A method for simultaneously determining vitamin A and vitamin E using liquid chromatography

Abstract

The present invention relates to a method for simultaneously determining vitamin A and vitamin E by liquid chromatography, and the chromatographic conditions at least include: chromatographic column: C8-C30 reverse phase chromatographic column, and polar groups are embedded in the alkyl group; wherein the embedded polar groups are amide groups, sulfonamide groups, urea groups or carbamate groups; mobile phase: phase A: water, phase B: methanol; isocratic method or gradient method is adopted; under isocratic method, the column temperature is 25-50°C, and the mobile phase is 90-92% methanol aqueous solution; under gradient method, the column temperature is 40-50°C, the initial mobile phase is 90% methanol aqueous solution, and the termination mobile phase is 91-94% methanol aqueous solution. The present invention can simultaneously determine all-trans retinol and 13-cis retinol of vitamin A, α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol of vitamin E, and the chromatographic separation can achieve the baseline separation effect, thereby improving the precision of simultaneous determination of vitamin A and vitamin E by liquid chromatography.

Description

Technical Field

[0001] The invention relates to the field of vitamin determination, and in particular to a method for simultaneously determining vitamin A and vitamin E by liquid chromatography. Background Art

[0002] Vitamin A is a cyclohexene plus conjugated polyene structure, and is mostly fortified in infant or adult formula nutrition products in the form of palmitate or acetate. Most of them exist as natural all-trans isomers (all-trans retinol), but under the action of heating or physical and chemical factors, various cis isomers will be produced, among which 13-cis retinol is the most common and main active isomer (activity is about 75% of all-trans), and other isomers have different potencies. Therefore, establishing a separation and analysis method for vitamin A isomers is of great significance for scientific evaluation of vitamin A activity. The structural formulas of the two are as follows:

[0003] All-trans Retinol:

[0004] 13-cis-Retinol:

[0005] Tocopherol, commonly known as vitamin E, is a fat-soluble vitamin. Vitamin E has important physiological functions for the human body, including anti-oxidation, improving the body's immunity and delaying aging. In addition, vitamin E inhibits the growth and proliferation of human tumor cells by adjusting human body functions. Vitamin E has the function of promoting fertility and good antioxidant properties. It is an excellent antioxidant and nutrient and has been widely used in medicine, food, cosmetics and feed industries. According to the number and position of methyl groups, it is divided into four different forms: α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol. The four tocopherols have their own unique physiological activities, among which α-tocopherol has the strongest physiological activity and γ-tocopherol has strong antioxidant ability. The structural formulas of the four tocopherols are as follows:

[0006] Alpha-Tocopherol:

[0007] Beta-Tocopherol:

[0008] Gamma-Tocopherol:

[0009] Delta-Tocopherol:

[0010] High performance liquid chromatography is a common method for analyzing vitamins A and E. In my country's standard method for vitamins A and E in food (GB5009.82-2016), C30 column reversed phase liquid chromatography is used for determination. However, since all-trans retinol and 13-cis retinol in vitamin A are isomers, this method cannot separate the two, so the actual measurement result is only the total content of vitamin A. In addition, from a chemical structure point of view, β-tocopherol and γ-tocopherol are also positional isomers, and chromatographic separation also poses certain challenges. Summary of the invention

[0011] In order to solve the above technical problems, the present invention provides a method for simultaneously determining vitamin A and vitamin E by liquid chromatography, which can simultaneously determine all-trans retinol and 13-cis retinol of vitamin A, and α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol of vitamin E. The chromatographic separation can achieve a baseline separation effect, thereby improving the accuracy of simultaneous determination of vitamin A and vitamin E by liquid chromatography.

[0012] The technical solution adopted by the present invention to solve its technical problem is:

[0013] A method for simultaneously determining vitamin A and vitamin E by liquid chromatography, wherein the vitamin A contains all-trans retinol and 13-cis retinol; and the vitamin E contains α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol;

[0014] In the method, the chromatographic conditions at least include:

[0015] Chromatographic column: C8-C30 reverse phase chromatographic column, wherein a polar group is embedded in the alkyl group; wherein the embedded polar group is an amide group, a sulfonamide group, a urea group or a carbamate group;

[0016] Mobile phase: Phase A: water, Phase B: methanol; isocratic or gradient method is used;

[0017] Under the isocratic method, the column temperature was 25°C-50°C, and the mobile phase was 90%-92% methanol in water;

[0018] Under the gradient method, the column temperature is 40°C-50°C, the initial mobile phase is 90% methanol aqueous solution, and the terminal mobile phase is 91%-94% methanol aqueous solution.

[0019] Preferably, the chromatographic column is a C18 reverse phase chromatographic column with silica gel as the matrix and polar groups embedded in the alkyl group; silica gel includes but is not limited to: fully porous silica gel, core-shell surface porous silica gel, hybrid silica gel; the particle size of silica gel is 1.5-10μm; the pore size of silica gel is 8-50nm.

[0020] Preferably, the chromatographic column is ChromCore Polar C18.

[0021] Preferably, the column length of the chromatographic column is 50-300 mm; the inner diameter of the column is 2.1-10 mm; and the column tube material is stainless steel or PEEK (polyetheretherketone).

[0022] Preferably, the chromatographic column is ChromCore Polar C18, 3 μm, 4.6 mm×250 mm.

[0023] Preferably, the column temperature is 45° C., an isocratic method is used, and the mobile phase is 90% methanol in water.

[0024] Preferably, the column temperature is 45°C and a gradient method is used, wherein the total gradient elution time includes a 25-minute period during which the mobile phase A continuously changes from 10% to 9%, and the mobile phase B continuously changes from 90% to 91%; wherein phase A is water and phase B is methanol.

[0025] Preferably, the chromatographic column is ChromCore Polar C18, 3 μm, 4.6 mm×150 mm.

[0026] Preferably, the column temperature is 45° C., an isocratic method is used, and the mobile phase is 90% methanol in water.

[0027] Preferably, the column temperature is 45°C and a gradient method is used, wherein the total gradient elution time includes a 15-minute period during which mobile phase A is continuously changed from 10% to 9%, and mobile phase B is continuously changed from 90% to 91%; wherein phase A is water and phase B is methanol.

[0028] Advantages of the present invention: The present invention provides a method for simultaneously determining vitamin A and vitamin E by liquid chromatography, optimizes several factors such as the stationary phase, mobile phase components, isocratic or gradient, and column temperature, and can simultaneously determine all-trans retinol and 13-cis retinol of vitamin A, and α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol of vitamin E. Chromatographic separation can achieve a baseline separation effect, thereby improving the accuracy of simultaneous determination of vitamin A and vitamin E by liquid chromatography. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the liquid chromatogram of Example 1 and 2;

[0030] Figure 2 are the liquid chromatograms of Test Examples 1 to 7;

[0031] Figure 3 are the liquid chromatograms of Test Examples 8 to 14;

[0032] Figure 4 are the liquid chromatograms of Test Examples 15 to 18;

[0033] Figure 5 are the liquid chromatograms of Test Examples 19 to 22;

[0034] Figure 6 The liquid chromatograms of Example 4 and Example 5 are shown;

[0035] Figure 7 are the liquid chromatograms of Test Examples 23 to 29;

[0036] Figure 8 are the liquid chromatograms of Test Examples 30 to 32;

[0037] Fig. 9 are the liquid chromatograms of Test Examples 33 and 34;

[0038] Fig.10 It is the liquid chromatogram of comparative example 1 and 2;

[0039] Fig.11 It is the liquid chromatogram of comparative example three. DETAILED DESCRIPTION

[0040] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0041] In order to facilitate the subsequent implementation, the preparation of vitamin A mixed standard solution, vitamin E mixed standard solution, and vitamin A and vitamin E mixed standard solution is now prepared, specifically as follows:

[0042] Preparation of vitamin A mixed standard solution:

[0043] Take appropriate amounts of 13-cis retinol and all-trans retinol reference substances, dissolve and dilute with methanol to prepare a mixed solution containing 4 μg of 13-cis retinol and 10 μg of all-trans retinol per 1 mL.

[0044] Preparation of vitamin E mixed standard solution:

[0045] Take appropriate amount of α-tocopherol (3 μg / mL), β-tocopherol (1 μg / mL), γ-tocopherol (3 μg / mL) and δ-tocopherol (1 μg / mL) reference substances, dissolve them in methanol and dilute them to make a mixed solution containing 6 μg of α-tocopherol, 2 μg of β-tocopherol, 6 μg of γ-tocopherol and 2 μg of δ-tocopherol per 1 mL.

[0046] Preparation of mixed standard solution of vitamin A and vitamin E:

[0047] Take appropriate amounts of vitamin A mixed standard solution, α-tocopherol (3 μg / mL), β-tocopherol (1 μg / mL), γ-tocopherol (3 μg / mL) and δ-tocopherol (1 μg / mL) reference substances, dissolve in methanol and dilute to make a mixed solution containing 0.8 μg of 13-cis-retinol, 2 μg of all-trans-retinol, 6 μg of α-tocopherol, 2 μg of β-tocopherol, 6 μg of γ-tocopherol and 2 μg of δ-tocopherol per 1 mL.

[0048] Embodiment 1

[0049] This embodiment provides a method for simultaneously determining vitamin A and vitamin E using liquid chromatography, and the specific chromatographic conditions are as follows:

[0050] Chromatographic column: ChromCore Polar C18 (4.6×250 mm, 3 μm) (a monodisperse high-purity silica microsphere chromatographic column with amide groups embedded in octadecyl groups);

[0051] Mobile phase: 90% methanol in water, isocratic method;

[0052] Flow rate: 0.8 mL / min;

[0053] Column temperature: 45°C;

[0054] Injection volume: 1 μL;

[0055] Detection wavelength: UV 325nm (0-15min, determination of cis- and trans-vitamin A);

[0056] UV 294nm (15-60min, determination of four tocopherols);

[0057] Sample: Vitamin A and E mixed standard solution;

[0058] Peak names: 1: 13-cis-retinol, 2: all-trans-retinol, 3: δ-tocopherol, 4: β-tocopherol, 5: γ-tocopherol, 6: α-tocopherol.

[0059] Embodiment 2

[0060] The difference between this embodiment and the first embodiment is that:

[0061] Chromatographic column: ChromCore Polar C18 (4.6×150 mm, 3 μm).

[0062] For comparison of the liquid chromatograms of Examples 1 and 2, please refer to Figure 1 For the test data, please refer to Table 1.

[0063] Table 1

[0064]

[0065] from Figure 1 It can be concluded from Table 1 that in the isocratic method, when using fillers with a particle size of 3 μm, both 150 mm and 250 mm long columns can achieve baseline separation (resolution not less than 1.5).

[0066] Embodiment 3

[0067] This embodiment provides a method for simultaneously determining vitamin A and vitamin E using liquid chromatography, and the specific chromatographic conditions are as follows:

[0068] Chromatographic column: ChromCore Polar C18 (4.6×250 mm, 3 μm);

[0069] Mobile phase: 90% methanol in water, isocratic method;

[0070] Flow rate: 0.8 mL / min;

[0071] Column temperature: 45°C;

[0072] Injection volume: 10 μL;

[0073] Detection wavelength: UV 325nm (0-15min, determination of cis- and trans-vitamin A);

[0074] UV 294nm (15-60min, determination of four tocopherols);

[0075] Sample: Mixed standard solution of vitamins A and E (diluted 10 times with methanol).

[0076] The sample was injected 6 times in total. Please refer to Table 2 for the test data.

[0077] Table 2

[0078]

[0079] It can be concluded from Table 2 that under the isocratic method, the four tocopherols of cis- and trans-vitamin A and vitamin E were continuously analyzed with good sample detection reproducibility.

[0080] Test Example 1

[0081] The chromatographic conditions were as follows:

[0082] Chromatographic column: ChromCore Polar C18 (4.6×250 mm, 3 μm);

[0083] Mobile phase: 92% methanol in water, isocratic method;

[0084] Flow rate: 0.8 mL / min;

[0085] Column temperature: 20°C;

[0086] Injection volume: 1 μL;

[0087] Detector: UV 325nm;

[0088] Sample: Vitamin A mixed standard solution;

[0089] Peak names: 1: 13-cis-retinol, 2: all-trans-retinol.

[0090] Test Example 2

[0091] The difference from Experimental Example 1 is: Column temperature: 25°C.

[0092] Test Example 3

[0093] The difference from Experimental Example 1 is: Column temperature: 30°C.

[0094] Test Example 4

[0095] The difference from Experimental Example 1 is: Column temperature: 35°C.

[0096] Test Example 5

[0097] The difference from Experimental Example 1 is: Column temperature: 40°C.

[0098] Test Example 6

[0099] The difference from Experimental Example 1 is: Column temperature: 45°C.

[0100] Test Example 7

[0101] The difference from Experimental Example 1 is: Column temperature: 50°C.

[0102] For comparison of the chromatograms of Experimental Examples 1 to 7, please refer to Figure 2 For the test data, please refer to Table 3.

[0103] Table 3

[0104]

[0105] from Figure 2 As can be seen from Table 3, the temperature study on the separation of cis- and trans-vitamin A shows that temperature has a significant effect on the retention time and separation degree of cis- and trans-vitamin A: the higher the temperature, the shorter the elution time and the lower the separation degree, but baseline separation can be achieved within the range of 20-50°C.

[0106] Test Example 8

[0107] The chromatographic conditions were as follows:

[0108] Chromatographic column: ChromCore Polar C18 (4.6×250 mm, 3 μm);

[0109] Mobile phase: 92% methanol in water;

[0110] Flow rate: 0.8 mL / min;

[0111] Column temperature: 20°C;

[0112] Injection volume: 1 μL;

[0113] Detector: UV 294nm;

[0114] Sample: Vitamin E mixed standard solution;

[0115] Peak names: 1: δ-tocopherol, 2: β-tocopherol, 3: γ-tocopherol, 4: α-tocopherol.

[0116] Test Example 9

[0117] The difference from Experimental Example 8 is: Column temperature: 25°C.

[0118] Test Example 10

[0119] The difference from Experimental Example 8 is: Column temperature: 30°C.

[0120] Test Example 11

[0121] The difference from Experimental Example 8 is: Column temperature: 35°C.

[0122] Test Example 12

[0123] The difference from Experimental Example 8 is: Column temperature: 40°C.

[0124] Test Example 13

[0125] The difference from Experimental Example 8 is: Column temperature: 45°C.

[0126] Test Example 14

[0127] The difference from Experimental Example 8 is: Column temperature: 50°C.

[0128] For comparison of the liquid chromatography diagrams of Experimental Examples 8 to 14, please refer to Figure 3 For the test data, please refer to Table 4.

[0129] Table 4

[0130]

[0131] from Figure 3As can be seen from Table 4, the temperature study on the separation of the four tocopherols shows that temperature has a significant effect on the retention time and separation degree of the four tocopherols: the higher the temperature, the shorter the elution time, the separation degree of β-tocopherol and γ-tocopherol continues to improve, reaching baseline separation (separation degree = 1.69) at 25°C, and reaching a maximum value (separation degree greater than 1.9) at 40-45°C.

[0132] Test Example 15

[0133] The chromatographic conditions were as follows:

[0134] Chromatographic column: ChromCore Polar C18 (4.6×250 mm, 3 μm);

[0135] Mobile phase: 89% methanol, isocratic method;

[0136] Flow rate: 0.8 mL / min;

[0137] Column temperature: 45°C;

[0138] Injection volume: 1 μL;

[0139] Detector: UV 325nm;

[0140] Sample: Vitamin A mixed standard solution;

[0141] Peak names: 1: 13-cis-retinol, 2: all-trans-retinol.

[0142] Test Example 16

[0143] The difference from Experimental Example 15 is that: mobile phase: 90% methanol, isocratic method.

[0144] Test Example 17

[0145] The difference from Experimental Example 15 is that: mobile phase: 91% methanol, isocratic method.

[0146] Test Example 18

[0147] The difference from Experimental Example 15 is that: mobile phase: 92% methanol, isocratic method.

[0148] For comparison of the liquid chromatography diagrams of Experimental Examples 15 to 18, please refer to Figure 4 For the test data, please refer to Table 5.

[0149] Table 5

[0150]

[0151] from Figure 4As can be seen from Table 5, the study on the methanol content in the mobile phase for the separation of cis- and trans-vitamin A shows that the elution time and separation degree of cis- and trans-vitamin A increase with the decrease of the organic phase in the mobile phase, and baseline separation can be achieved within the research range.

[0152] Test Example 19

[0153] The chromatographic conditions were as follows:

[0154] Chromatographic column: ChromCore Polar C18 (4.6×250 mm, 3 μm);

[0155] Mobile phase: 89% methanol;

[0156] Flow rate: 0.8 mL / min;

[0157] Column temperature: 45°C;

[0158] Injection volume: 1 μL;

[0159] Detector: UV 294nm;

[0160] Sample: Vitamin E mixed standard solution;

[0161] Peak names: 1: δ-tocopherol, 2: β-tocopherol, 3: γ-tocopherol, 4: α-tocopherol.

[0162] Test Example 20

[0163] The difference from Experimental Example 19 is that: mobile phase: 90% methanol, isocratic method.

[0164] Test Example 21

[0165] The difference from Experimental Example 19 is that: mobile phase: 91% methanol, isocratic method.

[0166] Test Example 22

[0167] The difference from Experimental Example 19 is that: mobile phase: 92% methanol, isocratic method.

[0168] For comparison of the liquid chromatography diagrams of Experimental Examples 19 to 22, please refer to Figure 5 For the test data, please refer to Table 6.

[0169] Table 6

[0170]

[0171] from Figure 5As can be seen from Table 6, the study on the methanol content in the mobile phase for the separation of four tocopherols showed that the elution time and separation degree of β-tocopherol and γ-tocopherol increased with the decrease of the organic phase in the mobile phase, and baseline separation could be achieved within the studied range.

[0172] Embodiment 4

[0173] This embodiment provides a method for simultaneously determining vitamin A and vitamin E using liquid chromatography, and the specific chromatographic conditions are as follows:

[0174] Chromatographic column: ChromCore Polar C18 (4.6×150 mm, 3 μm);

[0175] Mobile phase: Phase A, water; Phase B, methanol; gradient method;

[0176] Gradient elution program:

[0177]

[0178] Flow rate: 0.8 mL / min;

[0179] Column temperature: 45°C;

[0180] Injection volume: 1 μL;

[0181] Detection wavelength: UV 325nm (0-15min, determination of cis- and trans-vitamin A);

[0182] UV 294nm (15-60min, determination of four tocopherols);

[0183] Sample: Vitamin A and E mixed standard solution;

[0184] Peak names: 1: 13-cis-retinol, 2: all-trans-retinol, 3: δ-tocopherol, 4: β-tocopherol, 5: γ-tocopherol, 6: α-tocopherol.

[0185] Embodiment 5

[0186] The difference between this embodiment and the fourth embodiment is that:

[0187] Chromatographic column: ChromCore Polar C18 (4.6×250 mm, 3 μm);

[0188] Mobile phase: Phase A, water; Phase B, methanol; gradient method;

[0189] Gradient elution program:

[0190]

[0191] For comparison of the liquid chromatograms of Examples 4 and 5, please refer to Figure 6 For the test data, please refer to Table 7.

[0192] Table 7

[0193]

[0194] from Figure 6 It can be concluded from Table 7 that in the gradient method, when using fillers with a particle size of 3 μm, both 150 mm and 250 mm long columns can achieve baseline separation (resolution not less than 1.5).

[0195] Embodiment 6

[0196] This embodiment provides a method for simultaneously determining vitamin A and vitamin E using liquid chromatography, and the specific chromatographic conditions are as follows:

[0197] Chromatographic column: ChromCore Polar C18 (4.6×150 mm, 3 μm);

[0198] Mobile phase: Phase A, water; Phase B, methanol; gradient method;

[0199] Gradient elution program:

[0200]

[0201] Flow rate: 0.8 mL / min;

[0202] Column temperature: 45°C;

[0203] Injection volume: 10 μL;

[0204] Detection wavelength: UV 325nm (0-15min, determination of cis- and trans-vitamin A), UV 294nm (15-50min, determination of four tocopherols);

[0205] Sample: Vitamin A and E mixed standard solution (diluted 10 times with methanol)

[0206] Peak names: 1: 13-cis-retinol, 2: all-trans-retinol, 3: δ-tocopherol, 4: β-tocopherol, 5: γ-tocopherol, 6: α-tocopherol;

[0207] The sample was injected 6 times in total. Please refer to Table 8 for the test data.

[0208] Table 8

[0209]

[0210] It can be concluded from Table 8 that under the gradient method, the four tocopherols of cis- and trans-vitamin A and vitamin E were continuously analyzed with good sample detection reproducibility.

[0211] Test Example 23

[0212] The chromatographic conditions were as follows:

[0213] Chromatographic column: ChromCore Polar C18 (4.6×250 mm, 3 μm);

[0214] Mobile phase: Phase A, water; Phase B, methanol; Gradient method; Gradient elution program:

[0215]

[0216] Flow rate: 0.8 mL / min;

[0217] Column temperature: 20°C;

[0218] Injection volume: 1 μL;

[0219] Detection wavelength: UV 294nm;

[0220] Sample: Vitamin E mixed standard solution;

[0221] Peak names: 1: δ-tocopherol, 2: β-tocopherol, 3: γ-tocopherol, 4: α-tocopherol.

[0222] Test Example 24

[0223] The difference from Experimental Example 23 is: Column temperature: 25°C.

[0224] Test Example 25

[0225] The difference from Experimental Example 23 is: Column temperature: 30°C.

[0226] Test Example 26

[0227] The difference from Experimental Example 23 is: Column temperature: 35°C.

[0228] Test Example 27

[0229] The difference from Experimental Example 23 is: Column temperature: 40°C.

[0230] Test Example 28

[0231] The difference from Experimental Example 23 is: Column temperature: 45°C.

[0232] Test Example 29

[0233] The difference from Experimental Example 23 is: Column temperature: 50°C.

[0234] For comparison of the liquid chromatograms of Experimental Examples 23 to 29, please refer to Figure 7For the test data, please refer to Table 9.

[0235] Table 9

[0236]

[0237] from Figure 7 As can be seen from Table 9, the study on the effect of temperature on the separation of four tocopherols under the gradient method showed that temperature had a significant effect on the retention time and separation degree of the four tocopherols: the higher the temperature, the shorter the elution time, the better the separation degree of β-tocopherol and γ-tocopherol, and baseline separation could be achieved in the range of 40-50°C.

[0238] Test Example 30

[0239] The chromatographic conditions were as follows:

[0240] Chromatographic column: ChromCore Polar C18 (4.6×150 mm, 3 μm);

[0241] Mobile phase: Phase A, water; Phase B, methanol; Gradient method; Gradient elution program:

[0242]

[0243] Flow rate: 0.8 mL / min;

[0244] Column temperature: 45°C;

[0245] Injection volume: 1 μL;

[0246] Detection wavelength: UV 294nm;

[0247] Sample: Vitamin E mixed standard solution;

[0248] Peak names: 1: δ-tocopherol, 2: β-tocopherol, 3: γ-tocopherol, 4: α-tocopherol.

[0249] Test Example 31

[0250] The difference from Test Example 30 is that:

[0251] Mobile phase: Phase A, water; Phase B, methanol; Gradient method; Gradient elution program:

[0252]

[0253] Test Example 32

[0254] The difference from Test Example 30 is that:

[0255] Mobile phase: phase A, water; phase B, methanol; gradient method;

[0256] Gradient elution program:

[0257]

[0258] For comparison of the chromatograms of Experimental Examples 30 to 32, please refer to Figure 8 For the test data, please refer to Table 10.

[0259] Table 10

[0260]

[0261] from Figure 8 As can be seen from Table 10, the study on the separation of four tocopherols by the termination organic phase ratio under the gradient method showed that the elution time and separation degree of β-tocopherol and γ-tocopherol increased with the decrease of the termination organic phase, and baseline separation could be achieved within the research range.

[0262] Test Example 33

[0263] The chromatographic conditions were as follows:

[0264] Chromatographic column: ChromCore Polar C18 (4.6×150 mm, 3 μm);

[0265] Mobile phase: phase A, water; phase B, methanol; gradient method;

[0266] Gradient elution program:

[0267]

[0268] Flow rate: 0.8 mL / min;

[0269] Column temperature: 45°C;

[0270] Injection volume: 1 μL;

[0271] Detection wavelength: UV 294nm;

[0272] Sample: Vitamin E mixed standard solution;

[0273] Peak names: 1: δ-tocopherol, 2: β-tocopherol, 3: γ-tocopherol, 4: α-tocopherol.

[0274] Test Example 34

[0275] The difference from Test Example 33 is that:

[0276] Mobile phase: phase A, water; phase B, methanol; gradient method;

[0277] Gradient elution program:

[0278]

[0279] For comparison of the liquid chromatograms of Experimental Examples 33 and 34, please refer to Fig. 9 For the test data, please refer to Table 11.

[0280] Table 11

[0281]

[0282] from Fig. 9 As can be seen from Table 11, the study on the effect of gradient time on the separation of four tocopherols under the gradient method showed that a shorter gradient time under the same slope would not affect the separation of β-tocopherol and γ-tocopherol, but could save analysis costs.

[0283] Comparative Example 1

[0284] The specific chromatographic conditions of GB5009.82-2016 high performance liquid chromatography are as follows:

[0285] Chromatographic column: ChromCoreC30 (4.6×250 mm, 3 μm) (this column is not embedded with polar groups);

[0286] Mobile phase: Phase A, water; Phase B, methanol; Gradient method; Gradient elution program:

[0287]

[0288] Flow rate: 0.8 mL / min;

[0289] Column temperature: 20°C;

[0290] Injection volume: 1 μL;

[0291] Detection wavelength: UV 325nm (0-15min, determination of cis- and trans-vitamin A);

[0292] UV 294nm (15-40min, determination of four tocopherols of vitamin E);

[0293] Sample: Vitamin A and E mixed standard solution;

[0294] Peak names: 1: 13-cis-retinol, 2: all-trans-retinol, 3: δ-tocopherol, 4: γ-tocopherol, 5: β-tocopherol, 6: α-tocopherol.

[0295] Comparative Example 2

[0296] The difference from Comparative Example 1 is that:

[0297] Chromatographic column: ChromCoreC18 (4.6×250 mm, 3 μm) (no polar group embedded).

[0298] For the liquid chromatography diagrams of comparative examples 1 and 2, please refer to Fig.10 For the test data, please refer to Table 12.

[0299] Table 12

[0300]

[0301] from Fig.10 It can be concluded from Table 12 that, using the GB5009.82-2016 HPLC method, the C30 reverse phase column can separate the four tocopherols, but can only partially separate cis- and trans-vitamin A; the C18 reverse phase column can only partially separate cis- and trans-vitamin A, while β-tocopherol and γ-tocopherol co-elute (not separated).

[0302] Comparative Example 3

[0303] The difference from Comparative Example 2 is: Chromatographic column: ChromCore Polar C18 (4.6×250 mm, 3 μm) (embedded polar group).

[0304] For the liquid chromatogram of comparative example 3, please refer to Fig.11 For the test data, please refer to Table 13.

[0305] Table 13

[0306]

[0307] from Fig.11 As can be seen from Table 13, using the GB5009.82-2016 high performance liquid chromatography method, a polar embedded reversed phase C18 column (ChromCore Polar C18 (4.6×250 mm, 3 μm)) was used to separate cis- and trans-vitamin A from four tocopherols: cis- and trans-vitamin A could be baseline separated, while β-tocopherol and γ-tocopherol were partially separated, and the peak elution order was opposite to that of the comparative example 1 detected by ChromCoreC30.

[0308] The above embodiments should not limit the present invention in any way, and any technical solutions obtained by equivalent replacement or equivalent conversion shall fall within the protection scope of the present invention.

Claims

1. A method for simultaneously determining vitamin A and vitamin E using liquid chromatography, characterized in that: The vitamin A contains all-trans retinol and 13-cis retinol; the vitamin E contains α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol. In the method, the chromatographic conditions at least include: The chromatographic column was ChromCore Polar C18, embedded with amide groups; Using isocratic or gradient methods: Under the isocratic method, the column temperature was 25°C-50°C, and the mobile phase was 90%-92% methanol in water; Under the gradient method, the column temperature was 40℃-50℃, the mobile phases were: phase A: water, phase B: methanol; the proportion of phase B in the initial mobile phase was 90%, which increased continuously, and the proportion of phase B in the termination mobile phase was 91%-94%; The wavelength for detecting all-trans retinol and 13-cis retinol was UV 325nm; The wavelength for detecting α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol is ultraviolet 294nm.

2. The method for simultaneously determining vitamin A and vitamin E by liquid chromatography according to claim 1, characterized in that: The column length of the chromatographic column is 50-300 mm; the inner diameter of the column is 2.1-10 mm; the column tube material is stainless steel or PEEK.

3. The method for simultaneously determining vitamin A and vitamin E by liquid chromatography according to claim 2, characterized in that: The chromatographic column is ChromCore Polar C18, 3 μm, 4.6 mm × 250 mm.

4. The method for simultaneously determining vitamin A and vitamin E by liquid chromatography according to claim 3, characterized in that: The column temperature was 45 °C, the isocratic method was used, and the mobile phase was 90% methanol in water.

5. The method for simultaneously determining vitamin A and vitamin E by liquid chromatography according to claim 3, characterized in that: The column temperature was 45°C, and a gradient method was used, wherein, in the total gradient elution time, there was a period of 25 minutes, during which the mobile phase A was continuously changed from 10% to 9%, and the mobile phase B was continuously changed from 90% to 91%.

6. The method for simultaneously determining vitamin A and vitamin E by liquid chromatography according to claim 2, characterized in that: The chromatographic column is ChromCore Polar C18, 3 μm, 4.6 mm × 150 mm.

7. The method for simultaneously determining vitamin A and vitamin E by liquid chromatography according to claim 6, characterized in that: The column temperature was 45 °C, the isocratic method was used, and the mobile phase was 90% methanol in water.

8. The method for simultaneously determining vitamin A and vitamin E by liquid chromatography according to claim 6, characterized in that: The column temperature was 45°C, and a gradient method was used, wherein, in the total gradient elution time, there was a 15-minute period during which mobile phase A was continuously changed from 10% to 9%, and mobile phase B was continuously changed from 90% to 91%.

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