WO2021082452A1 - Method for determining composition of lutein - Google Patents

Abstract

A method for determining the composition of lutein, in which high performance liquid chromatography is employed for measurement. The chromatographic conditions comprise: using YMC Carotenoid C30 as a chromatographic column; using methanol/water as mobile phase A and methyl tert-butyl ether as mobile phase B; the elution procedure comprises 0-30 min with 0% to 50% B, and 30-40 min with 50% B, where the ratio of methanol to water is 88-100:12-0. The method uses a high-active ingredient all-trans lutein as a measurement index, and can accurately and effectively evaluate the quality of a lutein product. The chromatographic conditions used can achieve effective separation of all-trans lutein and main cis-isomers and isomers thereof and accurate quantification of the all-trans lutein.

Description

A method for determining the composition of lutein Technical field

The present invention relates to the technical field of analytical chemistry, in particular to a method for determining the components of lutein.

Background technique

Lutein is a yellow substance belonging to carotenoids. It is widely present in nature. In the human body, it exists in the blood plasma and the macular area of the eye. It can absorb a large amount of blue light and avoid photo-oxidative damage to the retina. This kind of antioxidant can scavenge free radicals and protect the optic nerve from free radical damage, so it is widely used in various health foods, foods and medicines. Because of its multiple conjugated double bond structure, the molecular structure of lutein is easily affected by factors such as light, oxygen, and high temperature. In theory, there can be multiple isomers, including cis-isomer and its isomeric corn. Xanthin etc.

In organisms, the biological activities of different isomers are different, and the biological activity of the trans conformation is much higher than that of the cis conformation, resulting in significant differences in their biological functions or potency. Once lutein is subjected to the process of preparation and application The influence of light, heating, oxygen and other factors will inevitably affect the quality of products with lutein as the main active ingredient. At present, few people monitor the changes of cis-isomers during the extraction and preparation of lutein, especially health foods and foods that use lutein as raw materials to evaluate the quality of products based on the content of total lutein. It is difficult to guarantee the quality of the product. The existing national standards use a reference substance with purity as the total lutein content, and use the sum of trans-lutein and its cis-isomer and isomers as a quantitative indicator, and trans-lutein and its The cis-isomer and the isomers are not ideally separated, as shown in Figure 1. In the chromatogram of the isomerized standard solution, only three chromatographic peaks can be seen, and there is no theoretical existence or related literature. The multiple cis-isomers and isomers of lutein make the existing methods have great limitations in monitoring the quality of lutein. Not only that, the preparation method of the test product in the standard is quite cumbersome, which increases the cost of lutein quality control.

Summary of the invention

In view of this, the present invention aims to provide a qualitative or quantitative method for all-trans lutein and isomers in health foods, foods, medicines and lutein raw materials using lutein as raw materials. Specifically, the present invention proposes a method for determining the components of lutein, which adopts high performance liquid chromatography for detection, wherein the chromatographic conditions include: using YMC Carotenoid C30 as the chromatographic column; using methanol/water as the mobile phase A, Base tert-butyl ether is the mobile phase B; the elution procedure is 0min-30min, 0%-50%B, 30min-40min, 50%B; the methanol/water ratio is 88-100:12-0.

Preferably, the ratio of methanol/water is 92-100:8-0; more preferably, the ratio of methanol/water is 95:5.

Further, the mobile phase A and/or the mobile phase B contains 2,6-di-tert-butyl-p-cresol.

Preferably, the concentration of the 2,6-di-tert-butyl-p-cresol is 0.1%.

Further, the chromatographic conditions include: a flow rate of 0.7 mL/min to 1.1 mL/min; and a column temperature of 25°C to 40°C.

Furthermore, in the aforementioned method for determining the composition of lutein, the chromatographic conditions include: using YMC Carotenoids C30 with a specification of 250mm×4.6mm, 5μm as the chromatographic column; using 0.1% 2,6-di-tert-butyl-p-cresol , A methanol/water solution with a ratio of 95:5 is used as mobile phase A, and methyl tert-butyl ether is used as mobile phase B. The elution procedure is 0min～30min, 0%～50%B, 30min～40min, 50%B; The flow rate is 0.8mL/min, the column temperature is 30°C, and the detection wavelength is 445nm.

Optionally, the lutein component as described above includes, but is not limited to, all-trans lutein.

The present invention also provides a method for detecting lutein raw materials (such as lutein particles) or samples containing lutein, characterized in that the method includes:

Preparation of test solution: grind lutein particles or samples containing lutein, take the powder into a volumetric flask, add water, sonicate at room temperature, then add absolute ethanol containing BHT, sonicate , Cooled to room temperature, constant volume;

The above-mentioned test solution is tested by any one of the aforementioned methods for determining lutein components.

Specifically, the preparation of the test solution includes: taking lutein particles or a sample containing lutein, grinding it, taking about 300 mg of the powder, accurately weighing it, placing it in a 100 mL brown volumetric flask, adding 5 mL of water, and keeping it at room temperature. Under ultrasonic treatment for 20 minutes, then add 0.1% BHT-containing absolute ethanol to close to the mark, sonicate for 5 minutes, cool to room temperature, dilute to the mark with 0.1% BHT-containing absolute ethanol, shake well, and centrifuge to get it. (Lutein microcapsules need to be accurately measured 1mL to 10mL brown volumetric flask, and 0.1% BHT absolute ethanol is made up to the mark).

Furthermore, the aforementioned method for detecting lutein particles or lutein-containing samples includes:

Preparation of reference substance solution: take lutein reference substance, add absolute ethanol containing BHT to make a stock solution containing lutein, and use the stock solution to prepare reference substance solutions of various concentrations;

Preparation of test solution: grind lutein particles or samples containing lutein, take the powder into a volumetric flask, add water, sonicate at room temperature, then add absolute ethanol containing BHT, sonicate , Cooled to room temperature, constant volume;

The above-mentioned reference solution and test solution are tested by the method described in any one of claims 1-8. Based on this, the standard curve of the reference substance can be established, and the content of the active ingredient can be calculated based on the test result of the test substance.

More specifically, the reference solution can be prepared as follows: Take an appropriate amount of the lutein reference substance, accurately weigh it, and add 0.1% BHT (2,6-di-tert-butyl-p-cresol) in absolute ethanol to prepare it. Obtain a reference substance stock solution containing 100μg of lutein per 1mL; accurately measure 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0mL of the stock solution, add 0.1% BHT absolute ethanol and dilute to a concentration of 2, 4 , 8, 12, 16, 20μg/mL series of concentration reference solution.

The qualitative or quantitative analysis method of each component of lutein provided by the present invention has the following advantages:

1. This method includes the high-active ingredient all-trans lutein as the detection index, which can more truly and effectively evaluate the quality of lutein products.

2. The chromatographic conditions of this method can realize the effective separation of all-trans lutein and its main cis-isomer and isomers, and can also accurately quantify all-trans lutein.

3. This method qualifies lutein and its isomers through comparison of reference substances and the UV spectrum characteristics of the compounds, and multiple compounds are detected.

4. The treatment method of the test product adopts the method of water and absolute ethanol extraction, which is easy to operate and the solvent is economical, green and safe.

5. This method has been systematically verified by methodology. This method is simple and fast, has good specificity, accurate results, good repeatability, and has a certain promotion value.

Description of the drawings

Figure 1 is the HPLC chart of the isomerization of the lutein reference substance solution in the national standard;

Figure 2 is the HPLC chart of the test solution of the mobile phase program I of the present invention;

Figure 3 is the HPLC chart of the test solution of the mobile phase program II of the present invention;

Figure 4 is the HPLC chart of the test solution of the mobile phase program III of the present invention;

Figure 5 is the HPLC chart of the test solution of the mobile phase program IV of the present invention;

Figure 6 is the HPLC chart of the test solution of the mobile phase program V of the present invention;

Fig. 7 is the HPLC chart of the test solution solution of the mobile phase methanol/water=86:14 of the present invention;

Fig. 8 is an HPLC chart of the test solution solution of the mobile phase methanol/water=88:12 of the present invention;

Fig. 9 is an HPLC chart of the test solution solution of the mobile phase methanol/water=90:10 of the present invention;

Fig. 10 is an HPLC chart of the test solution solution of the mobile phase methanol/water=92:8 of the present invention;

Figure 11 is the HPLC chart of the test solution solution of the mobile phase methanol/water=95:5 of the present invention;

Figure 12 is the HPLC chart of the test solution solution with the mobile phase methanol/water=100:0 of the present invention;

Fig. 13 is an HPLC chart of the test solution solution with a flow rate of 0.8 ml/min according to the present invention;

Fig. 14 is an HPLC chart of the test solution solution with a column temperature of 30°C according to the present invention;

Figure 15 is the verification result of the purity of trans-lutein of the present invention;

Figure 16 is the HPLC chart of the trans-lutein reference substance solution of the present invention;

Figure 17 is the HPLC chart of the trans-zeaxanthin reference substance solution of the present invention;

Figure 18 is the HPLC chart of the lutein isomerization reference substance solution of the present invention;

Figure 19 is an HPLC chart of the test solution of the present invention;

Figure 20 is an ultraviolet spectrum of peak 1 of the present invention;

Figure 21 is an ultraviolet spectrum of peak 2 of the present invention;

Figure 22 is an ultraviolet spectrum of peak 3 of the present invention;

Figure 23 is an ultraviolet spectrum of peak 4 of the present invention;

Figure 24 is an ultraviolet spectrum of peak 5 of the present invention;

Figure 25 is an ultraviolet spectrum of peak 6 of the present invention;

Figure 26 is an ultraviolet spectrum of peak 7 of the present invention;

Figure 27 is an ultraviolet spectrum of peak 8 of the present invention;

Figure 28 is an ultraviolet spectrum of peak 9 of the present invention;

Figure 29 is an ultraviolet spectrum of peak 10 of the present invention;

Figure 30 is an ultraviolet spectrum of peak 11 of the present invention;

Figure 31 is an ultraviolet spectrum of peak 12 of the present invention;

Fig. 32 is an HPLC chart of the trans-lutein reference substance solution of the specificity experiment of the present invention;

Figure 33 is an HPLC diagram of the negative test solution of the specificity test of the present invention;

Figure 34 is an HPLC chart of the test solution for the specificity experiment of the present invention.

Detailed ways

In view of the excellent activity of all-trans lutein, the present invention aims to propose a qualitative or qualitative analysis of all-trans lutein and isomers in health foods, foods, medicines, and lutein particles using lutein as raw materials. For quantitative methods, those skilled in the art can learn from the content of this article and appropriately improve the parameter implementation. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The drugs, reagents, and instruments used in the determination method provided by the present invention, without the manufacturer's indication, are all conventional products that can be purchased commercially. If the specific experimental conditions are not specified, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer.

Instruments: High performance liquid chromatograph (Dionex Ultimate 3000, Agilent 1260, Shimadzu LC-20AT, equipped with DAD detector or UV detector); electronic analytical balance (METTLER XP6, METTLER TOLEDO MS 105DU, Sartorius BSA224S-CW); desktop high-speed Centrifuge (H1650-W, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); CNC ultrasonic cleaner (KQ-500DB, Kunshan Ultrasonic Instrument Co., Ltd.); ultrapure water instrument (Milli-Q, Millipore, USA); Light stability test box (ICH-110 stability test box MEMMERT, Germany).

Reagents: methanol (chromatographically pure, American Merida and Merck); absolute ethanol (chromatographically pure, American Merida); methyl tert-butyl ether (chromatographically pure, American Merida); acetonitrile (chromatographically pure, American Merida) Pure, American Tiandi Company); Anhydrous ethanol (Analytical Pure, Nanjing Chemical Reagent Co., Ltd.); DMF (Analytical Pure, Beijing Bailingwei Technology Co., Ltd.); Water (ultra-pure water, self-made); 2,6-Di-tert-butyl P-cresol (BHT, chemically pure CP, Sinopharm Chemical Reagent Co., Ltd.); iodine (analytical pure, Sinopharm Chemical Reagent Co., Ltd.)

Reference substance and samples: Lutein reference substance (ie trans-lutein) (content based on 85.6%, batch number: LOT LRAB3708, purchased from Sigma-Aldrich); Zeaxanthin reference substance (ie trans-zeaxanthin) ) (CH13190320, Chengdu Croma Biotechnology Co., Ltd.); Lutein microparticles (2017120607, 2017120608, 2018010602 batch), Zhejiang Pharmaceutical Co., Ltd.); Lutein microparticles negative sample (from sodium starch octenyl succinate, Prepared by mixing white sugar, edible corn starch and other auxiliary materials), Meiling tablets samples (20190101, 20190102, 20190103 batches), provided by the health products department of Jiangsu Kangyuan Pharmaceutical Co., Ltd.

Example 1 Selection of chromatographic conditions

Preparation of reference substance solution: Take an appropriate amount of lutein reference substance, accurately weigh it, and add anhydrous ethanol containing 0.1% BHT to make a solution containing 100 μg of lutein per 1 mL to obtain the reference substance stock solution. Precisely measure 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0 mL of the stock solution in a 10 mL measuring flask, and dilute with 0.1% BHT absolute ethanol to a concentration of 2, 4, 8, 12, 16, 20 μg/ mL of the serial concentration of the reference solution.

Preparation of test solution: Take about 300 mg of lutein particles (2017120608 batch), accurately weigh it, place it in a 100 mL brown volumetric flask, add 5 mL of water, and treat it with ultrasound (power 100W) at room temperature for 20 minutes, and then add 0.1% BHT absolute ethanol is close to the mark, ultrasonic (power 100W) treatment for 5min, cool to room temperature, dilute to the mark with 0.1% BHT absolute ethanol, then accurately measure 1mL to 10mL brown volumetric flask, use 0.1% BHT Dilute the absolute ethanol to the mark, shake well, and centrifuge to get it.

1 Wavelength selection

A DAD detector was used to scan the lutein reference solution at a full wavelength in the range of 200nm～500nm. The results showed that the maximum absorption wavelength of lutein was at 444nm, which was consistent with the chromatographic conditions in the national standard, so 445nm was chosen. As the detection wavelength.

2 Selection of mobile phase

2.1 Selection of mobile phase ratio

First, refer to the chromatographic conditions in the national standard, that is, use YMC Carotenoid C30 as the chromatographic column, column temperature of 30°C, flow rate of 1.0 mL/min, detection wavelength of 445 nm, and methanol/water (88:12) as mobile phase A. Methyl tert-butyl ether is mobile phase B, both phases contain 0.1% BHT, gradient elution, program I is 0～18min, 0%～90%B, 18～18.1min, 90%～0%B, 18.1 ~28min, 0% B, the result is shown in Figure 2. It can be seen from the figure that the trans-lutein peak 7 and peak 6 basically coincide with the conditions in the national standard, and the resolution with peak 8 is about 1.45, which does not reach the baseline separation. Therefore, first consider adjusting the ratio of A and B phases to investigate the difference. The separation effect between trans-lutein and each isomer under the gradient elution program. The further set gradient program is as follows: Gradient program II is 0～20min, 0%→70%B, 20～20.1min, 70%～0%B, 20.1～30min, 0%B; Program Ⅲ is 0～25min, 0 %～60%B, 25～25.1min, 60%～0%B, 25.1～35min, 0%B; Program IV is 0～30min, 0%～50%B, 30～30.1min, 50%～0% B, 30.1-40min, 0%B; Program V: 0-35min, 0%-40%B, 35-45min, 40%B. The results are shown in Figures 3 to 6. It can be seen from the figure that as the proportion of A increases, the resolution between peak 7 of trans-lutein and peaks 6 and 8 of the adjacent isomers has increased, peak 7 and peak 8 Baseline separation can be achieved in program III, but peak 7 and peak 6 cannot achieve baseline separation no matter which program they are in, and the retention time is extended from 16min to 32min. It can be seen that changing the ratio of the two phases A and B has a certain effect on improving the separation effect of peak 7 and peak 6, but the effect is not very obvious. In comparison, program IV is used as a mobile phase gradient, and the separation effect is slightly better. The retention time is about 25min. The resolution of peak 7 and peak 6 can reach 1.40-1.50, which has a certain separation effect.

2.2 Selection of mobile phase methanol/water ratio

Use YMC Carotenoid C30 as the chromatographic column, column temperature of 30℃, flow rate of 1.0mL/min, detection wavelength of 445nm, methanol/water as mobile phase A, methyl tert-butyl ether as mobile phase B, both phases contain 0.1% BHT, gradient elution 0min～30min, 0%～50%B, 30min～40min, 50%B. Under these conditions, the methanol/water ratio of phase A is mainly investigated as 86:14, 88:12, 90 The separation effect of trans-lutein peak 7 and adjacent isomer peaks 6 and 8 at 10, 92:8, 95:5 and 100:0, the results are shown in Figures 7-12. It can be seen from the figure that as the methanol ratio increases, the resolution gradually increases, the resolution of peak 7 and peak 8 increases from 1.5 to 5.0, the resolution from peak 6 increases from 1.0 to 2.5, and the retention time decreases from 28min to 14min, methanol/water 95:5 to 100:0 peak 7 and adjacent isomer peaks can achieve baseline separation. The methanol/water ratio of 92-100:8-0 has a good separation effect. Considering the separation of other peaks and the economy of the solvent, the methanol/water ratio is preferred to be 95:5. Under these conditions, the resolution is about 1.9 and the retention time is about 22 minutes. In addition, the two phases A and B in the national standard contain 0.1% BHT, which is complicated to prepare. Therefore, the subsequent process only contains 0.1% BHT in phase A or B. It is found that under the conditions of the present invention, the separation effect is not affected, and the specific concentration It can be adjusted according to the actual situation.

3 Investigation of different flow rates

Use YMC Carotenoid C30 as the chromatographic column, methanol/water (95:5, containing 0.1% BHT) as mobile phase A, methyl tert-butyl ether as mobile phase B, gradient elution, 0min～30min, 0%～50 %B, 30min～40min, 50%B, column temperature is 30℃, detection wavelength is 445nm, flow rate is 0.7, 0.8, 0.9, 1.0, 1.1mL/min to separate trans-lutein and its isomer Effect and content, the results show that trans-lutein can achieve baseline separation from neighboring isomers at different flow rates, and there is no significant difference in content, indicating that the flow rate of this method is 0.7mL/min～1.1mL/min Good durability in the range. Considering the resolution, retention time and the separation of other isomers, the flow rate of 0.8mL/min is preferred, as shown in Figure 13. Under this condition, the retention time is 23.6min, the resolution is 1.93, the symmetry factor is 1.06, theoretically The number of boards is 79684.

4Inspection of different column temperatures

Under the above conditions, the effects of column temperature of 25℃, 30℃, 35℃ and 40℃ on the separation effect and content of trans-lutein and its adjacent isomers were investigated, and the results showed that trans-lutein It has good separation effect from adjacent isomers at different column temperatures, and there is no significant difference in content, indicating that the method has good durability within the column temperature range of 25°C to 40°C. The usual temperature of 30°C is preferred. As shown in Figure 14, the retention time under this condition is 23.5 min, the resolution is 1.92, the symmetry factor is 1.06, and the number of theoretical plates is 82,472.

5Inspection of different instruments

Under the above conditions, the separation effect and content of three brands of Shimadzu (SHIMADZU-20AT), Agilent (Agilent-1260), and Dionex (Dionex Ultimate 3000) on the separation effect and content of trans-lutein and its isomers were investigated. As a result, using three brands of instruments, lutein can be separated better, and there is no significant difference in content, indicating that the instrument has better durability.

6 Chromatographic peak purity verification

The chromatographic conditions of lutein obtained through the above investigation are: chromatographic column is YMC Carotenoid C30 (250mm×4.6mm, 5μm); methanol/water (95:5, containing 0.1% BHT) is used as mobile phase A, and methyl tertiary Butyl ether is mobile phase B, gradient elution, 0min～30min, 0%～50%B, 30min～40min, 50%B; flow rate is 0.8mL/min; column temperature is 30℃; detection wave is 445nm; sample injection The amount is 10ul. Under these conditions, the DAD detector was used to verify the peak purity of the trans-lutein peak 7 in the chromatogram of the test product. The result is shown in Figure 15. From the result, it can be seen that the purity angle is less than the purity threshold, and the lutein chromatographic peak purity meets the requirements. .

Example 2 Selection of the preparation method of the test product

Firstly, the test solution was prepared by the method of 2.1 national standard. As a result, the peak of lutein could not be detected. It is speculated that the lutein particles are microcapsule inclusion compounds, resulting in the use of national standard low-polarity organic solvents to be unable to extract lutein. Therefore, considering the use of a suitable solvent to destroy the microcapsules, so that the lutein can be released, and then using absolute ethanol for extraction, the extraction conditions are optimized under this idea.

1 Selection of membrane breaking solvent

The effects of water, DMF, and absolute ethanol as the membrane breaking solvent on the content of all-trans lutein were investigated. Take about 300 mg of lutein particles (2017120608 batch), accurately weigh them, place them in a 100 mL brown volumetric flask, add 5 mL each of water, DMF, and absolute ethanol, and treat them with ultrasound (power 100W) at room temperature for 20 minutes, and then add the 0.1% BHT absolute ethanol to close to the mark, ultrasonic (power 100W) treatment for 5 minutes, cool to room temperature, dilute to the mark with 0.1% BHT absolute ethanol, and then accurately measure 1mL to 10mL brown volumetric flask, 0.1% Dilute the BHT absolute ethanol to the mark, shake well, and centrifuge to get it. The results are shown in Table 1. The results show that when water is used as the membrane breaking solvent, the content of all-trans lutein is significantly higher than that of DMF and absolute ethanol, and the cost is low. Therefore, water is preferred as the membrane breaking solvent.

Table 1 The effect of different membrane breaking solvents on the content of all-trans lutein

2 Selection of membrane breaking temperature

The effects of ultrasonic temperatures of 20°C, 30°C, 40°C, and 60°C on the content of all-trans lutein were investigated. The results are shown in Table 2. The results show that different membrane breaking temperatures have no significant difference in the content of all-trans lutein, but as the temperature increases, the content has a downward trend. Considering the sensitivity of temperature to lutein and the convenience of operation, room temperature is preferred. As the ultrasonic temperature.

Table 2 The effect of membrane rupture temperature on the content of all-trans lutein

3 Selection of the amount of solvent to break the membrane

The effects of the volume of rupture solvent water of 2mL, 5mL, 7.5mL, and 10mL on the content of all-trans lutein were investigated. The results are shown in Table 3. The results showed that the amount of different rupture solvents had no significant difference in the content of all-trans lutein. In order to ensure sufficient rupture and the solubility of all-trans lutein, 5mL was finally selected as the rupture solvent.

Table 3 The effect of different solvents for breaking membranes on the content of lutein

4 Choice of rupture time

The effect of rupture time of 5min, 10min, 20min, 30min on the content of all-trans lutein was investigated. The results are shown in Table 4. The results show that different rupture time has no significant effect on the content of all-trans lutein, but the rupture time is 10min. The content of 20min is relatively high. In order to ensure sufficient membrane rupture, 20min is finally selected as the membrane rupture time.

Table 4 The effect of different membrane rupture time on the content of lutein

5 Selection of the extraction time of absolute ethanol

The effects of extraction time of 0min, 5min, 10min, and 20min with absolute ethanol on the content of all-trans lutein were investigated. The results are shown in Table 5. The results showed that the different extraction times of absolute ethanol had no significant effect on the content of lutein. Considering that ultrasound helps to disperse lutein uniformly and save time, 5min was finally selected as the absolute ethanol extraction time.

Table 5 The effect of absolute ethanol extraction time on the content of lutein

Through the above investigation, the preparation method of the test solution is as follows: take about 300 mg of lutein particles, accurately weigh them, place them in a 100 mL brown volumetric flask, add 5 mL of water, treat with ultrasound (power 100W) at room temperature for 20 minutes, and then add 0.1% BHT absolute ethanol to close to the mark, ultrasonic (power 100W) treatment for 5 minutes, cool to room temperature, dilute to the mark with 0.1% BHT absolute ethanol, and then accurately measure 1mL to 10mL brown volumetric flask, 0.1% Dilute the BHT absolute ethanol to the mark, shake well, and centrifuge to get it.

Example 3 Qualitative study of lutein related compounds

1 Preparation of Lutein Reference Substance Solution

Take about 5mg of the reference substance of lutein (ie trans-lutein), accurately weigh it, and place it in a 50mL brown volumetric flask. Add anhydrous ethanol containing 0.1% BHT to prepare a reference substance containing 100μg of lutein per 1mL. As for the stock solution, accurately measure 1.2 mL of the stock solution and place it in a 10 mL brown volumetric flask. Dilute with 0.1% BHT and absolute ethanol to prepare a reference solution with a concentration of 12 μg/mL.

2 Preparation of zeaxanthin reference substance solution

Take about 5mg of zeaxanthin (ie trans-zeaxanthin) reference substance, accurately weigh it, and place it in a 50mL brown volumetric flask. Add absolute ethanol containing 0.1% BHT to prepare a reference substance containing 100μg of lutein per 1mL As for the stock solution, accurately measure 1.0mL of the stock solution and place it in a 50mL brown volumetric flask. Dilute with 0.1% BHT and absolute ethanol to prepare a reference solution with a concentration of 2μg/mL.

3 Preparation of Lutein Isomerization Reference Substance Solution

Take about 5mg of lutein reference substance, accurately weigh it in a 50mL transparent volumetric flask, add absolute ethanol to prepare a reference substance stock solution containing 100μg of lutein per 1mL, take 1mL of this stock solution and place it in a 10mL transparent volumetric flask. Add 0.1 mL of iodine in ethanol, dilute to the mark with absolute ethanol, and place in a light box for 1 hour to obtain a mixed solution of reference substance for lutein isomerization.

4 Preparation of test solution

The test solution was prepared according to the optimized preparation method of the test solution in Example 2.

5 determination

Precisely draw the above-mentioned reference solution and test solution respectively, and use HPLC with DAD detector for determination.

6 results

The HPLC spectra of Lutein Reference Substance, Zeaxanthin Reference Substance, Lutein Isomerization Reference Substance Solution and Test Substance Solution are shown in Figure 16-19 (the attached figure is a partial enlarged view), and the DAD full-wave scan of each compound The spectrum is shown in Figure 20-31. The isomers can be preliminarily identified by the characteristics of ultraviolet spectroscopy: Compared with all-trans lutein, the maximum absorption wavelength of the mono-cis isomer usually has a blue shift of 4 to 6 nm, and the double-cis isomer Then there is a blue shift of 8-12nm; secondly, the mono-cis isomer has a cis absorption between 330 and 340nm, and the closer the cis double bond is to the center of the molecule, the greater the cis absorption (usually expressed by the Q value) The intensity of the cis absorption peak); Finally, both lutein and B-carotene are carotenoids, and both have a common isoprene structure, so the elution order of their corresponding positional isomers is consistent according to this. Therefore, according to the comparison of the reference substance, the UV spectrum characteristics and the elution order, the structure of 9 compounds was determined and three compounds that were not reported in the literature were inferred. The UV absorption wavelength, Q value, peak area and other parameters of each peak are shown in Table 6. The compound structure is shown in Figure 32. details as follows:

Peak 7: First, it is consistent with the HPLC retention time of the lutein reference solution, which can be preliminarily judged to be all-trans lutein; secondly, the measured absorption peaks at 420nm, 444nm, and 472nm are consistent with the reported heights of 423nm, 444nm, and 472nm; In addition, the measured Q value of 0.06 is highly consistent with the reported 0.05. Therefore, it is comprehensively judged that peak 7 is the compound all-trans lutein, and the structure is shown in e.

Peak 8: Firstly it is consistent with the HPLC retention time of the zeaxanthin reference solution, which can be preliminarily judged to be all-trans zeaxanthin; secondly, the measured values of absorption peaks are 335nm, 425nm, 450nm, 478nm, and the reported heights of 427nm, 450nm, 477nm Consistent; in addition, the measured Q value is 0.05, which is basically consistent with the reported 0.06. Therefore, it is comprehensively judged that peak 8 is the compound all-trans zeaxanthin, and the structure is shown in f.

Peak 3: First, the measured values of absorption peaks are 330nm, 412nm, 438nm, 464nm. Compared with the maximum absorption peak of trans-lutein at 444nm, the maximum absorption peak at 438nm has a blue shift of 6nm. According to the literature report, single-cis The maximum absorption wavelength of the isomer usually has a blue shift of 4-6nm, so it is inferred that the compound is mono-cis-lutein; the next four absorption peaks are highly consistent with the reported 330nm, 412nm, 436nm, and 462nm; in addition, the Q value is determined The value of 0.47 is highly consistent with the reported 0.46; finally, referring to the elution sequence of carotene isomers, it is comprehensively judged that peak 3 is compound 13-cis-lutein, and the structure is shown in a.

Peak 5: First, the measured values of the absorption peaks are 330nm, 414nm, 438nm, and 466nm. The maximum absorption peak of 438nm is 6nm blue-shifted compared with the maximum absorption peak of trans-lutein at 444nm. The maximum absorption wavelength of the structure usually has a blue shift of 4-6nm, so it is inferred that the compound is mono-cis-lutein; the next four absorption peaks are consistent with the reported height of 330nm, 414nm, 438nm, and 464nm; In addition, the measured value of Q value 0.49 is highly consistent with the reported 0.45; finally, referring to the elution sequence of carotene isomers, it is comprehensively judged that peak 5 is compound 13'-cis-lutein, and the structure is shown in b.

Peak 6: First, the measured absorption peaks are 330nm, 420nm, 444nm, 474nm. Compared with the maximum absorption peak of trans-zeaxanthin at 450nm, the maximum absorption peak at 444nm has a blue shift of 6nm. The blue shift of 4～6nm is preliminarily judged to be mono-cis zeaxanthin; the next four absorption peaks are highly consistent with the reported 331nm, 444nm, 473nm and the reported 338nm, 444nm, 470nm; in addition, the measured Q value of 0.10 is consistent with the reported The 0.43 and the reported 0.49 are basically the same; finally, referring to the elution sequence of carotene isomers, it is comprehensively judged that the peak 6 is 13 or 13'-cis-zeaxanthin. See c and d for structure.

Peak 9: The first measured values of the absorption peaks are 332nm, 420nm, 438nm, 468nm, of which the maximum absorption peak 438 is 6nm blue-shifted compared with the maximum absorption peak of trans-lutein at 444nm, due to mono-cis isomerization The maximum absorption wavelength of the body usually has a blue shift of 4-6nm, so it is inferred that the compound is mono-cis-lutein; the next four absorption peaks are consistent with the known heights of 332nm, 418nm, 440nm, and 466nm; in addition, the measured value of Q value The value of 0.09 is consistent with the reported 0.10 height; finally combined with the elution order, it is comprehensively judged that the peak 9 is compound 9-cis-lutein, and the structure is shown in h.

Peak 10: First, the measured values of the absorption peaks are 330nm, 420nm, 442nm, 466nm. The maximum absorption peak of 442nm is 2nm blue-shifted compared with the maximum absorption peak of trans-lutein at 444nm. The maximum absorption wavelength of the cis isomer usually has a blue shift of 4 to 6 nm, which is basically the same. It is inferred that the compound is mono-cis lutein; the next four absorption peaks are with the known 332nm, 420nm, 444nm, 472nm and known The heights of 330nm, 422nm, 440nm, and 468nm are the same; in addition, the measured value of Q value 0.09 is consistent with the reported 0.05 and the reported 0.11 height; finally, combined with the elution order, it is comprehensively judged that the peak 10 is the compound 9'-cis-lutein, See i for structure.

Peak 11: First, the measured values of the absorption peaks are 338nm, 446nm, 474nm. Compared with the 450nm maximum absorption peak of trans-zeaxanthin, the maximum absorption peak 446 has a blue shift of 4nm, because the mono-cis isomer has 4 The blue shift of ~6nm is preliminarily judged to be mono-cis zeaxanthin; the next three absorption peaks are highly consistent with the reported 337nm, 445nm, 473nm and the reported 338nm, 445nm, 472nm; in addition, the measured Q value is 0.09, which is consistent with the reported The 0.14 and the reported 0.10 are highly consistent; finally, combined with the elution order, it is comprehensively judged that the peak 11 is 9-cis zeaxanthin.

Peak 12: First, the measured absorption peaks are 338nm, 446nm, 472nm. Compared with the 450nm maximum absorption peak of trans-zeaxanthin, the maximum absorption peak 446 has a blue shift of 4nm, due to the 4nm single-cis isomer. The blue shift of ~6nm is preliminarily judged to be mono-cis zeaxanthin; the next three absorption peaks are highly consistent with the reported 337nm, 445nm, 473nm and the reported 338nm, 445nm, 472nm; in addition, the measured Q value of 0.06 is consistent with the reported 0.14 And 0.10 are basically the same; finally, combined with the elution order, it is comprehensively judged that the peak 12 is 9'-cis zeaxanthin.

Peak 1: The measured value of the absorption peak is 330nm, 410nm, 430nm, 458nm. Compared with the maximum absorption peak of trans-lutein at 444nm, the maximum absorption peak of 430nm has a blue shift of 14nm. The blue shift of 8～12nm, combined with the peak sequence, is preliminarily judged as dicis-lutein.

Peak 2: The measured absorption peaks are 332nm, 410nm, 432nm, 458nm. Compared with the maximum absorption peak of trans-lutein at 444nm, the maximum absorption peak of 432nm has a blue shift of 12nm. The blue shift of 8～12nm, combined with the peak sequence, is preliminarily judged to be dicis-lutein.

Peak 4: The measured values of absorption peaks are 330nm, 418nm, 440nm, 470nm. Compared with the maximum absorption peak of trans-zeaxanthin at 450nm, the maximum absorption peak of 440nm has a blue shift of 10nm. The blue shift of 8～12nm, combined with the peak sequence, is preliminarily judged to be dicis-zeaxanthin.

Table 6 Identification of lutein and its isomers

The structure of each compound is as follows:

Example 4 Verification of trans-lutein quantitative methodology

1 System suitability

According to the method of Example 3, the lutein reference solution and the test solution were prepared respectively. Take the reference solution for 5 consecutive injections, record the peak area of the component to be tested, and calculate the RSD; take the test solution for 5 consecutive injections, and record the number of theoretical plates, resolution, and symmetry factor of the component to be tested. The results show that the resolution of lutein is greater than 1.8, which has reached the baseline separation, the symmetry factor is 1.07 (basically meets the requirements of 0.95 to 1.05), and the number of theoretical plates is greater than 80,000 to ensure the accuracy of quantitative analysis. It is determined to be no less than 5000. The RSD of the area is 0.26%, which is less than 2%, indicating that the system has good applicability.

2 Exclusive investigation

Preparation of the reference solution: according to the preparation method of the lutein reference solution in Example 3.

Preparation of the test solution: According to the optimized preparation method of the test solution in Example 2.

Preparation of negative test solution: Take the negative sample powder, and prepare the negative test solution according to the preparation method of the test solution in Example 2.

Take the reference solution, the test solution, and the negative test solution according to the preferred chromatographic conditions in Example 1, and the results are shown in Figure 32-34. It can be seen from the figure that the test product chromatogram presents a chromatographic peak consistent with the retention time of the main peak of the reference product chromatogram, and the blank solvent and negative test product chromatograms basically have no impurity peaks at the retention time of the component to be tested. Shows that this method has good specificity.

3 Linearity and range

Take an appropriate amount of lutein reference substance, accurately weigh it, and add anhydrous ethanol containing 0.1% BHT to prepare a reference substance stock solution containing 96.5739 μg lutein per 1 mL. Precisely measure 0.2mL, 0.4mL, 0.8mL, 1.2mL, 1.6mL, and 2.0mL of the stock solution respectively in a 10mL brown volumetric flask, add 0.1% BHT absolute ethanol and dilute to 1.93, 3.86, 7.73, 11.59, 15.45 , 19.31μg/mL series of concentration reference solution.

Separately draw 10 μL of the above-mentioned solution, and draw the standard curve according to the chromatographic conditions of Example 2. Use the concentration of the reference solution (μg/mL) as the abscissa (X) and the peak area as the ordinate (Y) to plot the standard curve. The regression equation is: Y=156255X-7493.5 (R ² =1), and the result shows that within the linear range of 1.93～19.31μg/mL, the linear relationship is good.

4 Detection limit and quantification limit

Take the reference solution (1.93μg/mL) and dilute it step by step, with the concentration corresponding to 10 times the baseline noise (S/N=10) as the limit of quantification, and the concentration corresponding to 3 times the baseline noise (S/N=3) For the detection limit, the limit of quantification of trans-lutein in this method is 0.019μg/mL, and the limit of detection is 0.004μg/mL.

5 Precision test

Take the linear intermediate concentration (11.59μg/mL) reference substance solution and the test substance solution for 6 consecutive injections, measure the peak area, calculate the RSD, and the results of the reference substance and the test substance peak area precision RSD are less than 2% , The RSD of retention time precision is also less than 2%, indicating that the precision of the instrument is good.

6 Stability test

Take the same reference substance solution and the same test substance solution and inject samples once at 0h, 1h, 2h, 3h, 4h, 5h, 6h, 9h, 12h, 15h, 19h, 24h, measure the peak area, and calculate the RSD. The RSD of the peak area of the reference solution was 0.44%, and the RSD of the peak area of the test solution was 0.52%, both of which were less than 2%. Shows that the reference solution and the test solution are stable within 24 hours.

7 Repeatability test

Take the same batch of samples, prepare 6 test solution solutions according to the method in Example 2, determine and calculate the sample content and RSD, and the results show that the average content of trans-lutein is 5.71%, and the RSD is 0.92%, which is less than 2%. Shows that the method has good repeatability.

8 Sample recovery test

Take an appropriate amount of lutein reference substance and add 0.1% BHT absolute ethanol to make a control solution containing 0.6048 mg of trans-lutein per 1 mL.

Take 150mg of this product powder, accurately weigh it, place it in a 100mL brown volumetric flask, add 1mL of the above-mentioned reference substance (the amount of lutein in the reference substance is equivalent to the test product), and then prepare and measure according to the preparation method of the test product solution The peak area of trans-lutein was calculated, and the sample recovery and RSD were calculated. The results are shown in Table 7. The average recovery of trans-lutein was 101.46%, and the RSD was 0.82%, which was less than 2%. Shows that the accuracy of the method is better.

Example 5 Quantitative determination of trans-lutein in three batches of lutein microparticles

1 Preparation of reference solution

Take an appropriate amount of the lutein reference substance, accurately weigh it, and add an absolute ethanol containing 0.1% BHT to make a solution containing 100 μg of lutein per 1 mL to obtain the reference substance stock solution. Take 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0 mL of the stock solution in a 10 mL volumetric flask, and dilute with 0.1% BHT absolute ethanol to a concentration of 2, 4, 8, 12, 16, and 20 μg/mL. Concentration of the reference solution.

2 Preparation of test solution

Take three batches of lutein microparticles (2017120607, 2017120608, 2018010602) about 300mg, accurately weigh them, place them in a 100mL brown volumetric flask, add 5mL water, and ultrasonic (power 100W) at room temperature for 20min, then add 0.1% BHT The absolute ethanol is close to the mark, ultrasonic (power 100W) treatment for 5min, cool to room temperature, dilute to the mark with 0.1% BHT absolute ethanol, and then accurately measure 1mL to 10mL brown volumetric flask, 0.1% BHT anhydrous Dilute ethanol to the mark, shake well, and centrifuge to get it.

3 Chromatographic conditions

Use YMC Carotenoid C30 (250mm×4.6mm, 5μm) as the chromatographic column; use methanol/water (95:5, containing 0.1% BHT) as mobile phase A, use methyl tert-butyl ether as mobile phase B, gradient elution , Namely 0min～30min, 0%～50%B, 30min～40min, 50%B; flow rate is 0.8mL/min, column temperature is 30℃, and detection wavelength is 445nm. The number of theoretical plates should not be less than 5000 calculated based on the trans-lutein peak.

4 determination

Precisely draw 10 μL each of the reference solution and the test solution, and inject them into the liquid chromatograph for determination.

5 results

The determination results of the three batches of lutein particles were: the average content of the 2017120607 batch was 6.05%, the average content of the 2017120608 batch was 5.71%, and the average content of the 2018010602 batch was 5.68%.

Example 6 Quantitative determination of trans-lutein in three batches of Meiling tablets

1 Preparation of reference solution

The preparation of the lutein reference solution in Example 5 is the same.

2 Preparation of test solution

Take three batches of Meiling tablets (batch numbers: 20190101, 20190102, 20190103), remove the coating, grind, and take the powder about 300mg, accurately weigh them, place them in a 100mL brown volumetric flask, add 5mL of water, and ultrasound at room temperature (Power 100W) Treat for 20 minutes, then add 0.1% BHT-containing absolute ethanol to close to the mark, ultrasonic (power 100W) treatment for 5 minutes, cool to room temperature, dilute to the mark with 0.1% BHT-containing absolute ethanol, shake well, Centrifugal, that's it.

3 Chromatographic conditions

The same chromatographic conditions as in Example 5.

4 determination

Precisely draw 10 μL each of the reference solution and the test solution, and inject them into the liquid chromatograph for determination.

5 results

The determination results of the three batches of Meiling tablets were: the average content of the 20190101 batch was 4.15 mg/g, the average content of the 20190102 batch was 4.17 mg/g, and the average content of the 20190103 batch was 4.20 mg/g.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be considered This is the protection scope of the present invention.

Claims (10)

1. A method for determining the components of lutein, characterized in that the method adopts high performance liquid chromatography for detection, wherein the chromatographic conditions include: using YMC Carotenoids C30 as a chromatographic column; using methanol/water as mobile phase A, methyl tert-butyl The base ether is the mobile phase B; the elution procedure is 0min-30min, 0%-50%B, 30min-40min, 50%B; the methanol/water ratio is 88-100:12-0.
2. The method according to claim 1, wherein the ratio of methanol/water is 92-100:8-0.
3. The method according to claim 1, wherein the ratio of methanol/water is 95:5.
4. The method according to claim 1, wherein the mobile phase A and/or the mobile phase B contains 2,6-di-tert-butyl-p-cresol.
5. The method according to claim 1, wherein the concentration of the 2,6-di-tert-butyl-p-cresol is 0.1%.
6. The method according to claim 1, wherein the chromatographic conditions further comprise: a flow rate of 0.7 mL/min to 1.1 mL/min; and a column temperature of 25°C to 40°C.
7. The method of claim 1, wherein:

   Use YMC Carotenoid C30 with a specification of 250mm×4.6mm, 5μm as the chromatographic column; use 0.1% 2,6-di-tert-butyl-p-cresol and methanol/water solution with a ratio of 95:5 as mobile phase A, Base tert-butyl ether is mobile phase B, elution procedure is 0min～30min, 0%～50%B, 30min～40min, 50%B; flow rate is 0.8mL/min, column temperature is 30℃, detection wavelength is 445nm .
8. The method of claim 1, wherein the lutein component comprises all-trans lutein.
9. A method for detecting lutein raw materials or samples containing lutein, characterized in that the method comprises:

   Preparation of test solution: grind the lutein raw material or the sample containing lutein, take the powder into a volumetric flask, add water, sonicate at room temperature, then add absolute ethanol containing BHT, sonicate , Cooled to room temperature, constant volume;

   The test solution is tested by any one of claims 1-8.
10. The method according to claim 9, characterized in that the method further comprises:

    Preparation of reference substance solution: take lutein reference substance, add absolute ethanol containing BHT to make a stock solution containing lutein, and use the stock solution to prepare reference substance solutions of various concentrations;

    The above-mentioned reference solution is tested by the method described in any one of claims 1-8.

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