CN110879257A - Method for determining xanthophyll component - Google Patents

Abstract

The invention discloses a method for measuring lutein components, which is characterized by adopting high performance liquid chromatography for detection, wherein the chromatographic conditions comprise: YMC Carotenoid C30 is used as a chromatographic column; methanol/water is taken as a mobile phase A, and methyl tert-butyl ether is taken as a mobile phase B; the elution procedure is 0min to 30min, 0 percent to 50 percent of B, 30min to 40min and 50 percent of B; the ratio of methanol to water is 88-100: 12-0. The method comprises the steps of taking the high-activity component of the all-trans lutein as a detection index, more truly and effectively evaluating the quality of lutein products, effectively separating the all-trans lutein and main cis-isomers and isomers thereof under the used chromatographic conditions, and accurately quantifying the all-trans lutein.

Description

Method for determining xanthophyll component

Technical Field

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

Background

Lutein (lutein) is a yellow substance belonging to carotenoid, widely exists in nature, exists in blood plasma and macula lutea area of eyes in human body, can absorb a large amount of blue light to avoid photooxidation injury of retina, and can clear free radicals to protect optic nerve from being damaged by free radicals as an antioxidant, so that the lutein (lutein) is widely applied to various health-care foods, foods and medicines. The molecular structure of lutein has multiple conjugated double bond structures, and is easily influenced by factors such as light, oxygen, high temperature and the like, so that theoretically, multiple isomers can exist, including cis-isomer and isomer thereof, such as zeaxanthin and the like.

In organisms, the biological activities of different isomers are different, and the biological activity of trans-conformation is much higher than that of cis-conformation, so that the biological functions or potency of the xanthophyll are remarkably different, and once the xanthophyll is influenced by factors such as light, heating, oxygen and the like in the preparation and application processes, the quality of a product taking the xanthophyll as a main active ingredient is inevitably influenced. At present, few people monitor the change of cis-isomer in the process of lutein extraction and preparation, and especially health food and food taking lutein as a raw material generally evaluate the quality of the product according to the content of total lutein, so that the quality of the product is difficult to guarantee. The existing national standard takes a reference substance with the purity as the total lutein content, takes the sum of trans-lutein, cis-isomer and isomer thereof as a quantitative index, and the trans-lutein, cis-isomer and isomer thereof are not separated ideally, as shown in figure 1, in the chromatogram of the isomerized standard solution, only three chromatographic peaks can be seen, and a plurality of cis-isomers and isomers in theory or related documents are not seen, so that the existing method has great limitation on the quality monitoring of the lutein. Moreover, the preparation method of the test sample in the standard is rather complicated, thereby increasing the cost of the quality control of the lutein.

Disclosure of Invention

In view of the above, the present invention aims to provide a qualitative or quantitative method for all-trans lutein and isomers in health food, medicine and lutein raw material using lutein as raw material. Specifically, the invention provides a method for measuring a lutein component, which adopts high performance liquid chromatography for detection, wherein the chromatographic conditions comprise: YMC Carotenoid C30 is used as a chromatographic column; methanol/water is taken as a mobile phase A, and methyl tert-butyl ether is taken as a mobile phase B; the elution procedure is 0min to 30min, 0 percent to 50 percent of B, 30min to 40min and 50 percent of B; the ratio of methanol to water is 88-100: 12-0.

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

Further, the mobile phase A and/or the mobile phase B contain 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: the flow rate is 0.7mL/min to 1.1 mL/min; the column temperature is 25-40 ℃.

Further, in the aforementioned method for measuring a xanthophyll component, the chromatographic conditions include: using YMC Carotenoid C30 with specification of 250mm × 4.6mm and 5 μm as chromatographic column; taking methanol/water solution containing 0.1 percent of 2, 6-di-tert-butyl-p-cresol and the ratio of 95:5 as a mobile phase A and methyl tert-butyl ether as a mobile phase B, and performing elution procedures of 0 min-30 min, 0 percent-50 percent B, 30 min-40 min and 50 percent B; the flow rate was 0.8mL/min, the column temperature was 30 ℃ and the detection wavelength was 445 nm.

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

The invention also provides a method for detecting the lutein raw material (such as lutein particles) or the sample containing the lutein, which is characterized by comprising the following steps:

preparation of a test solution: grinding xanthophyll microparticles or xanthophyll-containing samples, placing the powder into a volumetric flask, adding water, performing ultrasonic treatment at room temperature, adding absolute ethyl alcohol containing BHT, performing ultrasonic treatment, cooling to room temperature, and fixing the volume;

the test solution is tested by any one of the above methods for measuring the xanthophyll component.

Specifically, the preparation of the test solution comprises: taking xanthophyll particles or a sample containing xanthophyll, grinding, taking about 300mg of powder, precisely weighing, placing in a 100mL brown volumetric flask, adding 5mL of water, carrying out ultrasonic treatment at room temperature for 20min, adding absolute ethyl alcohol containing 0.1% BHT to approximate scale, carrying out ultrasonic treatment for 5min, cooling to room temperature, fixing the volume to the scale by using the absolute ethyl alcohol containing 0.1% BHT, shaking up, and centrifuging to obtain the xanthophyll compound. (the lutein microcapsule needs to be precisely measured in a brown volumetric flask with 1-10 mL, and the absolute ethyl alcohol of 0.1% BHT is metered to the scale).

Still further, the aforementioned method for detecting xanthophyll microparticles or a sample containing xanthophyll comprises:

preparation of control solutions: taking a lutein reference substance, adding absolute ethyl alcohol containing BHT to prepare stock solution containing lutein, and preparing reference substance solutions with various concentrations by using the stock solution;

preparation of a test solution: grinding xanthophyll microparticles or xanthophyll-containing samples, placing the powder into a volumetric flask, adding water, performing ultrasonic treatment at room temperature, adding absolute ethyl alcohol containing BHT, performing ultrasonic treatment, cooling to room temperature, and fixing the volume;

the above-mentioned reference solution and test solution are tested by the method of any one of claims 1 to 8. Thus, a standard curve of the reference substance can be established, and the content of the effective components can be calculated according to the detection result of the test substance.

More specifically, the control solution may be formulated as follows: taking a proper amount of a lutein reference substance, precisely weighing, and adding absolute ethyl alcohol containing 0.1% of BHT (2, 6-di-tert-butyl-p-cresol) to prepare a reference substance stock solution containing 100 micrograms of lutein per 1 mL; the stock solutions were measured precisely at 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0mL, and diluted with 0.1% BHT in absolute ethanol to give control solutions of 2, 4, 8, 12, 16, and 20 μ g/mL concentrations.

The qualitative or quantitative analysis method for the components of the lutein provided by the invention has the following advantages:

1. the method comprises the step of taking the high-activity component of the all-trans lutein as a detection index, so that the quality of lutein products can be more truly and effectively evaluated.

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

3. The method qualitatively detects multiple compounds by comparing reference substances and ultraviolet spectrum characteristics of the compounds.

4. The method for treating the test sample adopts a mode of extracting water and absolute ethyl alcohol, is simple and convenient to operate, and is economic, green and safe in solvent.

5. The method is verified by systematic methodology, and is simple, rapid, good in specificity, accurate in result, good in repeatability and worthy of popularization.

Drawings

FIG. 1 is an HPLC profile of isomerization of a lutein control solution in national standards;

FIG. 2 is a HPLC chromatogram of a test solution of mobile phase procedure I according to the present invention;

FIG. 3 is a HPLC chromatogram of a sample solution of mobile phase procedure II according to the present invention;

FIG. 4 is a HPLC chromatogram of a sample solution of mobile phase procedure III according to the present invention;

FIG. 5 is a HPLC chromatogram of a sample solution of mobile phase procedure IV according to the present invention;

FIG. 6 is a HPLC chromatogram of a test solution of mobile phase procedure V according to the present invention;

fig. 7 is a mobile phase methanol/water 86:14 sample solution HPLC profile of the present invention;

fig. 8 is a mobile phase methanol/water 88:12 sample solution HPLC profile of the present invention;

fig. 9 is a sample solution HPLC profile of mobile phase methanol/water 90:10 of the present invention;

fig. 10 is a sample solution HPLC profile of the present invention with a mobile phase of 92:8 methanol/water;

fig. 11 is a mobile phase methanol/water 95:5 sample solution HPLC profile of the present invention;

fig. 12 is a mobile phase methanol/water 100:0 HPLC profile of a test sample solution of the present invention;

FIG. 13 is an HPLC chromatogram of a test solution of the present invention with a flow rate of 0.8 ml/min;

FIG. 14 is an HPLC chromatogram of a sample solution at a column temperature of 30 ℃ according to the present invention;

FIG. 15 shows the verification of purity of trans-lutein according to the present invention;

FIG. 16 is an HPLC profile of a trans-lutein control solution of the present invention;

FIG. 17 is an HPLC chromatogram of a control solution of trans-zeaxanthin in accordance with the present invention;

FIG. 18 is an HPLC profile of a lutein isomerization control solution of the present invention;

FIG. 19 is an HPLC chromatogram of a test solution of the present invention;

FIG. 20 is a UV spectrum of peak 1 of the present invention;

FIG. 21 is a UV spectrum of peak 2 of the present invention;

FIG. 22 is a UV spectrum of peak 3 of the present invention;

FIG. 23 is a UV spectrum of peak 4 of the present invention;

FIG. 24 is a UV spectrum of peak 5 of the present invention;

FIG. 25 is a UV spectrum of peak 6 of the present invention;

FIG. 26 is a UV spectrum of peak 7 of the present invention;

FIG. 27 is a UV spectrum of peak 8 of the present invention;

FIG. 28 is a UV spectrum of peak 9 of the present invention;

FIG. 29 is a UV spectrum of peak 10 of the present invention;

FIG. 30 is a UV spectrum of peak 11 of the present invention;

FIG. 31 is a UV spectrum of peak 12 of the present invention;

FIG. 32 is an HPLC plot of a control solution of trans-lutein for a proprietary experiment of the present invention;

FIG. 33 is an HPLC plot of a solution of a proprietary experimental negative test article of the present invention;

FIG. 34 is an HPLC chart of a specific experimental test solution of the present invention.

Detailed Description

In view of the excellent activity of all-trans-lutein, the invention aims to provide a qualitative or quantitative method for all-trans-lutein and isomers in health food, medicine and lutein particles which take lutein as a raw material, and a person skilled in the art can use the content to realize the qualitative or quantitative method by properly improving parameters. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The medicines, reagents and instruments used in the determination method provided by the invention are all conventional products which can be obtained commercially without indicating manufacturers. Those who do not specify the particular experimental conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer.

The instrument comprises the following steps: high performance liquid chromatography (Dionex Ultimate 3000, Agilent 1260, Shimadzu LC-20AT, equipped with DAD detector or UV detector); electronic analytical balance (METTLER XP6, METTLER TOLEDO MS105DU, Sartorius BSA 224S-CW); a desk-top high-speed centrifuge (H1650-W, Hunan instruments laboratory development Co., Ltd.); a numerical control ultrasonic cleaner (KQ-500DB, ultrasonic instruments Co., Ltd., Kunshan); ultra pure water instruments (Milli-Q, Milli Ribo, USA); light stability test box (ICH-110 type stability test box MEMMERTER, Germany).

Reagent: methanol (chromatographically pure, Merrida, USA, Merck); absolute ethanol (chromatographically pure, mai ruida corp., usa); methyl tert-butyl ether (chromatographically pure, Merrida, USA); acetonitrile (chromatographically pure, Tiandi, USA); absolute ethanol (analytical grade, Nanjing chemical reagents Co., Ltd.); DMF (analytical grade, carbofuran technologies ltd, beijing); water (ultrapure water, self-made); 2, 6-di-tert-butyl-p-cresol (BHT, chemically pure CP, chemical reagents of national drug group, Ltd.); iodine (analytically pure, chemical reagents of national medicine group, Ltd.)

Control and sample: a lutein control (i.e., trans-lutein) (85.6% by weight, lot: lotfra 3708, available from Sigma-Aldrich company); zeaxanthin control (i.e., trans-zeaxanthin) (CH13190320, chengdu clomae biotechnology limited); lutein fine particles (2017120607, 2017120608, 2018010602 batches), zhejiang medical limited); the negative sample of xanthophyll particles (prepared by mixing starch sodium octenylsuccinate, white sugar, edible corn starch and other auxiliary materials) and the Meiling tablet sample (20190101, 20190102 and 20190103 batches) are provided by the health product department of Jiangsu Kangyuan pharmaceutical industry GmbH

Example 1 selection of chromatographic conditions

Preparation of control solutions: taking a proper amount of the lutein reference substance, precisely weighing, and adding absolute ethanol containing 0.1% BHT to prepare a solution containing 100 μ g of lutein per 1mL to obtain the reference substance stock solution. Precisely measuring the stock solutions 0.2mL, 0.4mL, 0.8mL, 1.2mL, 1.6mL and 2.0mL respectively, adding 0.1% BHT absolute ethanol, and diluting to obtain control solutions with concentration of 2, 4, 8, 12, 16 and 20 μ g/mL respectively.

Preparation of a test solution: weighing lutein particles (2017120608 batches) about 300mg, precisely weighing, placing in a 100mL brown volumetric flask, adding 5mL of water, carrying out ultrasonic treatment (power 100W) for 20min at room temperature, adding absolute ethyl alcohol containing 0.1% BHT to approximate scale, carrying out ultrasonic treatment (power 100W) for 5min, cooling to room temperature, carrying out constant volume treatment to scale by using absolute ethyl alcohol containing 0.1% BHT, precisely weighing 1mL to 10mL brown volumetric flask, diluting to scale by using absolute ethyl alcohol containing 0.1% BHT, shaking up, and centrifuging to obtain the lutein nanoparticle.

Selection of 1 wavelength

A DAD detector is adopted to carry out full-wavelength scanning on the lutein reference substance solution within the range of 200 nm-500 nm, and the result shows that the maximum absorption wavelength of lutein is at 444nm, which is consistent with the chromatographic condition in the national standard, so 445nm is selected as the detection wavelength.

2 selection of the Mobile phase

2.1 selection of the proportions of the mobile phases

Firstly, referring to the chromatographic conditions in the national standard, namely, taking YMC Carotenoid C30 as a chromatographic column, the column temperature is 30 ℃, the flow rate is 1.0mL/min, the detection wavelength is 445nm, methanol/water (88:12) as a mobile phase A, methyl tert-butyl ether as a mobile phase B, the two phases both contain 0.1% of BHT, and the results are shown in figure 2, wherein the procedure I is 0-18 min, 0-90% of B, 18-18.1 min, 90-0% of B, 18.1-28 min and 0% of B. As can be seen from the figure, under the condition of national standard, the peak 7 of trans-lutein is basically coincident with the peak 6, the separation degree from the peak 8 is about 1.45, and the baseline separation is not achieved, so that firstly, the separation effect between trans-lutein and each isomer under different gradient elution procedures is considered by adjusting A, B two-phase ratio. The further gradient program set up is as follows: the gradient program II is 0-20 min, 0% → 70% B, 20-20.1 min, 70% -0% B, 20.1-30 min and 0% B; the procedure III is 0-25 min, 0-60% B, 25-25.1 min, 60-0% B, 25.1-35 min and 0% B; the procedure IV is 0-30 min, 0-50% B, 30-30.1 min, 50-0% B, 30.1-40 min and 0% B; the procedure V is 0-35 min, 0-40% B, 35-45 min and 40% B. The results are shown in fig. 3-6, and it can be seen from the graphs that as the phase a proportion increases, the separation degree of the trans-lutein peak 7 from the adjacent isomer peaks 6 and 8 increases, the baseline separation of the peak 7 and the peak 8 can be achieved in the procedure iii, but the baseline separation of the peak 7 and the peak 6 cannot be achieved at all times in any procedure, and the retention time is prolonged from 16min to 32 min. It can be seen that the effect of improving the separation of peaks 7 and 6 is not very significant, although some effect is obtained by changing the ratio of the two phases A, B. In comparison, when the procedure iv is used as a mobile phase gradient, the separation effect is slightly better, the retention time is about 25min, and the separation degree of the peak 7 and the peak 6 can reach 1.40 to 1.50, which has a certain separation effect.

2.2 selection of the ratio methanol/Water of the Mobile phase

YMC Carotenoid C30 is used as a chromatographic column, the column temperature is 30 ℃, the flow rate is 1.0mL/min, the detection wavelength is 445nm, methanol/water is used as a mobile phase A, methyl tert-butyl ether is used as a mobile phase B, both phases contain 0.1% BHT, gradient elution is carried out for 0 min-30 min, 0% -50% B, 30 min-40 min and 50% B, under the condition, the separation effect of a trans-lutein peak 7 and an adjacent isomer peak 6 and a peak 8 is mainly examined when the methanol/water ratio of the phase A is respectively 86:14, 88:12, 90:10, 92:8, 95:5 and 100:0, and the result is shown in a figure 7-12. As can be seen from the figure, the separation degree is gradually increased along with the increase of the methanol ratio, the separation degree of the peak 7 and the peak 8 is increased from 1.5 to 5.0, the separation degree of the peak 6 is increased from 1.0 to 2.5, the retention time is reduced from 28min to 14min, the separation of the peak 7 and the adjacent isomer peak can reach the baseline separation of methanol/water from 95:5 to 100:0, the methanol/water ratio is 92-100: 8-0, the good separation effect is achieved, the methanol/water ratio is preferably selected to be 95:5 by comprehensively considering the separation of other peaks and the economical efficiency of the solvent, the separation degree is about 1.9 under the condition, and the retention time is about 22 min. In addition, A, B two phases in the national standard both contain 0.1% of BHT and are complex to prepare, so that the subsequent process only contains 0.1% of BHT in the phase A or the phase B, and the separation effect is not influenced under the condition of the invention, and the specific concentration can be adjusted according to the actual situation.

3 investigation of different flow rates

YMC Carotenoid C30 is used as a chromatographic column, methanol/water (95:5, containing 0.1% BHT) is used as a mobile phase A, methyl tert-butyl ether is used as a mobile phase B, gradient elution is carried out, the time is 0-30 min, the time is 0-50% B, the time is 30min, the detection wavelength is 445nm, the influence of the flow rates of 0.7, 0.8, 0.9, 1.0 and 1.1mL/min on the separation effect and the content of trans-lutein and isomers thereof is respectively considered, the result shows that the trans-lutein can achieve baseline separation with adjacent isomers under different flow rates, and the content has no obvious difference, which indicates that the method has good durability in the flow rate range of 0.7 mL/min-1.1 mL/min. Considering the separation degree, retention time and the separation of the remaining isomers in combination, the flow rate of 0.8mL/min is preferably selected, as shown in FIG. 13, under the conditions, the retention time is 23.6min, the separation degree is 1.93, the symmetry factor is 1.06 and the number of theoretical plates is 79684.

4 investigation of different column temperatures

Under the conditions, the influence of the column temperature of 25 ℃, 30 ℃, 35 ℃ and 40 ℃ on the separation effect and content of trans-lutein and adjacent isomers is respectively considered, and the results show that the trans-lutein has better separation effect with the adjacent isomers under different column temperatures, and the content has no obvious difference, which indicates that the method has good durability in the range of 25 ℃ to 40 ℃ of the column temperature, the common temperature of 30 ℃ is preferably selected, as shown in figure 14, the retention time under the conditions is 23.5min, the separation degree is 1.92, the symmetry factor is 1.06, and the theoretical plate number is 82472.

5 investigation of different instruments

Under the conditions, the influence of instruments of three brands of Shimadzu (Shimadzu-20AT), Agilent-1260 and Dionex Ultimate 3000 on the separation effect and content of trans-lutein and isomers thereof is respectively considered, and as a result, the lutein can be well separated by adopting the instruments of the three brands, and the content has no obvious difference, which indicates that the instrument has good durability.

6 chromatographic peak purity verification

The chromatographic conditions for lutein obtained by the above investigation were: the chromatographic column is YMC Carotenoid C30(250 mm. times.4.6 mm,5 μm); gradient elution is carried out by taking methanol/water (95:5, containing 0.1 percent of BHT) as a mobile phase A and methyl tert-butyl ether as a mobile phase B for 0 min-30 min, 0 percent-50 percent of B, 30 min-40 min and 50 percent of B; the flow rate is 0.8 mL/min; the column temperature is 30 ℃; the detection wave is 445 nm; the sample size is 10 ul. Under the condition, the DAD detector is adopted to verify the peak purity of the trans-lutein peak 7 in the chromatogram of the test sample, the result is shown in figure 15, the purity angle is smaller than the purity threshold value, and the purity of the lutein chromatogram peak meets the requirement.

EXAMPLE 2 selection of test article preparation methods

Firstly, a 2.1 national standard method is adopted to prepare a sample solution, the result shows that no peak of lutein is detected, and it is presumed that lutein particles are likely to be microcapsule inclusion compounds, so that lutein components cannot be extracted by adopting a national standard low-polarity organic solvent.

1 selection of Membrane rupture solvent

And (3) investigating the influence of water, DMF and absolute ethyl alcohol as a membrane breaking solvent on the content of all-trans lutein. Weighing lutein particles (2017120608 batches) about 300mg, precisely weighing, placing in a 100mL brown volumetric flask, respectively adding 5mL of water, DMF and absolute ethyl alcohol, carrying out ultrasonic treatment (power 100W) at room temperature for 20min, adding absolute ethyl alcohol containing 0.1% BHT to approach to the scale, carrying out ultrasonic treatment (power 100W) for 5min, cooling to room temperature, carrying out constant volume treatment to the scale by using the absolute ethyl alcohol containing 0.1% BHT, precisely weighing 1mL to 10mL brown volumetric flask, diluting the absolute ethyl alcohol containing 0.1% BHT to the scale, shaking up, and centrifuging to obtain the lutein nanoparticle. The results are shown in Table 1. The result shows that when water is used as a membrane breaking solvent, the content of all-trans-lutein is obviously higher than that of DMF and absolute ethyl alcohol, and the cost is low. Therefore, water is preferred as the membrane breaking solvent.

TABLE 1 Effect of different membrane-breaking solvents on the content of all-trans-lutein

2 selection of Membrane rupture temperature

The influence of the ultrasonic temperatures of 20 ℃, 30 ℃, 40 ℃ and 60 ℃ on the content of the all-trans lutein is examined, and the results are shown in Table 2. The results show that different membrane rupture temperatures have no obvious difference on the content of the all-trans-lutein, but the content tends to decrease with the increase of the temperature, and the room temperature is preferably used as the ultrasonic temperature by comprehensively considering the sensitivity of the temperature to the lutein and the convenience of operation.

TABLE 2 Effect of Membrane rupture temperature on Total Trans lutein content

3 selection of the amount of Membrane-rupturing solvent

The influence of the volumes of the membrane breaking solvent water, namely 2mL, 5mL, 7.5mL and 10mL, on the content of all-trans lutein was examined, and the results are shown in Table 3. The result shows that different amounts of the membrane breaking solvent have no obvious difference on the content of the all-trans lutein, and 5mL is finally selected as the amount of the membrane breaking solvent in order to ensure sufficient membrane breaking and the solubility of the all-trans lutein.

TABLE 3 Effect of different amounts of membrane-breaking solvent on the lutein content

4 selection of Membrane rupture time

And (3) investigating the influence of the membrane breaking time of 5min, 10min, 20min and 30min on the content of the all-trans-lutein, wherein the results are shown in table 4, and the results show that different membrane breaking times have no obvious influence on the content of the all-trans-lutein, but the content of the membrane breaking time of 10min and 20min is relatively high, and finally 20min is selected as the membrane breaking time in order to ensure sufficient membrane breaking.

TABLE 4 Effect of different Membrane rupture times on the lutein content

5 selection of Anhydrous ethanol extraction time

The influence of the absolute ethanol extraction time of 0min, 5min, 10min and 20min on the content of all-trans lutein is examined, and the results are shown in Table 5. The results show that different extraction times of absolute ethanol have no significant effect on the lutein content. Considering that the ultrasound helps the lutein to disperse evenly and save time, 5min is finally selected as the extraction time of the anhydrous ethanol.

TABLE 5 Effect of Anhydrous ethanol extraction time on lutein content

Through the investigation, the preparation method of the test solution comprises the following steps: taking about 300mg of lutein particles, precisely weighing, placing in a 100mL brown volumetric flask, adding 5mL of water, carrying out ultrasonic treatment (power 100W) for 20min at room temperature, adding absolute ethyl alcohol containing 0.1% BHT to approximate scale, carrying out ultrasonic treatment (power 100W) for 5min, cooling to room temperature, carrying out constant volume to scale by using absolute ethyl alcohol containing 0.1% BHT, precisely weighing 1mL to 10mL brown volumetric flask, diluting with absolute ethyl alcohol containing 0.1% BHT to scale, shaking up, and centrifuging to obtain the lutein nanoparticle.

EXAMPLE 3 qualitative study of lutein-related Compounds

1 preparation of lutein reference solution

Weighing about 5mg of xanthophyll (trans-xanthophyll) as a reference substance, precisely weighing, placing in a 50mL brown measuring bottle, adding anhydrous ethanol containing 0.1% BHT to obtain a reference substance stock solution containing 100 μ g of xanthophyll per 1mL, precisely weighing 1.2mL of the stock solution, placing in a 10mL brown measuring bottle, and diluting with anhydrous ethanol containing 0.1% BHT to obtain a reference substance solution with a concentration of 12 μ g/mL.

2 preparation of zeaxanthin control solution

Taking a zeaxanthin (namely trans-zeaxanthin) reference substance of about 5mg, precisely weighing, placing in a 50mL brown measuring flask, adding absolute ethyl alcohol containing 0.1% BHT to obtain a reference substance stock solution containing 100 mug of lutein per 1mL, precisely weighing 1.0mL of the stock solution, placing in a 50mL brown measuring flask, and diluting with absolute ethyl alcohol containing 0.1% BHT to obtain a reference substance solution with the concentration of 2 mug/mL.

3 preparation of lutein isomerization control solution

Taking about 5mg of lutein reference substance, precisely weighing the lutein reference substance in a 50mL transparent volumetric flask, adding absolute ethanol to prepare a reference substance stock solution containing 100 micrograms of lutein per 1mL, taking 1mL of the stock solution, placing the stock solution in a 10mL transparent volumetric flask, adding 0.1mL of iodine ethanol solution, diluting to scale with absolute ethanol, placing the solution in a light box, and placing the solution for 1 hour to obtain the lutein isomerized reference substance mixed solution.

4 preparation of test solutions

The test solution was prepared according to the method for preparing the test solution optimized in example 2.

5 determination of

The above-mentioned control solutions and test solutions were each precisely aspirated and measured by HPLC with a DAD detector.

6 results

HPLC spectra of the xanthophyll reference substance, zeaxanthin reference substance, xanthophyll isomerization reference substance solution and test solution are shown in figures 16-19 (the figure is a partial enlarged view), and DAD full-wave scanning spectrum of each compound is shown in figures 20-31. Isomers can be preliminarily identified by ultraviolet spectral characteristics: compared with all-trans lutein, the maximum absorption wavelength of the single cis-isomer generally has a blue shift of 4-6 nm, and the maximum absorption wavelength of the double cis-isomer has a blue shift of 8-12 nm; secondly, the single cis-isomer has cis-absorption between 330nm and 340nm, and the closer the cis-double bond is to the center of the molecule, the larger the cis-absorption (usually, Q value represents the intensity of cis-absorption peak); finally, lutein and B-carotene are carotenoids, all sharing a common isoprene structure, so that the elution order of their respective positional isomers is consistent. Therefore, based on the comparison of the control, the ultraviolet spectral characteristics and the elution order, 9 compound structures were determined and 3 compounds not reported in the literature were estimated, and parameters such as the ultraviolet absorption wavelength, the Q value, and the peak area of each peak are shown in table 6, and the structures of the compounds are shown in fig. 32. The method comprises the following specific steps:

peak 7: firstly, the HPLC retention time of the sample solution is consistent with that of a lutein reference solution, and the sample solution can be preliminarily judged to be all-trans lutein; secondly, the measured values of the absorption peaks are 420nm, 444nm and 472nm, and the heights of the measured values are consistent with those of reported 423nm, 444nm and 472 nm; further, Q value measurement of 0.06 is highly consistent with the reported value of 0.05. Therefore, the peak 7 is comprehensively judged to be the compound all-trans lutein, and the structure is shown in e.

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

Peak 3: firstly, the measured values of absorption peaks are 330nm, 412nm, 438nm and 464nm, wherein the maximum absorption peak 438nm is subjected to blue shift of 6nm compared with the maximum absorption peak 444nm of trans-lutein, and the maximum absorption wavelength of the single cis-isomer is generally subjected to blue shift of 4-6 nm according to the report of the literature, so that the compound is concluded to be the single cis-lutein; the heights of the four absorption peaks are consistent with the reported heights of 330nm, 412nm, 436nm and 462 nm; additionally, Q value measurement of 0.47 is highly consistent with reported 0.46; finally, referring to the elution order of carotene isomers, peak 3 was judged comprehensively as compound 13-cis-lutein with structure a.

Peak 5: firstly, the measured values of absorption peaks are 330nm, 414nm, 438nm and 466nm, wherein the maximum absorption peak 438nm generates 6nm blue shift compared with the maximum absorption peak 444nm of trans-lutein, and the maximum absorption wavelength of the monocistronic isomer generally has 4-6 nm blue shift, so that the compound is concluded to be monocistronic lutein; the heights of the four absorption peaks are consistent with those of reported 330nm, 414nm, 438nm and 464 nm; additionally, Q value measurement of 0.49 is highly consistent with reported 0.45; finally, the peak 5 is comprehensively judged to be the compound 13' -cis-lutein according to the elution sequence of carotene isomers, and the structure is shown in b.

Peak 6: firstly, the measured values of absorption peaks are 330nm, 420nm, 444nm and 474nm, wherein the maximum absorption peak 444nm is compared with the maximum absorption peak 450nm of trans-zeaxanthin, and the single cis-zeaxanthin is preliminarily judged as the single cis-zeaxanthin because the single cis-isomer has a blue shift of 4-6 nm; secondly, the heights of the four absorption peaks are consistent with those of the reported 331nm, 444nm and 473nm and those of the reported 338nm, 444nm and 470 nm; in addition, Q value measurement of 0.10 substantially agreed with reported 0.43 and reported 0.49; finally, the peak 6 is determined to be 13 or 13' -cis-zeaxanthin comprehensively according to the elution order of carotene isomers. The structure is shown in c and d.

Peak 9: firstly, the measured values of absorption peaks are 332nm, 420nm, 438nm and 468nm, wherein the maximum absorption peak 438 is subjected to blue shift of 6nm compared with the maximum absorption peak 444nm of trans-lutein, and the compound is concluded to be the monochamlutein due to the fact that the maximum absorption wavelength of the monochamisomer is generally subjected to blue shift of 4-6 nm; the second four absorption peaks are consistent with the known heights of 332nm, 418nm, 440nm and 466 nm; further, a Q value of 0.09 was found to be highly consistent with the reported 0.10; finally, the elution sequence is combined, and the peak 9 is comprehensively judged to be the compound 9-cis-lutein, and the structure is shown in h.

Peak 10: firstly, the measured values of absorption peaks are 330nm, 420nm, 442nm and 466nm, wherein the maximum absorption peak 442nm is subjected to blue shift of 2nm compared with the maximum absorption peak 444nm of trans-lutein, and is basically consistent with the blue shift of 4-6 nm of the maximum absorption wavelength of the known single cis-isomer, and the compound is concluded to be the single cis-lutein; the second four absorption peaks are consistent with the known heights of 332nm, 420nm, 444nm and 472nm and the known heights of 330nm, 422nm, 440nm and 468 nm; further, Q value measurement of 0.09 is highly consistent with reported 0.05 and reported 0.11; finally, the elution sequence is combined, and the peak 10 is comprehensively judged to be the compound 9' -cis-lutein with the structure shown in the specification i.

Peak 11: firstly, the measured value of an absorption peak is 338nm, 446nm and 474nm, wherein the maximum absorption peak 446 generates a blue shift of 4nm compared with the maximum absorption peak 450nm of trans-zeaxanthin, and the single cis-zeaxanthin is preliminarily judged as the single cis-zeaxanthin because the single cis-isomer has the blue shift of 4-6 nm; the heights of the second three absorption peaks are consistent with those of reported 337nm, 445nm and 473nm and those of reported 338nm, 445nm and 472 nm; further, the Q value was found to be 0.09, highly consistent with the reported 0.14 and the reported 0.10; finally, the elution order is combined, and the peak 11 is comprehensively judged to be 9-cis zeaxanthin.

Peak 12: firstly, the measured value of an absorption peak is 338nm, 446nm and 472nm, wherein the maximum absorption peak 446 generates a blue shift of 4nm compared with the maximum absorption peak 450nm of trans-zeaxanthin, and the single cis-zeaxanthin is preliminarily judged as the single cis-zeaxanthin because the single cis-isomer has the blue shift of 4-6 nm; the heights of the second three absorption peaks are consistent with those of reported 337nm, 445nm and 473nm and those of reported 338nm, 445nm and 472 nm; further, Q value measurement of 0.06 substantially agreed with reported values of 0.14 and 0.10; finally, the peak 12 is comprehensively judged to be 9' -cis-zeaxanthin by combining the elution sequence.

Peak 1: the measured values of the absorption peaks are 330nm, 410nm, 430nm and 458nm, wherein the maximum absorption peak 430nm is subjected to blue shift of 14nm compared with the maximum absorption peak 444nm of trans-lutein, and the bi-cis-lutein is preliminarily judged to be bi-cis-lutein by combining the peak appearance sequence due to the blue shift of 8-12 nm of the bi-cis-isomer.

Peak 2: the measured values of the absorption peaks are 332nm, 410nm, 432nm and 458nm, wherein the maximum absorption peak 432nm is compared with the maximum absorption peak 444nm of trans-lutein, a 12nm blue shift occurs, and the bicis-form lutein is preliminarily judged by combining the peak appearance sequence due to the 8-12 nm blue shift of the bicis-form isomer.

Peak 4: the measured values of the absorption peaks are 330nm, 418nm, 440nm and 470nm, wherein the maximum absorption peak 440nm is subjected to 10nm blue shift compared with the maximum absorption peak 450nm of trans-zeaxanthin, and the bicistronic isomer has 8-12 nm blue shift, so that the bicistronic zeaxanthin is preliminarily judged by combining the peak appearance sequence.

TABLE 6 identification of lutein and its isomers

Each compound has the following structure:

example 4 validation of the quantitative methodology of trans-lutein

1 system applicability

A lutein control solution and a test solution were prepared according to the method of example 3, respectively. Continuously feeding a reference substance solution for 5 times for determination, recording peak areas of components to be measured, and calculating RSD; continuously feeding sample solution for 5 times, and recording theoretical plate number, separation degree and symmetry factor of the component to be measured. The results show that the separation degree of the lutein is more than 1.8, the baseline separation is achieved, the symmetry factor is 1.07 (basically meeting the requirement of 0.95-1.05), the number of theoretical plates is more than 80000, the accuracy of quantitative analysis is ensured, the RSD of the peak area is not less than 5000, the RSD of the peak area is 0.26 percent and less than 2 percent, and the system applicability is better.

2 specialization examination

Preparation of control solutions: the preparation of the lutein control solution of example 3 was followed.

Preparation of a test solution: the test solution was prepared according to the method optimized in example 2.

Preparation of negative test solution: a negative sample powder was taken and a negative sample solution was prepared according to the method for preparing a sample solution in example 2.

The control solution, the test solution and the negative test solution were taken and subjected to the chromatographic conditions as preferred in example 1, as shown in FIGS. 32 to 34. As can be seen from the figure, the chromatogram of the test sample shows a chromatographic peak which is consistent with the retention time of the main peak of the chromatogram of the reference sample, and the chromatograms of the blank solvent and the negative test sample basically have no impurity peak at the retention time of the component to be detected. The method is proved to have good specificity.

3 linearity and Range

Taking a proper amount of lutein reference substance, precisely weighing, and adding anhydrous ethanol containing 0.1% BHT to obtain reference substance stock solution containing 96.5739 μ g of lutein per 1 mL. The stock solutions were precisely measured in 10mL brown flasks at 0.2mL, 0.4mL, 0.8mL, 1.2mL, 1.6mL, and 2.0mL, and diluted with 0.1% BHT in absolute ethanol to give control solutions of the respective concentrations of 1.93, 3.86, 7.73, 11.59, 15.45, and 19.31. mu.g/mL.

Respectively sucking 10 μ L of the above solutions, injecting sample according to the chromatographic conditions of example 2, plotting with the concentration of the control solution (μ g/mL) as abscissa (X) and the peak area as ordinate (Y), and drawing a standard curve to obtain a regression equation: y is 156255X-7493.5 (R2 is 1), and the result shows that the linear relation is good in a linear range of 1.93-19.31 mu g/mL.

4 detection limit and quantification limit

A control solution (1.93 mu g/mL) is diluted step by step, the concentration corresponding to 10 times of baseline noise (S/N is 10) is taken as a quantification limit, the concentration corresponding to 3 times of baseline noise (S/N is 3) is taken as a detection limit, the quantification limit of trans-lutein obtained by the method is 0.019 mu g/mL, and the detection limit is 0.004 mu g/mL.

5 precision test

And continuously feeding a linear intermediate concentration (11.59 mu g/mL) reference substance solution and a test sample solution for 6 times, measuring peak areas, calculating RSD, and indicating that the RSD of the reference substance and the test sample with peak area precision is less than 2% and the RSD of retention time precision is less than 2%, thus the instrument precision is good.

6 stability test

Respectively taking the same reference substance solution and the same test substance solution, respectively carrying out sample injection once in 0h, 1h, 2h, 3h, 4h, 5h, 6h, 9h, 12h, 15h, 19h and 24h, measuring the peak area, and calculating the RSD, wherein the RSD of the reference substance solution peak area is 0.44%, and the RSD of the test substance solution peak area is 0.52%, and are all less than 2%. The stability of the reference solution and the test solution is good within 24 hours.

7 repeatability test

Taking the same batch of samples, preparing 6 test sample solutions according to the method of example 2, measuring, and calculating the sample content and RSD, wherein the average content of the trans-lutein is 5.71%, the RSD is 0.92%, and is less than 2%. Indicating that the method has good repeatability.

8 sample recovery test

Taking appropriate amount of xanthophyll control, adding 0.1% BHT anhydrous alcohol to make into control solution containing 0.6048mg trans-xanthophyll per 1 mL.

Taking 150mg of the product powder, precisely weighing, placing in a 100mL brown volumetric flask, adding 1mL of the above reference substance (the amount of the lutein added in the reference substance is equivalent to that of the sample), preparing according to the preparation method of the sample solution, measuring the peak area of trans-lutein, and calculating the sample recovery rate and RSD, wherein the results are shown in Table 7. The average recovery of trans-lutein was 101.46% with an RSD of 0.82%, less than 2%. The method is proved to be good in accuracy.

EXAMPLE 5 quantitative determination of Trans-lutein in three batches of lutein microparticles

1 preparation of control solutions

Taking a proper amount of the lutein reference substance, precisely weighing, and adding absolute ethanol containing 0.1% BHT to prepare a solution containing 100 μ g of lutein per 1mL to obtain the reference substance stock solution. 0.2, 0.4, 0.8, 1.2, 1.6 and 2.0mL of the stock solutions are respectively taken and put into a 10mL measuring flask, and 0.1 percent of absolute ethyl alcohol of BHT is added to dilute the stock solutions into control solution with the concentration of 2, 4, 8, 12, 16 and 20 mu g/mL respectively.

2 preparation of test solution

Weighing about 300mg of three batches of lutein particles (2017120607, 2017120608 and 2018010602), precisely weighing, placing in a 100mL brown volumetric flask, adding 5mL of water, carrying out ultrasonic treatment (power 100W) at room temperature for 20min, adding absolute ethyl alcohol containing 0.1% BHT to approach the scale, carrying out ultrasonic treatment (power 100W) for 5min, cooling to room temperature, fixing the volume to the scale by using the absolute ethyl alcohol containing 0.1% BHT, precisely weighing 1mL to 10mL brown volumetric flask, diluting the absolute ethyl alcohol containing 0.1% BHT to the scale, shaking up and centrifuging to obtain the lutein nanoparticle.

3 chromatographic conditions

Using YMC Carotenoid C30(250mm multiplied by 4.6mm,5 μm) as a chromatographic column; gradient elution is carried out by taking methanol/water (95:5, containing 0.1 percent of BHT) as a mobile phase A and methyl tert-butyl ether as a mobile phase B, wherein the gradient elution is carried out for 0min to 30min, 0 percent to 50 percent of B, 30min to 40min and 50 percent of B; the flow rate was 0.8mL/min, the column temperature was 30 ℃ and the detection wavelength was 445 nm. The theoretical plate number is not less than 5000 calculated according to trans-lutein peak.

4 determination of

Precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

5 results

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

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

1 preparation of control solution the same preparation of lutein control solution as in example 5 was carried out.

2 preparation of test solution

Taking three batches of Meiling tablets (the batches are 20190101, 20190102 and 20190103 respectively), removing coatings, grinding, respectively taking about 300mg of powder, precisely weighing, placing in a 100mL brown volumetric flask, adding 5mL of water, carrying out ultrasonic treatment (power of 100W) for 20min at room temperature, adding absolute ethyl alcohol containing 0.1% BHT to approach the scale, carrying out ultrasonic treatment (power of 100W) for 5min, cooling to room temperature, carrying out constant volume treatment to the scale by using the absolute ethyl alcohol containing 0.1% BHT, shaking up and centrifuging to obtain the Meiling tablets.

3 chromatographic conditions

The same chromatographic conditions as in example 5.

4 determination of

Precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

5 results

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

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (10)

1. A method for measuring a lutein component is characterized by adopting high performance liquid chromatography detection, wherein the chromatographic conditions comprise: YMC Carotenoid C30 is used as a chromatographic column; methanol/water is taken as a mobile phase A, and methyl tert-butyl ether is taken as a mobile phase B; the elution procedure is 0min to 30min, 0 percent to 50 percent of B, 30min to 40min and 50 percent of B; the ratio of methanol to water is 88-100: 12-0.

2. The method of claim 1, wherein the ratio of methanol to water is 92-100: 8-0.

3. The method of claim 1, wherein the methanol/water ratio is 95: 5.

4. The process according to claim 1, wherein the mobile phase a and/or the mobile phase B comprises 2, 6-di-tert-butyl-p-cresol.

5. The method of claim 1, wherein the concentration of 2, 6-di-tert-butyl-p-cresol is 0.1%.

6. The method of claim 1, wherein the chromatographic conditions further comprise: the flow rate is 0.7mL/min to 1.1 mL/min; the column temperature is 25-40 ℃.

7. The method of claim 1,

using YMC Carotenoid C30 with specification of 250mm × 4.6mm and 5 μm as chromatographic column; taking methanol/water solution containing 0.1 percent of 2, 6-di-tert-butyl-p-cresol and the ratio of 95:5 as a mobile phase A and methyl tert-butyl ether as a mobile phase B, and performing elution procedures of 0 min-30 min, 0 percent-50 percent B, 30 min-40 min and 50 percent B; the flow rate was 0.8mL/min, the column temperature was 30 ℃ and the detection wavelength was 445 nm.

8. The method of claim 1, wherein the xanthophyll constituent comprises all-trans-lutein.

9. A method for detecting a xanthophyll starting material or a sample containing xanthophylls, the method comprising:

preparation of a test solution: grinding a lutein raw material or a sample containing lutein, putting the powder into a volumetric flask, adding water, performing ultrasonic treatment at room temperature, adding absolute ethyl alcohol containing BHT, performing ultrasonic treatment, cooling to room temperature, and fixing the volume;

the test solution is tested by the method of any one of claims 1 to 8.

10. The method of claim 9, further comprising:

preparation of control solutions: taking a lutein reference substance, adding absolute ethyl alcohol containing BHT to prepare stock solution containing lutein, and preparing reference substance solutions with various concentrations by using the stock solution;

the control solution is tested by the method of any one of claims 1 to 8.

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