determination of vitamin A, D, E in milk powder
1. Preparation of a sample:
accurately weighing 2g of the uniformly mixed sample into a 150mL flat-bottomed flask, adding 20mL of distilled water, 30mL of absolute ethyl alcohol, 1g of ascorbic acid, 1g of BHT and 20mL of 50% KOH aqueous solution, heating in a water bath at 80 ℃ for 30min, cooling to room temperature after completion, diluting to 100mL with 50% ethanol aqueous solution, taking 2mL of the solution, filtering with a 0.45-micrometer filter membrane, and performing on a machine for analysis.
2. Setting instrument parameters:
the operating conditions of the on-line solid phase extraction section were:
(1) the enriching and purifying column for vitamin A, D and E adopts PLRP-S column with specification of 4.6 × 12.5mm and aperture of 15-20 um;
(2) the mobile phase is 40% ethanol water solution, and the running time is 22 min; flow rate of mobile phase: 0-4min is 1 mL/min; 4-15min is 0.2 mL/min; 1mL/min for 15-22min, and 200 μ L of sample injection volume;
(3) the flow path of the external six-way valve 3 is set as follows: 0-4min is 4 → 5 → 7 → 6; 8 → 9; 4-18min is 8 → 7 → 5 → 9; 4 → 6; 18-22min is 4 → 5 → 7 → 6; 8 → 9.
The operating conditions of the one-dimensional liquid chromatography are as follows:
(1) the liquid chromatographic column adopts a Poroshell 120EC-C8 column, the specification is 4.6 multiplied by 100mm, and the particle size is 4 mu m;
(2) the mobile phase is water and acetonitrile, gradient elution is adopted, and the running time is 22 min; the flow rate of the mobile phase is 1.5 mL/min;
(3) adopting an ultraviolet detector, setting the wavelength to be 325nm for 0-11min and setting the wavelength to be 294nm for 11-22 min;
(4) the flow path of the in-tank six-way valve 25 is set to: 0-12.4min is 17 → 16 → 19 → 18; 14 → 15; 12.4-13.1min is 14 → 19 → 16 → 15; 17 → 18; 13.1-22min is 17 → 16 → 19 → 18; 14 → 15.
The operating conditions of the two-dimensional liquid chromatography are as follows:
(1) the vitamin D capturing column adopts a Poroshell 120EC-C18 column, the specification is 4.6 x 5mm, and the particle size is 4 mu m;
(2) the liquid chromatographic column adopts an Eclipse PAH column, the specification is 2.1 x 100mm, and the particle size is 3.5 mu m;
(3) the mobile phase is methanol and acetonitrile, gradient elution is adopted, and the running time is 22 min; the flow rate of the mobile phase is 0.4 mL/min;
(4) an ultraviolet detector was used, with a wavelength set at 264 nm.
3. And (3) characterization:
and comparing the retention time of the sample peak with that of the standard sample peak to determine whether the retention time of the chromatographic peak is consistent, thereby determining whether the object to be detected is detected in the sample.
4. Quantification:
quantification was done using standard curve external standard method.
5. And (3) calculating:
and calculating the contents of the vitamins A, D and E in the sample according to the contents of the vitamins A, D and E in the sample extracting solution.
The present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Claims (6)
1. A method for measuring vitamins A, D and E in milk powder is characterized by comprising the following steps:
1) sample treatment: uniformly mixing milk powder samples, weighing a proper amount, adding water, absolute ethyl alcohol, ascorbic acid, BHT (butylated hydroxytoluene) and KOH (potassium hydroxide) aqueous solution, heating for saponification, and cooling to a constant volume after saponification is finished;
2) determining whether the solution obtained in the step 1) contains vitamin A, D and E by adopting a high performance liquid chromatograph with a double-valve three-pump system; if the one-dimensional liquid chromatography result shows that the sample is suspected to contain vitamin A and vitamin E, comparing the retention time of the sample with that of the vitamin A and vitamin E standard substances to confirm whether the sample contains the vitamin A and the vitamin E; if the two-dimensional liquid chromatography result shows that the sample is suspected to contain vitamin D, comparing the retention time of the sample with that of a vitamin D standard substance to confirm whether the sample contains the vitamin D or not;
3) if the sample is confirmed to contain the vitamins A, D and E, calculating the contents of the vitamins A, D and E in the sample through a formula;
in the step 2), in the determination conditions of the high performance liquid chromatograph equipped with the double-valve three-pump system, the operation conditions of the on-line solid phase extraction part are as follows:
(1) the enriching and purifying column for vitamin A, D and E adopts PLRP-S column with specification of 4.6 × 12.5mm and aperture of 15-20 um;
(2) the mobile phase is 40% ethanol water solution, and the running time is 22 min; flow rate of mobile phase: 0-4min is 1 mL/min; 4-15min is 0.2 mL/min; 1mL/min for 15-22min, and 200 μ L of sample injection volume;
(3) the flow path of the external six-way valve 3 is set as follows: 0-4min is 4 → 5 → 7 → 6; 8 → 9; 4-18min is 8 → 7 → 5 → 9; 4 → 6; 18-22min is 4 → 5 → 7 → 6; 8 → 9;
in the step 2), in the determination conditions of the high performance liquid chromatograph equipped with the double-valve three-pump system, the operation conditions of the one-dimensional liquid chromatograph are as follows:
(1) the liquid chromatographic column adopts a Poroshell 120EC-C8 column, the specification is 4.6 multiplied by 100mm, and the particle size is 4 mu m;
(2) the mobile phase is water and acetonitrile, gradient elution is adopted, the running time is 22min, and the flow rate of the mobile phase is 1.5 mL/min;
(3) adopting an ultraviolet detector, setting the wavelength to be 325nm for 0-11min and setting the wavelength to be 294nm for 11-22 min;
(4) the flow path of the in-tank six-way valve 25 is set to: 0-12.4min is 17 → 16 → 19 → 18; 14 → 15; 12.4-13.1min is 14 → 19 → 16 → 15; 17 → 18; 13.1-22min is 17 → 16 → 19 → 18; 14 → 15;
in the step 2), in the determination conditions of the high performance liquid chromatograph equipped with the double-valve three-pump system, the operation conditions of the two-dimensional liquid chromatograph are as follows:
(1) the vitamin D capturing column adopts a Poroshell 120EC-C18 column, the specification is 4.6 x 5mm, and the particle size is 4 mu m;
(2) the liquid chromatographic column adopts an Eclipse PAH column, the specification is 2.1 x 100mm, and the particle size is 3.5 mu m;
(3) the mobile phase is methanol and acetonitrile, gradient elution is adopted, the running time is 22min, and the flow rate is 0.4 mL/min;
(4) an ultraviolet detector was used, with a wavelength set at 264 nm.
2. The method of claim 1, wherein: in the step 1), weighing 2g of sample, adding 20mL of warm water, 30mL of absolute ethyl alcohol, 1g of ascorbic acid, 1g of BHT and 20mL of 50% KOH aqueous solution into the sample, heating in a water bath at 80 ℃ for 30min, cooling to room temperature after completion, diluting to 100mL with 50% ethanol aqueous solution, taking 2mL, filtering with a 0.45-micrometer filter membrane, and performing on-machine analysis.
3. The method of claim 1, wherein: in the step 1), if the sample contains starch, a proper amount of amylase needs to be added before saponification.
4. The method of claim 1, wherein: and 2) comparing the retention time of the sample peak with that of the standard sample peak, and determining whether the retention time of chromatographic peaks is consistent, thereby determining whether the vitamins A, D and E to be detected in the sample are detected, and if so, quantifying by adopting a standard curve external standard method.
5. The method of claim 1, wherein: in the step 2), whether the sample contains vitamin A and vitamin E is determined by comparing the retention time of the sample and the standard substance in the one-dimensional liquid chromatography; and (3) comparing the retention time of the sample and the standard substance in the two-dimensional liquid chromatography to confirm whether the sample contains vitamin D.
6. The method of claim 1, wherein: in the step 3), calculating the contents of the vitamins A, D and E in the sample after conversion according to the contents of the vitamins A, D and E in the sample extracting solution obtained in the step 2).
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