A method for detecting carotenoid isomers

Abstract

The invention discloses a method for detecting carotenoid isomers. The isomers include beta-carotene and alpha-carotene, lutein and zeaxanthin. The method comprises: for a sample to be tested, high performance liquid chromatography is used for detection, mobile phase A is acetonitrile, mobile phase B is a mixed solution of methanol and isopropanol, and the volume ratio of methanol to isopropanol in the mixed solution is 1-2:1; the elution process is as follows: at the beginning of elution, the mobile phase A is 100%, and the mobile phase B is 0%; at the 5th to 15th minute, the mobile phase A is 70% to 100%, and the mobile phase B is 0% to 30%; at the 25th to 35th minute, the mobile phase A is 0% to 50%, and the mobile phase B is 50% to 100%; at the 35th to 45th minute, the mobile phase A is 60% to 100%, and the mobile phase B is 0% to 40%.

Description

Technical Field

[0001] The invention belongs to the technical field of carotenoid detection in analytical chemistry, and in particular relates to a method for detecting carotenoid isomers. Background Art

[0002] Carotenoids are tetraterpenoids containing 40 carbon atoms, which are widely used in medicine, health care, food processing and cosmetics. Currently, more than 700 different carotenoids have been identified, all of which contain many double bonds. The number, position and structure of the double bonds on both sides of the double bonds result in the diversity of carotenoids and the differences in color and function.

[0003] Common carotenoids include β-carotene, lutein, zeaxanthin and α-carotene. Among them, the molecular formula of β-carotene is C 40 H 56 , with a β-ring at each end, such as Figure 1 As shown in a, it is an important source of vitamin A in animals and has functions such as anti-oxidation and cancer prevention.

[0004] α-carotene and β-carotene are isomers of each other. Figure 1 As shown in Figure 2, both have the same carbon skeleton, but α-carotene has a β-ring and an ε-ring at both ends. Studies have shown that α-carotene is more effective than β-carotene in preventing cardiovascular disease, diabetes, and inhibiting the proliferation of human neuroblastoma cells and skin tumor cells (Li et al., 2021; Zhao et al., 2022).

[0005] Lutein is a derivative of α-carotene, and zeaxanthin is a derivative of β-carotene. The chemical structure of both is C 40 H 56 O2, only the position of one double bond is different, and they are isomers, such as Figure 1 c, 1d. Currently, the health products and fruits and vegetables on the market are a mixture of lutein and zeaxanthin, which are the main components of retinal macular pigments and have important effects on human health, such as preventing retinal damage, regulating immune function, and delaying atherosclerosis (Age-Related Eye Disease Study 2Research, 2013; Stringham et al., 2010). Although the two have the same molecular formula, there are still certain differences in their specific functions.

[0006] Therefore, clarifying the content of various carotenoid isomers in the test samples will be of great help to further research, which will help to better study the characteristics and effects of carotenoids and contribute to disease prevention and human health.

[0007] The patent with publication number CN115236223A discloses a method for detecting carotenoids. The method comprises: for the sample to be tested, liquid chromatography is used for detection; wherein, mobile phase A is β-carotene and its auxiliary mobile phase; mobile phase B is methanol; the elution process is as follows: at 0 minutes, mobile phase A is 100%, and mobile phase B is 0%; at 30-34 minutes, mobile phase A is 80%-88%, and mobile phase B is 12%-20%; at 52-56 minutes, mobile phase A is 80%-88%, and mobile phase B is 12%-20%; at 56-58 minutes, mobile phase A is 100%, and mobile phase B is 0%. The present invention provides an accurate and efficient method for detecting carotenoids, which can accurately distinguish a variety of carotenoids with similar structures, especially lycopene, astaxanthin and β-carotene, through a specific dual mobile phase and gradient elution process.

[0008] The patent with publication number CN115078590A discloses a method for detecting carotenoids by two-dimensional liquid chromatography, which comprises the following steps: construction of a two-dimensional liquid chromatography platform; preparation of a standard solution; and a pretreatment method. The method proposed in the present invention achieves the separation of eight carotenoids: β-carotene, canthaxanthin, α-carotene, lycopene, β-cryptoxanthin, astaxanthin, lutein, and zeaxanthin, but the separation method is relatively complicated.

[0009] At present, the existing technology lacks a method that can be simple, fast, and simultaneously and accurately detect isomers of lutein and zeaxanthin, β-carotene and α-carotene. Summary of the invention

[0010] In view of the bottleneck problems in the current field, the present invention provides a method for detecting carotenoid isomers, which can easily and quickly detect lutein and zeaxanthin, β-carotene and α-carotene at the same time through a specific mobile phase and gradient elution process. In particular, the method provided by the present invention can accurately distinguish lutein from zeaxanthin, β-carotene from α-carotene.

[0011] The present invention is achieved through the following technical solutions:

[0012] A method for detecting carotenoid isomers, wherein the carotenoid isomers include β-carotene and α-carotene, lutein and zeaxanthin, and the method comprises:

[0013] The sample to be tested is detected by high performance liquid chromatography, and the mobile phase is a two-component mobile phase; wherein the mobile phase A is acetonitrile, and the mobile phase B is a mixture of methanol and isopropanol, and the volume ratio of methanol to isopropanol in the mixture of methanol and isopropanol is 1 to 2:1;

[0014] The elution process is as follows:

[0015] At the beginning of elution, mobile phase A is 100% and mobile phase B is 0%;

[0016] At 5 to 15 minutes, the mobile phase was changed to 70% to 100% mobile phase A and 0% to 30% mobile phase B;

[0017] At 25-35 minutes, the mobile phase was changed, with mobile phase A at 0%-50% and mobile phase B at 50%-100%;

[0018] At 35 to 45 minutes, the mobile phase was changed, with mobile phase A at 60% to 100% and mobile phase B at 0% to 40%.

[0019] Preferably, the volume ratio of methanol to isopropanol in the mixed solution of methanol and isopropanol is 3:2.

[0020] Preferably, the elution process is as follows:

[0021] At the beginning of elution, mobile phase A is 100% and mobile phase B is 0%;

[0022] At 6-14 minutes, the mobile phase was changed to 70%-90% mobile phase A and 10%-30% mobile phase B;

[0023] At 26-34 minutes, the mobile phase was changed, with mobile phase A at 0%-20% and mobile phase B at 80%-100%;

[0024] At 36-44 minutes, the mobile phase was changed, with mobile phase A at 80%-100% and mobile phase B at 0%-20%.

[0025] Further preferably, the elution process is as follows:

[0026] At the beginning of elution, mobile phase A is 100% and mobile phase B is 0%;

[0027] At 10 minutes, the mobile phase was changed to 80% mobile phase A and 20% mobile phase B;

[0028] At 30 minutes, the mobile phase was changed, with mobile phase A at 0% and mobile phase B at 100%;

[0029] At 40 minutes, the mobile phase was changed to 100% mobile phase A and 0% mobile phase B.

[0030] Preferably, the chromatographic purity of the acetonitrile, methanol and isopropanol is greater than or equal to 99.9%.

[0031] Preferably, the detection wavelength of the HPLC is 430 nm to 470 nm. More preferably, the detection wavelength of the HPLC is 450 nm.

[0032] Preferably, the chromatographic column for the high performance liquid chromatography is YMC C30.

[0033] Preferably, the injection volume of the HPLC method is 10 to 50 μL. More preferably, the injection volume of the HPLC method is 15 to 25 μL.

[0034] Preferably, the column temperature of the HPLC is 30-45°C.

[0035] Preferably, the flow rate of the HPLC method is 0.8 to 1.2 mL / min.

[0036] Preferably, one or more of acetone, acetonitrile, methanol and n-hexane are used to extract the sample to be tested.

[0037] The method provided by the present invention can simultaneously detect lutein, zeaxanthin, β-carotene and α-carotene in health products, cosmetics, fruits and vegetables, microbial fermentation products and other test objects. The method provided by the present invention is particularly suitable for the determination of carotenoid isomers in yeast fermentation cells.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention can accurately and quickly distinguish lutein from zeaxanthin, β-carotene from α-carotene by optimizing the high performance liquid chromatography detection process, using a specific mobile phase and gradient elution program. This is of great significance for the detection and separation of carotenoids in the sample to be tested and for refined research. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The molecular structures of β-carotene (a), α-carotene (b), lutein (c) and zeaxanthin (d);

[0040] Figure 2 The liquid chromatogram of the mixed solution of the lutein standard product (①), the zeaxanthin standard product (②), the α-carotene standard product (③) and the β-carotene standard product (④) measured in Example 1;

[0041] Figure 3This is a liquid chromatogram of the yeast fermentation strain extract measured in Example 2, wherein ① is lutein, ② is zeaxanthin, ③ is α-carotene, ④ is β-carotene, and ⑤ is other carotenoids. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0043] Example 1

[0044] 1.1 Preparation of standard solution:

[0045] Weigh 10 mg of β-carotene standard and dissolve it in 10 mL of n-hexane. Ultrasonicate for 10 min to fully mix it. Centrifuge at 12000 rpm for 2 min. Filter the supernatant with a 0.22 μm organic filter as mother solution 1.

[0046] Weigh 10 mg of α-carotene standard and dissolve it in 10 mL of n-hexane. Ultrasonicate for 10 min to fully mix it. Centrifuge at 12000 rpm for 2 min. Filter the supernatant with a 0.22 μm organic filter as mother solution 2.

[0047] Weigh 10 mg of lutein standard and dissolve it in 10 mL of n-hexane. Ultrasonicate for 10 min to fully mix it. Centrifuge at 12000 rpm for 2 min. Filter the supernatant with a 0.22 μm organic filter as mother solution 3.

[0048] Weigh 10 mg of zeaxanthin standard and dissolve it in 10 mL of n-hexane. Ultrasonicate for 10 min to fully mix it. Centrifuge at 12000 rpm for 2 min. Filter the supernatant with a 0.22 μm organic filter as mother solution 4.

[0049] Mix equal amounts of the above four stock solutions and dilute them to complete the preparation of the standard solution.

[0050] 1.2 Detection of carotenoid isomers

[0051] HPLC conditions were: chromatographic column YMC C30, detection wavelength 450 nm, flow rate 1.0 mL / min, injection volume 20 μL, column temperature 40 °C.

[0052] The mobile phase is a two-component mobile phase; wherein the mobile phase A is acetonitrile, and the mobile phase B is a mixture of methanol and isopropanol, wherein the volume ratio of methanol to isopropanol in the mixture of methanol and isopropanol is 3:2.

[0053] Perform gradient elution. The specific gradient elution conditions are as shown in Table 1:

[0054] Table 1

[0055] Time (min) Mobile phase A Mobile phase B 0 100％ 0％ 10 80％ 20％ 30 0％ 100％ 40 100％ 0％

[0056] Test results: Figure 2 As shown in the figure, lutein and zeaxanthin, β-carotene and α-carotene can be well separated. By adding a certain standard mother solution to the mixed sample and observing the change in peak area, it can be determined that peak ① is lutein, with an elution time of about 16.5 min, peak ② is zeaxanthin, with an elution time of about 18.9 min, peak ③ is α-carotene, with an elution time of about 32.9 min, and peak ④ is β-carotene, with an elution time of about 36.1 min.

[0057] Comparative Example 1

[0058] Detection of carotenoid isomers:

[0059] The only difference from the detection in Example 1 is that a constant mobile phase ratio is used for elution, wherein mobile phase A accounts for 50%, mobile phase B accounts for 50%, and the detection time is 80 minutes.

[0060] Test results: When the mobile phase was eluted at a constant ratio, the isomers could not be separated.

[0061] Comparative Example 2

[0062] Detection of carotenoid isomers:

[0063] The only difference from the detection in Example 1 is that gradient elution is used. The gradient elution conditions are shown in Table 2 below:

[0064] Table 2

[0065] Time (min) Mobile phase A Mobile phase B 0 100％ 0％ 20 0％ 100％ 40 100％ 0％

[0066] Test results: Under the gradient elution conditions, β-carotene and α-carotene can be well separated, but lutein and zeaxanthin cannot be completely separated.

[0067] Example 2

[0068] Determination of carotenoid isomers in yeast fermentation cells:

[0069] 2.1 Extraction of carotenoids from yeast

[0070] (1) Take 2 ml of yeast fermentation broth, centrifuge at 12000 rpm for 5 min, discard the supernatant, then wash once with 2 ml of distilled water, centrifuge and discard the supernatant, add 200 μL of distilled water and resuspend the bacteria.

[0071] (2) Add 500 ml of grinding beads (half 0.1 mm and half 0.5 mm zirconium oxide beads) to a 2 ml centrifuge tube, and then transfer all the resuspended bacterial solution to the 2 ml centrifuge tube.

[0072] (3) Place the centrifuge tube containing the beads and bacteria in a fully automatic sample rapid grinder at 65 Hz for 5 min.

[0073] (4) After grinding, place the centrifuge tube in a 4°C centrifuge at 12,000 rpm for 10 min and discard the supernatant.

[0074] (5) Add 1 ml of acetone to the centrifuge tube for extraction, mix thoroughly, and place under ultrasound for 10 minutes.

[0075] (6) After sonication, centrifuge at 12000 rpm for 2 min and aspirate the supernatant into a new 2 ml centrifuge tube.

[0076] (7) Add 1 ml of acetone to the original 2 ml centrifuge tube, re-extract until the cells turn white, mix thoroughly, sonicate for 10 min, centrifuge at 12,000 rpm for 2 min, and then aspirate the supernatant into a new centrifuge tube in step (6).

[0077] (8) The mixed extracts were filtered through a 0.22 μm organic filter and then subjected to HPLC detection.

[0078] 2.2 Detection of carotenoid isomers

[0079] The detection was performed using the same detection method and parameters as in Example 1, i.e., the gradient elution method shown in Table 1.

[0080] Test results: Figure 3 As shown, this detection method can be used to simultaneously detect lutein and zeaxanthin, β-carotene and α-carotene isomers in the sample, wherein peak ⑤ is the peak diagram of other carotenoids.

[0081] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for detecting carotenoid isomers, wherein the carotenoid isomers include β-carotene and α-carotene, lutein and zeaxanthin, characterized in that: The method comprises: For the samples to be tested, high performance liquid chromatography was used for detection, the chromatographic column was YMC C30, the detection wavelength was 430 nm~470 nm, and the mobile phase was a two-component mobile phase; wherein the mobile phase A was acetonitrile, and the mobile phase B was a mixture of methanol and isopropanol, and the volume ratio of methanol to isopropanol in the mixture of methanol and isopropanol was 1~2:1; The elution process is as follows: At the beginning of elution, mobile phase A is 100% and mobile phase B is 0%; At 5-15 minutes, the mobile phase was changed, with mobile phase A at 70%-100% and mobile phase B at 0%-30%; At 25-35 minutes, the mobile phase was changed, with mobile phase A at 0%-50% and mobile phase B at 50%-100%; At 35-45 minutes, the mobile phase was changed to 60%-100% for mobile phase A and 0%-40% for mobile phase B.

2. The method according to claim 1, characterized in that The volume ratio of methanol to isopropanol in the mixed liquid of methanol and isopropanol is 3:

2.

3. The method according to claim 1, characterized in that The elution process is as follows: At the beginning of elution, mobile phase A is 100% and mobile phase B is 0%; At 6-14 minutes, the mobile phase was changed, with mobile phase A at 70%-90% and mobile phase B at 10%-30%; At 26-34 minutes, the mobile phase was changed, with mobile phase A at 0%-20% and mobile phase B at 80%-100%; At 36-44 minutes, the mobile phase was changed, with mobile phase A at 80%-100% and mobile phase B at 0%-20%.

4. The method according to claim 1, characterized in that: The elution process is as follows: At the beginning of elution, mobile phase A is 100% and mobile phase B is 0%; At 10 minutes, the mobile phase was changed to 80% mobile phase A and 20% mobile phase B; At 30 minutes, the mobile phase was changed, with mobile phase A at 0% and mobile phase B at 100%; At 40 minutes, the mobile phase was changed to 100% mobile phase A and 0% mobile phase B.

5. The method according to claim 1, characterized in that The chromatographic purity of the acetonitrile, methanol and isopropanol is greater than or equal to 99.9%.

6. The method according to claim 1, characterized in that The high performance liquid chromatography method includes: an injection volume of 10 to 50 μL; a column temperature of 30 to 45° C.; and a flow rate of 0.8 to 1.2 mL / min.

7. The method according to claim 1, characterized in that One or more of acetone, acetonitrile, methanol and n-hexane are used to extract the sample to be tested.

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