Method for detecting content of lutein and / or zeaxanthine and application thereof

Abstract

The invention relates to a method for detecting the content of lutein and / or zeaxanthine, which comprises the following steps: taking a standard substance of a sample to be detected, and preparing a plurality of groups of standard substance solutions with different concentrations; testing the standard substance solution by adopting an online solid-phase extraction-liquid chromatography to obtain a standard working curve; pretreating a sample to be detected to obtain a sample solution, testing the sample solution by adopting an online solid-phase extraction-liquid chromatography, and substituting the obtained result into the standard working curve to obtain the content of xanthophyll and / or zeaxanthine. According to the method provided by the invention, the separation effect between the xanthophyll and the isomer thereof is improved, the content of the xanthophyll and the isomer thereof can be accurately measured, the repeatability is good, the accuracy is high, the overall analysis time is short, and the sample analysis and detection efficiency is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method for detecting the content of lutein and / or zeaxanthin and its application. Background Technology

[0002] Lutein and zeaxanthin are two important natural carotenoids widely found in vegetables, fruits, flowers, and other plants. Neither humans nor animals can synthesize these two substances themselves; they must be obtained through dietary intake or related supplements. Lutein is an excellent antioxidant with functions such as protecting eyesight, delaying arteriosclerosis, and anti-cancer properties. It is used as a food additive, added to food along with other beneficial complementary nutrients for health benefits, and is mainly used in the pharmaceutical, health product, food, cosmetic, and animal feed industries.

[0003] Lutein has a conjugated double bond in its molecular structure, which gives it strong light absorption properties. It has strong absorption in the range of 400-500 nm. It is unstable in the presence of oxygen, acid, strong light and high temperature, and is easily degraded, changed or isomerized. It is generally more stable under alkaline conditions. Alkaline saponification is a commonly used step in the extraction process of carotenoid components.

[0004] The current national standard GB5009.248-2016, "Determination of Lutein in Food," generally involves sample preparation steps such as saponification, liquid-liquid extraction, and water washing, followed by separation and analysis using high-performance liquid chromatography (HPLC) and a C30 column. This method quantifies lutein by summing the peak areas of all-trans lutein and the cis isomer, without specifying requirements for resolution. Furthermore, the sample preparation method is complex, resulting in low analytical efficiency.

[0005] The AOAC (2016.13) method for the determination of lutein in infant formula and adult nutritional products is similar in principle and process to the national standard method. However, AOAC uses a C30 column (YMC C30 3μm, 250×2.0mm) with higher column efficiency for analysis and imposes higher requirements on the analytical instrument (UHPLC). The system suitability test requires a resolution of >1.4 between 13-cis-lutein and 13'-cis-lutein, and a resolution of >2.2 between 13'-cis-lutein and all-trans-lutein. However, this method still suffers from drawbacks such as complex sample preparation and low analytical efficiency.

[0006] To simplify sample preparation, some literature has proposed using online solid-phase extraction-liquid chromatography (SPEC-LC) to determine the content of lutein and carotene in matcha or other foods. However, this method does not perform well in the overall separation of lutein and zeaxanthin, and it does not pay particular attention to the separation and determination of the cis isomer of lutein.

[0007] Therefore, providing a method for detecting the content of lutein and / or zeaxanthin, simplifying the sample preparation process, shortening the overall analysis time, and achieving good separation of lutein from its cis isomer and zeaxanthin, and thus accurately determining its content, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a method for detecting the content of lutein and / or zeaxanthin, and its application. The method provided by this invention improves the separation effect between lutein and its isomers, enabling accurate determination of their respective contents. It exhibits good repeatability, high accuracy, and a shorter overall analysis time, significantly improving the efficiency of sample analysis and detection.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a method for detecting the content of lutein and / or zeaxanthin, the detection method comprising the following steps:

[0011] Take the standard of the sample to be tested and prepare several sets of standard solutions with different concentrations; use online solid phase extraction-liquid chromatography to test the standard solutions and obtain the standard working curve;

[0012] The sample solution is obtained by pretreatment of the sample to be tested. The sample solution is tested by online solid phase extraction-liquid chromatography. The results are substituted into the standard working curve to obtain the content of lutein and / or zeaxanthin.

[0013] This invention employs an online solid-phase extraction-liquid chromatography (SPEC-LC) method, which effectively separates all-trans lutein, the cis isomer of lutein, and zeaxanthin from the sample to be tested. It enables precise determination of their respective contents with high accuracy and good repeatability, and can be widely used in the detection of food, pharmaceuticals, feed, and cosmetics.

[0014] Preferably, the preparation of the standard solution includes: mixing lutein and / or zeaxanthin, antioxidants and diluents to obtain several sets of standard solutions with different concentrations.

[0015] Preferably, the pretreatment includes: mixing the sample to be tested, antioxidant, diluent and alkaline reagent, and then saponifying to obtain a sample solution.

[0016] The method for detecting lutein content in the national standard GB5009.248-2016 requires saponifying, extracting, washing, concentrating, and adjusting the volume of the sample to obtain a sample solution. The preparation process is complex and prolongs the overall separation time.

[0017] In this invention, the pretreatment method eliminates the need for extraction and washing processes, reducing the overall analysis and detection time by nearly 30 minutes, greatly simplifying the analysis process and improving analysis efficiency; at the same time, it eliminates the need for liquid-liquid extraction processes using organic solvents, making it more environmentally friendly.

[0018] Preferably, the antioxidant comprises any one or a combination of two of 2,6-di-tert-butyl-p-cresol or ascorbic acid.

[0019] Preferably, the diluent comprises an aqueous ethanol-water solution or anhydrous ethanol with a volume concentration of 60-70% (e.g., 62%, 64%, 65%, 66%, 68%, etc.).

[0020] Preferably, the alkaline reagent includes potassium hydroxide.

[0021] Preferably, the concentration of the alkaline reagent in the sample solution is 0.1-1 g / mL (e.g., 0.1 g / mL, 0.2 g / mL, 0.3 g / mL, 0.4 g / mL, 0.5 g / mL, 0.8 g / mL, 0.9 g / mL, etc.).

[0022] Preferably, the saponification time is 15-45 minutes, for example, it can be 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, etc.

[0023] Preferably, the saponification temperature is 20-75℃, for example, it can be 25℃, 30℃, 40℃, 50℃, 60℃, etc.

[0024] Preferably, the saponification process further includes centrifugation and filtration.

[0025] Preferably, the centrifugation speed is 9500-10500 rpm, for example, it can be 9600 rpm, 9800 rpm, 10000 rpm, 10200 rpm, 10400 rpm, etc.

[0026] Preferably, the centrifugation time is 3-7 minutes, for example, it can be 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, 6.5 minutes, etc.

[0027] Preferably, the filter membrane used for filtration has a pore size of 0.2-0.45μm, such as 0.22μm, 0.25μm, 0.3μm, 0.35μm, 0.4μm, etc., and the filter membrane is preferably a nylon membrane.

[0028] Preferably, the online solid-phase extraction uses an extraction column comprising a polystyrene-divinylphenyl matrix extraction column or a polyvinylpyrrolidone matrix extraction column.

[0029] In this invention, the separation effect can be further improved by selecting an extraction column with specific packing material.

[0030] Preferably, the length of the extraction column is 5-100 mm, for example, it can be 15 mm, 20 mm, 25 mm, 50 mm, 80 mm, etc., and preferably 10-30 mm.

[0031] Preferably, the inner diameter of the extraction column is 2-10 mm, for example, it can be 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, etc., and more preferably 2-4.6 mm.

[0032] Preferably, the particle size of the extraction column is 5-50 μm, for example, it can be 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, etc., and is more preferably 10-40 μm.

[0033] Preferably, the extraction column comprises a ferrule column or a packed column.

[0034] Preferably, the analytical column used in the liquid chromatography comprises a column with a surface-polymerized C8-C18 alkyl-bonded silica matrix, and more preferably a column with a surface-polymerized octadecyl-bonded silica matrix.

[0035] In this invention, the use of chromatographic columns with specific packing materials can improve the precision and accuracy of detection.

[0036] Preferably, the silicone includes any one of fully porous silicone, core-shell surface porous silicone, or hybrid silicone.

[0037] Preferably, the particle size of the silica gel is 1.5-10μm, for example, it can be 2μm, 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm, etc., and is preferably 1.5-5μm.

[0038] Preferably, the pore size of the silicone is 8-50nm, for example, it can be 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, etc., and more preferably 8-18nm.

[0039] Preferably, the length of the analytical column is 50-600mm, for example, it can be 80mm, 100mm, 200mm, 300mm, 400mm, 500mm, etc., and preferably 100-300mm.

[0040] Preferably, the inner diameter of the analytical column is 2-50 mm, for example, it can be 3 mm, 4 mm, 10 mm, 20 mm, 30 mm, 40 mm, etc., and more preferably 2-4.6 mm.

[0041] Preferably, the mobile phase in the online solid-phase extraction includes phase A, phase B, phase C, and phase D, wherein phase A is water, phase B is acetonitrile, phase C is ethanol, and phase D is methyl tert-butyl ether.

[0042] Preferably, the online solid-phase extraction employs a gradient elution method, and the gradient elution procedure is as follows:

[0043] From 0 to 4 minutes, phase A accounts for 35-45% of the volume (e.g., 36%, 38%, 40%, 42%, 44%, etc.), and phase C accounts for 65-55% (e.g., 56%, 58%, 60%, 62%, 64%, etc.). Then, with a linear gradient change, by 5 minutes, phase B accounts for 100% of the volume. From 5 to 10 minutes, phase B accounts for 100% of the volume. Then, with a linear gradient change, by 11 minutes, phase B accounts for 45-55% of the volume (e.g., 46%, 48%, 50%, 52%, 54%, etc.), and phase D accounts for 55-45% of the volume (e.g., 46%, 48%, 50%, 52%, 54%, etc.). From 11 to 15 minutes, phase B accounts for 45-55% of the volume (e.g., 46%, 48%, 50%, 52%, 54%). The volume percentage of phase D is 55-45% (e.g., 46%, 48%, 50%, 52%, 54%, etc.); then, with a linear gradient change until min 16, phase B volume percentage is 100%; from min 16 to 20, phase B volume percentage is 100%; then, with a linear gradient change until min 21, phase A volume percentage is 35-45% (e.g., 36%, 38%, 40%, 42%, 44%, etc.), and phase C volume percentage is 65-55% (e.g., 56%, 58%, 60%, 62%, 64%, etc.); from min 21 to 25, phase A volume percentage is 35-45% (e.g., 36%, 38%, 40%, 42%, 44%, etc.), and phase C volume percentage is 65-55% (e.g., 56%, 58%, 60%, 62%, 64%, etc.).

[0044] In this invention, online solid-phase extraction employs a specific mobile phase and elution procedure. By adjusting the type and ratio of the mobile phase, the separation effect of all-trans lutein and the cis isomer can be improved, thereby increasing the precision of detection.

[0045] Preferably, the online solid-phase extraction has a linear flow rate of 0.8-10.0 mm / s, such as 1 mm / s, 2 mm / s, 4 mm / s, 6 mm / s, 8 mm / s, etc.

[0046] Preferably, the volumetric flow rate in the online solid-phase extraction is 0.5-2 mL / min, for example, it can be 0.8 mL / min, 1 mL / min, 1.2 mL / min, 1.4 mL / min, 1.6 mL / min, 1.8 mL / min, etc.

[0047] Preferably, the volumetric flow rate is 1.2-1.8 mL / min for 0-4 min (e.g., 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, 1.6 mL / min, 1.7 mL / min, etc.); then the flow rate is uniformly varied to 0.6-1 mL / min for 5 min (e.g., 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, etc.); and the volumetric flow rate is 0.6-1 mL / min for 5-25 min (e.g., 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, etc.).

[0048] Preferably, the mobile phase in the liquid chromatography includes phase A1, phase B1, phase C1 and phase D1, wherein phase A1 is water, phase B1 is acetonitrile, phase C1 is methanol and phase D1 is methyl tert-butyl ether.

[0049] Preferably, the liquid chromatography employs a gradient elution method, and the gradient elution procedure is as follows:

[0050] From 0 to 6 minutes, the volume percentage of phase A1 is 15-25% (e.g., 16%, 18%, 20%, 22%, 24%, etc.), and the volume percentage of phase B1 is 85-75% (e.g., 76%, 78%, 80%, 82%, 84%, etc.). Then, with a linear gradient change until 7 minutes, the volume percentage of phase A1 is 3-7% (e.g., 4%, 5%, 6%, etc.), and the volume percentage of phase C1 is 97-93% (e.g., 94%, 95%, 96%, etc.). Then, with a linear gradient change until 12 minutes, the volume percentage of phase C1 is 100%. Then, with a linear gradient change until 20 minutes, the volume percentage of phase C1 is 85-95% (e.g., 86%, 88%, 99%). The volume percentage of phase D1 is 15-5% (e.g., 6%, 8%, 12%, 14%). After a linear gradient change to 21 min, the volume percentage of phase C1 is 45-55% (e.g., 46%, 48%, 50%, 52%, 54%), and the volume percentage of phase D1 is 55-45% (e.g., 46%, 48%, 50%, 52%, 54%). From 21 to 25 min, the volume percentage of phase C1 is 45-55% (e.g., 46%, 48%, 50%, 52%, 54%), and the volume percentage of phase D1 is 55-45% (e.g., 46%, 48%, 50%, 52%, 54%).

[0051] In this invention, liquid chromatography employs a specific mobile phase and elution program. By adjusting the type and ratio of the mobile phase, the separation effect of all-trans lutein and the cis isomer can be improved, thereby enhancing the precision of detection.

[0052] Preferably, the linear flow rate in the liquid chromatograph is 0.8-3.0 mm / s, for example, it can be 1 mm / s, 1.2 mm / s, 1.5 mm / s, 1.8 mm / s, 2 mm / s, 2.2 mm / s, 2.5 mm / s, 2.8 mm / s, etc.

[0053] Preferably, the volumetric flow rate in the liquid chromatograph is 0.8-1.2 mL / min, for example, it can be 0.9 mL / min, 0.95 mL / min, 1 mL / min, 1.05 mL / min, 1.1 mL / min, 1.15 mL / min, etc.

[0054] Preferably, the column temperature in the liquid chromatograph is 20-45℃, for example, it can be 25℃, 30℃, 35℃, 40℃, etc.

[0055] Preferably, the valve-cutting time in the liquid chromatography is 4-6 min, for example, it can be 4-5 min, 4-5.5 min, 4-5.8 min, etc.

[0056] Preferably, the detection method includes the following steps:

[0057] Lutein and / or zeaxanthin, antioxidants and diluents were mixed to obtain several groups of standard solutions with different concentrations; the standard solutions were tested by online solid-phase extraction-liquid chromatography to obtain standard working curves.

[0058] Mix the sample to be tested, antioxidant, diluent, and alkaline reagent, saponify at 20-75℃ for 15-45 min, then centrifuge at 9500-10500 rpm for 3-7 min, and filter through a 0.2-0.45 μm filter membrane to obtain the sample solution; test the sample solution using online solid-phase extraction-liquid chromatography, and substitute the results into the standard working curve to obtain the content of lutein and / or zeaxanthin;

[0059] The concentration of the alkaline reagent in the sample solution is 0.1-1 g / mL;

[0060] The conditions for the online solid-phase extraction are as follows:

[0061] The extraction column comprises a polystyrene-divinylphenyl matrix extraction column or a polyvinylpyrrolidone matrix extraction column; the column length is 5-100 mm, the inner diameter is 2-10 mm, and the particle size is 5-50 μm; the mobile phase comprises phase A, phase B, phase C, and phase D, wherein phase A is water, phase B is acetonitrile, phase C is ethanol, and phase D is methyl tert-butyl ether; the gradient elution program is as follows: 0-4 min, phase A volume percentage 35-45%, phase C volume percentage 65-55%; then linear gradient change to phase B volume percentage 100% at 5 min; 5-10 min, phase B volume percentage 100%. 0%; then, with a linear gradient change to min 11, phase B volume percentage was 45-55%, and phase D volume percentage was 55-45%; from min 11 to 15, phase B volume percentage was 45-55%, and phase D volume percentage was 55-45%; then, with a linear gradient change to min 16, phase B volume percentage was 100%; from min 16 to 20, phase B volume percentage was 100%; then, with a linear gradient change to min 21, phase A volume percentage was 35-45%, and phase C volume percentage was 65-55%; from min 21 to 25, phase A volume percentage was 35-45%, and phase C volume percentage was 65-55%; the linear velocity was 0.8-10.0 mm / s;

[0062] The conditions for the liquid chromatography are as follows:

[0063] The analytical column comprises a surface-polymerized C8-C18 alkyl-bonded silica matrix; the silica has a particle size of 1.5-10 μm, a pore size of 8-50 nm, a column length of 50-600 mm, and an inner diameter of 2-50 mm; the mobile phase comprises phases A1, B1, C1, and D1, wherein phase A1 is water, phase B1 is acetonitrile, phase C1 is methanol, and phase D1 is methyl tert-butyl ether; the gradient elution program is as follows: 0-6 min, phase A1 volume percentage 15-25%, phase B1 volume percentage 85-75%; then linear gradient change until 7 min, phase A1 volume percentage 3... -7%, C1 phase volume percentage 97-93%; then linear gradient change to 12 min, C1 phase volume percentage 100%; then linear gradient change to 20 min, C1 phase volume percentage 85-95%, D1 phase volume percentage 15-5%; then linear gradient change to 21 min, C1 phase volume percentage 45-55%, D1 phase volume percentage 55-45%; 21-25 min, C1 phase volume percentage 45-55%, D1 phase volume percentage 55-45%; linear flow rate 0.8-3.0 mm / s, column temperature 20-45℃, valve cut-off time 4-6 min.

[0064] In a second aspect, the present invention provides an application of the method for detecting lutein and / or zeaxanthin content according to the first aspect in the detection of food, pharmaceuticals, feed or cosmetics.

[0065] Compared with the prior art, the present invention has at least the following beneficial effects:

[0066] (1) In the detection method provided by the present invention, the sample saponification solution is directly analyzed on the instrument. Within 26 minutes, the entire process of sample extraction, purification and analysis is completed online automatically, which simplifies the analysis process. Compared with the current standard method, the overall analysis time is shortened by nearly 32%.

[0067] (2) The detection method provided by the present invention has a good separation effect of lutein isomers. In the preferred embodiment, the separation degree between the main cis isomers is >1.9 and the separation degree between lutein and zeaxanthin is >4.0.

[0068] (3) The detection method provided by the present invention has good repeatability and high precision, and can accurately determine the content of lutein and its isomers. Attached Figure Description

[0069] Figure 1 This is the equipment setup diagram in Example 1.

[0070] Figure 2 This is the liquid chromatogram of the standard solution in Example 1.

[0071] Figure 3 This is the standard curve of lutein in Example 1.

[0072] Figure 4 This is the standard curve of zeaxanthin in Example 1.

[0073] Figure 5 This is a liquid chromatogram of the test solution used in the system in Example 1; wherein, peak 1 is 13-cis-lutein, peak 2 is 13'-cis-lutein, peak 3 is 9-cis-lutein, peak 4 is all-trans-lutein, and peak 5 is zeaxanthin.

[0074] Figure 6 This is a liquid chromatogram of the test solution used in the system in Example 2; wherein, peak 1 is 13-cis lutein, peak 2 is 13'-cis lutein, peak 3 is 9-cis lutein, peak 4 is all-trans lutein, and peak 5 is zeaxanthin.

[0075] Figure 7 This is a liquid chromatogram of the test solution used in the system in Example 3; wherein, peak 1 is 13-cis lutein, peak 2 is 13'-cis lutein, peak 3 is 9-cis lutein, peak 4 is all-trans lutein, and peak 5 is zeaxanthin.

[0076] Figure 8This is a liquid chromatogram of the test solution used in the system in Example 4; wherein, peak 1 is all-trans lutein, peak 2 is zeaxanthin, peak 3 is 13-cis lutein, peak 4 is 13'-cis lutein, peak 5 is 9-cis lutein, and peak 6 is other cis lutein.

[0077] Figure 9 This is a liquid chromatogram of the test solution used in the system in Example 5; wherein, peak 1 is 13-cis-lutein, peak 2 is 13'-cis-lutein, peak 3 is 9-cis-lutein, peak 4 is all-trans-lutein, and peak 5 is zeaxanthin.

[0078] Figure 10 This is a liquid chromatogram of the test solution used in the system in Example 6; wherein, peak 1 is 13-cis-lutein, peak 2 is 13'-cis-lutein, peak 3 is 9-cis-lutein, peak 4 is all-trans-lutein, and peak 5 is zeaxanthin.

[0079] Figure 11 This is a liquid chromatogram of the test solution used in the system in Example 7; wherein, peak 1 is 13-cis lutein, peak 2 is 13'-cis lutein, peak 3 is 9-cis lutein, peak 4 is all-trans lutein, and peak 5 is zeaxanthin.

[0080] Figure 12 This is a liquid chromatogram of the test solution used in the system in Example 8; wherein, peak 1 is all-trans lutein, peak 2 is zeaxanthin, peak 3 is 13-cis lutein, peak 4 is 13'-cis lutein, peak 5 is 9-cis lutein, and peak 6 is an unknown isomer.

[0081] Figure 13 This is a liquid chromatogram of the test solution used in the system in Example 9; wherein, peak 1 is zeaxanthin, peak 2 is lutein, and peak 3 is cis-lutein.

[0082] Figure 14 This is a liquid chromatogram of the test solution used in the system in Example 10; wherein, peak 1 is 13-cis-lutein, peak 2 is 13'-cis-lutein, peak 3 is 9-cis-lutein, peak 4 is all-trans-lutein, and peak 5 is zeaxanthin.

[0083] Figure 15 This is a liquid chromatogram of the test solution used in Comparative Example 1; where peak 1 is all-trans lutein, peak 2 is zeaxanthin, peak 3 is 13-cis lutein, and peak 4 is 13'-cis lutein.

[0084] Figure 16 This is the liquid chromatogram of the test solution used in Comparative Example 2; where peak 1 is all-trans lutein, peak 2 is zeaxanthin, peak 3 is 13-cis lutein, peak 4 is 13'-cis lutein, and peak 5 is 9-cis lutein.

[0085] Figure 17 This is the UV absorption spectrum of all-trans lutein in Example 4.

[0086] Figure 18 This is the UV absorption spectrum of zeaxanthin in Example 4.

[0087] Figure 19 This is the UV absorption spectrum of 13-cis-lutein in Example 4.

[0088] Figure 20 This is the UV absorption spectrum of 13'-cis-lutein in Example 4.

[0089] Figure 21 This is the UV absorption spectrum of 9-cis-lutein in Example 4.

[0090] Figure 22 These are the UV absorption spectra of other cis-lutein in Example 4.

[0091] Figure 23 This is the liquid chromatogram of the sample in Test Example 2; peak 1 is all-trans lutein and peak 2 is zeaxanthin.

[0092] Figure 24 This is the liquid chromatogram of the sample in Test Example 3.

[0093] Figure 25 This is the liquid chromatogram of the sample in Test Example 4.

[0094] Figure 26 The liquid chromatograms of different brands of infant formula milk powder tested in Test Example 5 using the method of Example 1 are shown. Peak 1 is all-trans lutein and peak 2 is zeaxanthin.

[0095] Figure 27 The test example 5 uses the method of Example 10 to test different brands of infant formula milk powder, where peak 1 is all-trans lutein and peak 2 is zeaxanthin.

[0096] Figure 28 The liquid chromatograms of different health foods tested in Test Example 5 using the method of Example 10 are shown below. Peak 1 is 13-cis-lutein, peak 2 is 13'-cis-lutein, peak 3 is all-trans-lutein, and peak 4 is zeaxanthin. Detailed Implementation

[0097] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0098] The instruments and reagents used in the following examples are shown below:

[0099] The liquid chromatograph was an Agilent 1260 Infinity II liquid chromatography system;

[0100] Lutein standard, purity 84.9%, purchased from Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.

[0101] Zeaxanthin standard was purchased from Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.

[0102] Infant formula milk powder 1-6 and children's formula milk powder 1 are respectively Meiyougao infant formula milk powder, Qizhi infant formula milk powder, Feihe infant formula milk powder, Jinlingguan infant formula milk powder, Feihe Jingcui Yijia formula milk powder, Feihe Zhenai Feifan formula milk powder, and Feihe children's formula milk powder.

[0103] Health food product 1 is Jamieson Lutein Soft Capsules;

[0104] The health food (capsule) is bilberry lutein soft capsules;

[0105] The health food (gel candy) is a lutein ester gel candy;

[0106] The nutrient premix is ​​a compound vitamin raw material containing lutein;

[0107] Other materials and reagents, unless otherwise specified, can be used if purchased from authorized retailers.

[0108] Preparation Example 1

[0109] Solution preparation

[0110] (1) Preparation of ascorbic acid (VC) aqueous solution: Take an appropriate amount of VC, add water to dissolve it and prepare a solution with a concentration of 0.2 g / mL;

[0111] (2) Preparation of 2,6-di-tert-butyl-p-cresol (BHT)-ethanol solution: Weigh 1g of BHT, add 100mL of ethanol to dissolve it, and prepare it before use to obtain a BHT ethanol solution with a concentration of 1.0g / 100mL.

[0112] (3) Preparation of potassium hydroxide solution: Weigh 50g of potassium hydroxide, dissolve it in 50mL of water, cool it and place it in a polyethylene bottle to obtain a sodium hydroxide solution with a mass concentration of 50%.

[0113] (4) Prepare an ethanol aqueous solution: Mix 70 mL of ethanol and 30 mL of water evenly to obtain an ethanol aqueous solution with a volume concentration of 70%.

[0114] Preparation Example 2

[0115] Preparation of standard solutions

[0116] Accurately weigh 1 mg of lutein and 1 mg of zeaxanthin, dissolve them in 0.1% BHT ethanol solution, and dilute to 10 mL in a brown volumetric flask to obtain standard stock solutions. Then, accurately measure appropriate amounts of each stock solution and dilute with 0.1% BHT ethanol to prepare mixed standard solutions with lutein and zeaxanthin concentrations of 0.8, 0.4, 0.2, 0.1, and 0.05 μg / mL, respectively. Take appropriate amounts of the stock solutions and correct the concentrations of lutein and zeaxanthin according to Appendix A of DB64 / T1514-2017 (Determination of Zeaxanthin, Carotene and Lutein in Lycium barbarum and Lycium barbarum Seed Oil).

[0117] Preparation Example 3

[0118] Preparation of solution for lutein system suitability test

[0119] Take lutein standard and prepare 50 mL of lutein standard solution with a concentration of 0.6 μg / mL using 0.1% BHT ethanol solution. Add 2 mL of iodine-ethanol solution (take 1 mg of iodine and dilute it with ethanol to 1 L). Shake well and place the mixture under sunlight or fluorescent light for 30 min to obtain the system suitability test solution containing the cis isomer.

[0120] Preparation Example 4

[0121] Sample solution preparation

[0122] Accurately weigh 2g of sample and place it in a 50mL brown centrifuge tube. Add 5mL of ascorbic acid aqueous solution (0.2g / mL) to dissolve the sample. Then add 20mL of BHT-ethanol solution (1.0g / 100mL) and vortex for 30s. Add 5mL of 50% potassium hydroxide solution and vortex for 30min. After cooling, add 70% ethanol and bring the volume to 50mL. Centrifuge at 10000rpm for 5min. Take the supernatant and pass it through a 0.22μm nylon membrane to obtain the sample solution.

[0123] Example 1

[0124] This embodiment provides a method for detecting the content of lutein and zeaxanthin, the detection method comprising the following steps:

[0125] The standard solution provided in Example 2 was tested using online solid-phase extraction-liquid chromatography to obtain a standard working curve; the sample solution was tested using online solid-phase extraction-liquid chromatography, and the results were substituted into the standard working curve to obtain the contents of lutein and zeaxanthin.

[0126] The conditions for online solid-phase extraction are as follows:

[0127] Extraction column: SelectCore HLB online SPE column (online solid-phase extraction column of polyvinylpyrrolidone hydrophilic-lipophilic balanced packing material, 25μm, 3.0mm×30mm);

[0128] The mobile phase, elution program, and flow rate are shown in the table below:

[0129]

[0130]

[0131] Injection volume: 100 μL;

[0132] The conditions for liquid chromatography are as follows:

[0133] Analytical column: ChromCore PAH (surface-polymerized octadecyl-bonded silica matrix column, 3 μm, 4.6 mm × 150 mm);

[0134] The mobile phase, elution program, and flow rate are shown in the table below:

[0135]

[0136] Column temperature: 25℃;

[0137] UV detection wavelength: 445nm;

[0138] Equipment setup, such as Figure 1 The valve switching process and explanation are shown in the table below:

[0139] time Valve position illustrate 0.0-4.0 1-6 Sample loading and purification 4.0-6.0 1-2 Target analyte transferred to analytical column 6.0-end 1-6 Analytical column separation analysis

[0140] The elution positions of lutein and zeaxanthin in the standard solution are as follows: Figure 2 As shown, with the target peak area as the ordinate and concentration as the abscissa, the following results are obtained: Figure 3 , Figure 4 The graph shows the linear relationship between the relative peak area and concentration of lutein and zeaxanthin.

[0141] The standard curve equation for lutein is: y = 49.6821x - 0.9208, r = 0.99987. This indicates that within the concentration range of 0.05-0.8 μg / mL, the linear correlation coefficient is >0.999. Using S / N = 10, the limit of quantitation (LOQ) for the target analyte is 0.00061 μg / mL. Based on a sample size of 2 g and a final volume of 50 mL, the method-based limit of quantitation (MDQ) is 1.5 μg / 100g (the national standard method is 10 μg / 100g).

[0142] The standard curve equation for zeaxanthin is: y = 32.8116x - 0.2498, r = 0.99956. The limit of quantitation (S / N = 10) is 0.0028 μg / mL. Based on a sample size of 2 g and a volume of 50 mL, the method limit of quantitation (MDQ) is 7.14 μg / 100 g.

[0143] Example 2

[0144] This embodiment provides a method for detecting the content of lutein and zeaxanthin, which differs from Example 1 only in the following conditions of online solid-phase extraction and liquid chromatography.

[0145] The conditions for online solid-phase extraction are as follows:

[0146] Extraction column: SelectCore PSS online SPE (online solid-phase extraction column packed with polystyrene-divinylbenzene (PS-DVB), 40μm, 3.0mm×30mm);

[0147] The mobile phase, elution program, and flow rate are shown in the table below:

[0148]

[0149]

[0150] The conditions for liquid chromatography are as follows:

[0151] The mobile phase, elution program, and flow rate are shown in the table below:

[0152]

[0153] Column temperature: 20℃;

[0154] Other operations are described in Example 1.

[0155] Example 3

[0156] This embodiment provides a method for detecting the content of lutein and zeaxanthin, which differs from Example 1 only in the following conditions of online solid-phase extraction and liquid chromatography.

[0157] The conditions for online solid-phase extraction are as follows:

[0158] The mobile phase, elution program, and flow rate are shown in the table below:

[0159]

[0160] The conditions for liquid chromatography are as follows:

[0161] The mobile phase, elution program, and flow rate are shown in the table below:

[0162]

[0163]

[0164] Column temperature: 30℃;

[0165] Other operations are described in Example 1.

[0166] Example 4

[0167] This embodiment provides a method for detecting the content of lutein and zeaxanthin, which differs from Example 1 only in the following conditions of liquid chromatography.

[0168] Injection volume: 10 μL;

[0169] The conditions for liquid chromatography are as follows:

[0170] Analytical column: ChromCore C30 (stationary phase packing with triacontaneyl groups chemically bonded to a silica matrix, 3 μm, 4.6 mm × 150 mm);

[0171] The mobile phase, elution program, and flow rate are shown in the table below:

[0172]

[0173] Other operations are described in Example 1.

[0174] Example 5

[0175] This embodiment provides a method for detecting the content of lutein and zeaxanthin. The only difference between this method and Example 1 is that the mobile phase C of the online solid-phase extraction is methanol, while other operations are the same as in Example 1.

[0176] Example 6

[0177] This embodiment provides a method for detecting the content of lutein and zeaxanthin, which differs from Example 1 only in the elution procedure of online solid-phase extraction:

[0178]

[0179]

[0180] Other operations are described in Example 1.

[0181] Example 7

[0182] This embodiment provides a method for detecting the content of lutein and zeaxanthin, which differs from Example 1 only in the elution procedure of liquid chromatography:

[0183]

[0184] Other operations are described in Example 1.

[0185] Example 8

[0186] This embodiment provides a method for detecting the content of lutein and zeaxanthin. The only difference between this method and Example 1 is that the analytical column is different. The method uses ChromCore C30 (a stationary phase packing material with triacontyl groups chemically bonded to a silica matrix, 3 μm, 4.6 mm × 150 mm). Other operations are the same as in Example 1.

[0187] Example 9

[0188] This embodiment provides a method for detecting lutein and zeaxanthin content. The only difference between this method and Example 1 is the analytical column. A Poroshell 120PFP column (a pentafluorophenylsilane bonded phase on a silica matrix, 4.6 mm × 100 mm, 4 μm) is used. Other operations are the same as in Example 1.

[0189] Example 10

[0190] This embodiment provides a method for detecting the content of lutein and zeaxanthin. The only difference between this method and Example 1 is that the extraction column is different. SelectCore PSS online SPE (an online solid phase extraction column with polystyrene-divinylbenzene (PS-DVB) packing, 40 μm, 3.0 mm × 30 mm) is used. Other operations are the same as in Example 1.

[0191] Comparative Example 1

[0192] This comparative example provides a method for detecting the content of lutein and zeaxanthin, which is performed according to the method provided in national standard GB5009.248-2016. The detection method includes the following steps:

[0193] The sample to be tested was obtained after saponification, extraction, washing with water, solvent recovery, and volume adjustment. The specific operation followed the national standard GB5009.248-2016, using liquid chromatography for analysis and detection. The chromatographic conditions are as follows:

[0194] Analytical column: ChromCore C30 (stationary phase packing with triacontaneyl groups chemically bonded to a silica matrix, 3 μm, 4.6 mm × 150 mm);

[0195] The mobile phase, elution program, and flow rate are shown in the table below:

[0196]

[0197] Injection volume: 5 μL;

[0198] Column temperature: 30℃;

[0199] UV detection wavelength: 445nm.

[0200] Comparative Example 2

[0201] This comparative example provides a method for detecting the content of lutein and zeaxanthin, which differs from Comparative Example 1 only in that the following detection conditions of liquid chromatography are different.

[0202] The mobile phase and elution procedure are shown in the table below:

[0203]

[0204] Column temperature: 25℃;

[0205] For other operations, refer to Comparative Example 1.

[0206] Test Example 1

[0207] System suitability testing

[0208] The lutein and zeaxanthin content detection methods provided in Examples 1-10 and Comparative Examples 1-2 were used to test the system provided in Preparation Example 3 with the test solution. Each sample was tested three times and the average value was taken. The separation effect of the system applicability test solution of each example is compared in Table 1.

[0209] Table 1

[0210] Peak number Rs (lutein / zeaxanthin) Rs(13 / 13'-cis-lutein) Rs (lutein / 9-cis-lutein) Example 1 8 4.37 2.16 1.99 Example 2 8 3.59 2.81 1.5 Example 3 7 3.67 1.7 1.7 Example 4 8 2.07 0.64 NA Example 5 7 3.64 2.29 1.01 Example 6 7 3.81 2.25 1.15 Example 7 6 2.41 1.23 1.44 Example 8 7 1.73 0.48 NA Example 9 4 1.29 NA NA Example 10 8 4.51 2.42 2.05 Comparative Example 1 6 NA NA 3.14 Comparative Example 2 7 1.95 NA NA

[0211] Combination Figure 5 As can be seen, in the detection method provided in Example 1, the main component all-trans lutein (peak 4) has good separation from other isomers, with a separation degree of 1.99 with 13'-cis lutein (peak 2) and 4.37 with zeaxanthin (peak 5); the two main cis isomers, 13'-cis lutein (peak 2) and 13-cis lutein (peak 1), have a separation degree of 2.1, which is also good.

[0212] The results obtained from the detection methods in Examples 2-3 are as follows: Figure 6-7 As shown, the test results are similar to those of Example 1.

[0213] Combination Figure 8 As can be seen, in the detection method provided in Example 4, the resolution between zeaxanthin (peak 2) and all-trans lutein (peak 1) is 1.91, but the separation between zeaxanthin and the subsequent 13-cis lutein (peak 3) is poor, with a resolution <1.5, and the two main cis isomers, 13'-cis lutein (peak 4) and 13-cis lutein (peak 3), cannot be separated. The UV absorption spectra of each component are shown below. Figures 17-22 .

[0214] Combination Figure 9 It can be seen that, in the detection method provided in Example 5, although the separation of 13 / 13'-cis-lutein and all-trans-lutein from zeaxanthin is good, the separation of all-trans-lutein from the previously mentioned 9-cis-lutein is not good (Rs=1.01).

[0215] Combination Figure 10 It can be seen that in the detection method provided in Example 6, although the separation of each target peak is acceptable, the sum of the peak areas of the target peaks is reduced by 34% compared with Example 1, indicating that when the proportion of organic phase initially loaded is too high, it will cause the flow-through loss of the target peaks.

[0216] Combination Figure 11 As can be seen, in the detection method provided in Example 7, the analytical column adopts the gradient conditions of the current national standard (GB5009.248-2016). Except for all-trans lutein and zeaxanthin, which can meet the separation requirements, other target peaks cannot achieve baseline separation.

[0217] Combination Figure 12 As can be seen, in the detection method provided in Example 8, using the C30 chromatographic column recommended by the national standard combined with the gradient conditions in Example 1, only all-trans lutein and zeaxanthin could meet the separation requirements; the other target peaks were all poorly separated. The resolution was less than 1.5.

[0218] Combination Figure 13 As can be seen, in the detection method provided in Example 9, the perfluorophenyl chromatographic column was used as the analytical column for testing, and the results showed that the separation of all-trans lutein from zeaxanthin and the cis isomer of lutein was not good.

[0219] Combination Figure 14 It can be seen that in the detection method provided in Example 10, the PSS online SPE online solid phase extraction column is used, and the resolution of each target peak is comparable to that in Example 1, indicating that polystyrene-divinylbenzene (PS-DVB) packing can be used as an online solid phase extraction column.

[0220] Combination Figure 15 It can be seen that, in the detection method provided by Comparative Example 1, although the separation of all-trans lutein (peak 1) and the cis isomer is relatively good, the separation of all-trans lutein and zeaxanthin (peak 2) is relatively poor, and the separation of the two main cis isomers cannot be achieved.

[0221] Combination Figure 16 It can be seen that in the detection method provided in Comparative Example 2, the separation degree of zeaxanthin (peak 2) and all-trans lutein (peak 1) is 1.95, but the separation of zeaxanthin and the subsequent 13-cis lutein (peak 3) is poor, with a separation degree of <1.5, and the two main cis isomers 13'-cis lutein (peak 4) and 13-cis lutein (peak 3) cannot be separated.

[0222] The test results show that:

[0223] (1) As can be seen from Examples 1 to 10, the detection method provided by the present invention can detect the content of lutein and / or zeaxanthin. In the preferred embodiment, all-trans lutein, its cis isomer and zeaxanthin have good separation, which can realize the accurate determination of their respective contents, with high accuracy and good repeatability.

[0224] (2) By comparing Examples 1 and Examples 4-9, it can be seen that the present invention can further improve the separation of each component by further optimizing the type of analytical column, the mobile phase of solid phase extraction and liquid chromatography analysis and the elution procedure.

[0225] (3) As can be seen from the comparison between Example 1 and Comparative Examples 1-2, the present invention uses an online solid-phase extraction-liquid chromatography technique, which significantly improves the separation of all-trans lutein, its cis isomer and zeaxanthin in the sample compared with liquid chromatography analysis alone, and can achieve accurate determination of the content of each component.

[0226] Test Example 2

[0227] Precision testing

[0228] Using Feihe Yijia infant formula milk powder as a sample, the sample solution was prepared using the method described in Example 4, and tested using the detection method provided in Example 1. Each sample was injected five times consecutively, and the average value was taken. The test results are shown in Table 2. Figure 23 As shown.

[0229] Table 2

[0230]

[0231]

[0232] The test results showed that the retention time RSD% of the two target substances was <0.1, and the peak area RSD% was 0.57 and 2.06%, respectively, indicating that the method has good precision.

[0233] Test Example 3

[0234] Repeatability test

[0235] Five portions of Feihe Xingfeifan infant formula milk powder were taken as samples and processed according to the sample solution preparation method in Example 4 to obtain five sample solutions. The detection method provided in Example 1 was used for testing to obtain the test results of all-trans lutein. The results are shown in Table 3. Figure 24 As shown.

[0236] Table 3

[0237]

[0238] The test results showed that the retention time and peak area of ​​the all-trans lutein chromatographic peak had RSDs of 0.18% and 0.96%, respectively, indicating that the repeatability of this method was good and fully met the requirements of GB5009.248.2016 (RSD < 15%).

[0239] Test Example 4

[0240] Recovery rate test

[0241] Using Jinlingguan milk powder as a sample, three portions, each 2g, were taken. Two of these portions were added with 400μL and 800μL of 4μg / mL lutein standard stock solution (obtained by dissolving and diluting lutein standard with 0.1% BHT ethanol solution, respectively). An equal volume of lutein standard solution was taken and a blank solvent was added. The sample solutions were prepared according to the method described in Preparation Example 4 and tested using the preparation method provided in Example 1. The results are shown in Table 4. Figure 25 As shown.

[0242] Table 4

[0243]

[0244] The test results show that the average recovery rate of the detection method provided by this invention is 100.59%, indicating that the method is stable and reliable.

[0245] Test Example 5

[0246] Separation tests of different brands of infant formula milk powder and health food

[0247] The detection methods provided in Examples 1 and 10 were used to test different brands of infant formula milk powder, and the results are as follows: Figure 26 , Figure 27 As shown in the spectral separation results, the target peak and interfering peaks in each milk powder matrix are well separated, fully meeting the quantitative requirements of the method.

[0248] The health food was tested using the detection method provided in Example 10, and the results are as follows: Figure 28 As shown in the spectral separation results, it can be seen that there are no other impurity peaks interfering with lutein and zeaxanthin in the capsules, which can achieve accurate quantification of the two target analytes; the two main cis isomers 13'-cis-lutein and 13-cis-lutein in the gel candy sample are well separated from the main component, which can also achieve the determination of the main component and cis isomers.

[0249] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for detecting the content of lutein and / or zeaxanthin, characterized in that, The detection method includes the following steps: Take the standard of the sample to be tested and prepare several sets of standard solutions with different concentrations; use online solid phase extraction-liquid chromatography to test the standard solutions and obtain the standard working curve; The sample solution is obtained by pretreatment of the sample to be tested. The sample solution is tested by online solid phase extraction-liquid chromatography. The results are substituted into the standard working curve to obtain the content of lutein and / or zeaxanthin.

2. The detection method according to claim 1, characterized in that, The preparation of the standard solution includes: mixing lutein and / or zeaxanthin, antioxidants and diluents to obtain several sets of standard solutions with different concentrations; Preferably, the pretreatment includes: mixing the sample to be tested, antioxidant, diluent and alkaline reagent, and then saponifying to obtain a sample solution.

3. The detection method according to claim 2, characterized in that, The antioxidant includes any one or a combination of two of 2,6-di-tert-butyl-p-cresol or ascorbic acid; Preferably, the diluent comprises an aqueous ethanol-water solution or anhydrous ethanol with a volume concentration of 60-70%. Preferably, the alkaline reagent includes potassium hydroxide; Preferably, the concentration of the alkaline reagent in the sample solution is 0.1-1 g / mL; Preferably, the saponification time is 15-45 minutes; Preferably, the saponification temperature is 20-75°C.

4. The detection method according to claim 2 or 3, characterized in that, The saponification process also includes centrifugation and filtration. Preferably, the centrifugation speed is 9500-10500 rpm; Preferably, the centrifugation time is 3-7 minutes; Preferably, the filter membrane used for filtration has a pore size of 0.2-0.45 μm.

5. The detection method according to any one of claims 1-4, characterized in that, The online solid-phase extraction uses extraction columns including polystyrene-divinylphenyl matrix extraction columns or polyvinylpyrrolidone matrix extraction columns. Preferably, the length of the extraction column is 5-100 mm, more preferably 10-30 mm; Preferably, the inner diameter of the extraction column is 2-10 mm, more preferably 2-4.6 mm; Preferably, the particle size of the extraction column is 5-50 μm, more preferably 10-40 μm; Preferably, the extraction column comprises a ferrule column or a packed column.

6. The detection method according to any one of claims 1-5, characterized in that, The analytical column used in the liquid chromatography includes a column with a surface-polymerized C8-C18 alkyl-bonded silica matrix, preferably a column with a surface-polymerized octadecyl-bonded silica matrix. Preferably, the silicone includes any one of fully porous silicone, core-shell surface porous silicone, or hybrid silicone; Preferably, the silica gel has a particle size of 1.5-10 μm, more preferably 1.5-5 μm; Preferably, the pore size of the silicone is 8-50 nm, more preferably 8-18 nm; Preferably, the length of the analytical column is 50-600 mm, and more preferably 100-300 mm; Preferably, the inner diameter of the analytical column is 2-50 mm, and more preferably 2-4.6 mm.

7. The detection method according to any one of claims 1-6, characterized in that, The mobile phase in the online solid-phase extraction includes phase A, phase B, phase C, and phase D, wherein phase A is water, phase B is acetonitrile, phase C is ethanol, and phase D is methyl tert-butyl ether; Preferably, the online solid-phase extraction employs a gradient elution method, and the gradient elution procedure is as follows: From 0 to 4 min, phase A accounts for 35-45% of the volume, and phase C accounts for 55-65% of the volume; then, at a constant rate, phase B accounts for 100% of the volume from 5 to 10 min; from 5 to 10 min, phase B accounts for 100% of the volume; then, at a constant rate, phase B accounts for 45-55% of the volume, and phase D accounts for 55-45% of the volume from 11 to 15 min; from 11 to 15 min, phase B accounts for 45-55% of the volume, and phase D accounts for 55-45% of the volume; then, at a constant rate, phase B accounts for 100% of the volume from 16 to 20 min; from 16 to 20 min, phase B accounts for 100% of the volume; then, at a constant rate, phase A accounts for 35-45% of the volume, and phase C accounts for 65-55% of the volume from 21 to 25 min; from 21 to 25 min, phase A accounts for 35-45% of the volume, and phase C accounts for 65-55% of the volume. Preferably, the online solid-phase extraction has a linear flow rate of 0.8-10.0 mm / s; Preferably, the volumetric flow rate in the online solid-phase extraction is 0.5-2 mL / min; Preferably, the volumetric flow rate is 1.2-1.8 mL / min for 0-4 min; then the flow rate is uniformly changed to 0.6-1 mL / min for 5 min; and the volumetric flow rate is 0.6-1 mL / min for 5-25 min.

8. The detection method according to any one of claims 1-7, characterized in that, The mobile phase in the liquid chromatograph includes phase A1, phase B1, phase C1 and phase D1, wherein phase A1 is water, phase B1 is acetonitrile, phase C1 is methanol and phase D1 is methyl tert-butyl ether; Preferably, the liquid chromatography employs a gradient elution method, and the gradient elution procedure is as follows: From 0 to 6 min, the volume percentage of phase A1 was 15-25%, and that of phase B1 was 85-75%. Following a linear gradient change to 7 min, the volume percentage of phase A1 was 3-7%, and that of phase C1 was 97-93%. From 12 min, phase C1 reached 100%. From 20 min, phase C1 was 85-95%, and that of phase D1 was 15-5%. From 21 min, phase C1 was 45-55%, and that of phase D1 was 55-45%. From 21 to 25 min, phase C1 was 55-45%, and that of phase D1 was 45-55%. Preferably, the linear flow rate in the liquid chromatography is 0.8-3.0 mm / s; Preferably, the volumetric flow rate in the liquid chromatography is 0.8-1.2 mL / min; Preferably, the column temperature in the liquid chromatography is 20-45℃; Preferably, the valve-cutting time in the liquid chromatography is 4-6 minutes.

9. The detection method according to any one of claims 1-8, characterized in that, The detection method includes the following steps: Lutein and / or zeaxanthin, antioxidants and diluents were mixed to obtain several groups of standard solutions with different concentrations; the standard solutions were tested by online solid-phase extraction-liquid chromatography to obtain standard working curves. Mix the sample to be tested, antioxidant, diluent, and alkaline reagent, saponify at 20-75℃ for 15-45 min, then centrifuge at 9500-10500 rpm for 3-7 min, and filter through a 0.2-0.45 μm filter membrane to obtain the sample solution; test the sample solution using online solid-phase extraction-liquid chromatography, and substitute the results into the standard working curve to obtain the content of lutein and / or zeaxanthin; The concentration of the alkaline reagent in the sample solution is 0.01-1 g / mL; The conditions for the online solid-phase extraction are as follows: The extraction column comprises a polystyrene-divinylphenyl matrix extraction column or a polyvinylpyrrolidone matrix extraction column; the column length is 5-100 mm, the inner diameter is 2-10 mm, and the particle size is 5-50 μm; the mobile phase comprises phase A, phase B, phase C, and phase D, wherein phase A is water, phase B is acetonitrile, phase C is ethanol, and phase D is methyl tert-butyl ether; the gradient elution program is as follows: 0-4 min, phase A volume percentage 35-45%, phase C volume percentage 65-55%; then linear gradient change to phase B volume percentage 100% at 5 min; 5-10 min, phase B volume percentage 100%; then linear gradient change to phase D. The linear gradient changes until min 11, with phase B accounting for 45-55% of the volume and phase D accounting for 55-45%; from min 11 to 15, phase B accounts for 45-55% of the volume and phase D accounts for 55-45% of the volume; then, with a linear gradient change until min 16, phase B accounts for 100% of the volume; from min 16 to 20, phase B accounts for 100% of the volume; then, with a linear gradient change until min 21, phase A accounts for 35-45% of the volume and phase C accounts for 65-55% of the volume; from min 21 to 25, phase A accounts for 35-45% of the volume and phase C accounts for 65-55% of the volume; the linear velocity is 0.8-10.0 mm / s; The conditions for the liquid chromatography are as follows: The analytical column comprises a surface-polymerized C8-C18 alkyl-bonded silica matrix; the silica has a particle size of 1.5-10 μm, a pore size of 8-50 nm, a column length of 50-600 mm, and an inner diameter of 2-50 mm; the mobile phase comprises phases A1, B1, C1, and D1, wherein phase A1 is water, phase B1 is acetonitrile, phase C1 is methanol, and phase D1 is methyl tert-butyl ether; the gradient elution program is as follows: 0-6 min, phase A1 volume percentage 15-25%, phase B1 volume percentage 85-75%; then linear gradient change until 7 min, phase A1 volume percentage 3... -7%, C1 phase volume percentage 97-93%; then linear gradient change to 12 min, C1 phase volume percentage 100%; then linear gradient change to 20 min, C1 phase volume percentage 85-95%, D1 phase volume percentage 15-5%; then linear gradient change to 21 min, C1 phase volume percentage 45-55%, D1 phase volume percentage 55-45%; 21-25 min, C1 phase volume percentage 45-55%, D1 phase volume percentage 55-45%; linear flow rate 0.8-3.0 mm / s, column temperature 20-45℃, valve cut-off time 4-6 min.

10. The application of a method for detecting the content of lutein and / or zeaxanthin according to any one of claims 1-9 in the detection of food, pharmaceuticals, feed or cosmetics.

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