CN112578047A - High performance liquid chromatography detection method for xanthophyll and zeaxanthin content - Google Patents
High performance liquid chromatography detection method for xanthophyll and zeaxanthin content Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention discloses a high performance liquid chromatography detection method for lutein and zeaxanthin content, which can realize better dissolution of lutein and zeaxanthin by dissolving a sample with dichloromethane, can completely dissolve the lutein and zeaxanthin in the sample at one time only by short ultrasonic treatment, and can fix the volume to the scale with the dichloromethane, thereby shortening the sample treatment time; the lutein and zeaxanthin with various structures can be effectively separated, and the separation degree is more than 1.5; the method for determining the contents of the lutein and the zeaxanthin by using the high performance liquid chromatography is used for simply and accurately determining the contents of the lutein and the zeaxanthin in a single-component sample and a complex-component sample by selecting a sample treatment process and chromatographic conditions. The method can replace the traditional national standard method to measure the contents of the lutein and the zeaxanthin, and is simple, convenient, rapid and accurate.
Description
Technical Field
The invention relates to the technical field of chromatographic detection methods, in particular to a high performance liquid chromatography detection method for lutein and zeaxanthin content.
Background
The lutein can absorb blue-violet light and can help retina of eye to resist ultraviolet. Lutein is an important antioxidant for the eye to play a role. The human body can maintain the vision persistence by supplementing a large amount of lutein, improve the visual reaction time and reduce the visual injury. For the myopia, the increase of the myopia degree can be delayed by supplementing lutein, and the damage of computer radiation to the human body can be effectively prevented by frequently using the lutein. But also widely used in the processing of seasonings, tobacco, pastry, candy and various feeds. In our country, lutein has been widely used as a colorant. The xanthophyll also has strong antioxidant ability, and can inhibit the activity of oxygen free radicals and prevent the damage of the oxygen free radicals to normal cells. In addition, the lutein has unique biological efficacy in the aspect of inhibiting tumor growth, and the mechanism of the lutein mainly comprises antioxidant activity, tumor vascular proliferation inhibition, cell proliferation inhibition and the like. The lutein can be used as an effective adjuvant to enhance the function of insulin in reducing blood sugar. Therefore, the risk of diabetes can be reduced by eating foods such as vegetables and fruits rich in lutein. The zeaxanthin has the health effects of resisting oxidation, preventing macular degeneration, treating cataract, preventing cardiovascular diseases, enhancing the immunity of the organism, relieving atherosclerosis and the like, and is closely related to the health of human beings.
In the known determination method of the contents of lutein and zeaxanthin, a GB5009.248-2016 method is mainly used, but the analysis of the national standard method has limitations, the detection process is relatively complicated, a neutral alumina solid phase extraction column is required for extraction and filtration, and in order to solve the problem, the method simplifies the detection process and rapidly and accurately analyzes the contents of lutein and zeaxanthin by using a high performance liquid chromatography.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a high performance liquid chromatography detection method for the contents of lutein and zeaxanthin.
The technical scheme of the invention is as follows:
a high performance liquid chromatography detection method for the contents of lutein and zeaxanthin comprises the following steps:
(1) preparing a reference substance solution:
preparing a standard stock solution: precisely weighing 125.0mg of lutein or zeaxanthin reference substance in a 50mL volumetric flask, adding 30mL of dichloromethane, ultrasonically dissolving, and diluting with dichloromethane to scale;
drawing a standard working curve: taking 0.2ml, 1.0ml, 2.0ml, 3.0ml and 4.0ml to 50ml volumetric flasks of the stock solution of the standard substance, diluting the flask by using 30ppm of BHT ethyl acetate solution as a diluent to prepare 5 standard working solutions with different concentrations, filtering the solution by using a filter membrane, and injecting the solution into HPLC;
(2) sample treatment:
weighing a sample with the concentration equivalent to the middle point of the curve, placing the sample in a 100ml volumetric flask, adding a proper amount of dichloromethane for ultrasonic dissolution, using dichloromethane for constant volume till the volume is scaled, and taking the obtained solution to be centrifuged in a 50ml centrifugal tube; precisely placing 5.0ml of supernatant in a 50ml volumetric flask, diluting to scale with 30ppmBHT ethyl acetate solution, and mixing; filtering with filter membrane, and injecting into HPLC;
(3) liquid chromatography detection:
respectively injecting the standard solution and the sample solution into a high performance liquid chromatography system to detect the measurement coefficient R of the standard curve2≥0.999；
(4) And (3) calculating a detection result:
and respectively calculating according to the standard curves of the lutein and the zeaxanthin to respectively obtain the concentrations of the lutein and the zeaxanthin in the sample.
Preferably, in the step of processing the sample, the rotation speed of 2500-.
More preferably, in the centrifugal treatment process, a rotating speed of 3000rpm is adopted, and the treatment time is 15 min.
Preferably, in the step of drawing the standard working curve and processing the sample, the filter membrane is 0.45um filter membrane.
Preferably, in the liquid chromatography detection process, the conditions of the liquid chromatography detection are as follows: a chromatographic column: YMCC30 column, 250X 4.6mm, 5 μm; flow rate: 1.5mL/min, detection wavelength: 450nm, sample size ((A)): 20 μ L, column temperature: at 30 ℃, mobile phase: methanol: dichloromethane =80: 20.
preferably, in the calculation process of the detection result, the following formulas are adopted to calculate the concentrations of lutein and zeaxanthin in the sample:
X=C*V*K/M*m
in the formula: x: the content of lutein or zeaxanthin in the sample, mg/g;
c: calculating the concentration of lutein or zeaxanthin in the obtained sample according to the corresponding standard curve, mg/mL;
v: the volume of the sample is determined;
k: sample dilution factor;
m: sample weighing, g;
m: and (4) loading the weight on average.
The invention has the advantages that:
the method for determining the contents of the lutein and the zeaxanthin by using the high performance liquid chromatography is adopted, and the selection of the sample treatment process and the chromatographic conditions finally achieves the purpose of simply, conveniently and accurately determining the contents of the lutein and the zeaxanthin in the samples with single components and complex components. The method can replace the traditional national standard method to measure the contents of the lutein and the zeaxanthin, and is simple, convenient, rapid and accurate.
The invention adopts a system of dichloromethane and methanol, and has the advantages that: since dichloromethane and methanol are mutually soluble. The method is suitable for detecting pure lutein and zeaxanthin, and is also suitable for detecting lutein, zeaxanthin concentrated solution and lutein and zeaxanthin containing other complex components in dosage forms such as oil, and dichloromethane is used as solution with good solubility, so that the content can be well dissolved, and the aim of completely extracting the main components can be achieved. And the addition of the methanol can well eliminate the interference generated by different polarities of a solution and a mobile phase required by sample dissolution in the analysis process of an instrument.
Compared with the prior art, the invention has the following remarkable advantages:
1. the high performance liquid chromatography detection method for the contents of lutein and zeaxanthin is suitable for any dosage form (soft capsules, tablets and the like);
2. according to the detection method, the sample is dissolved by the dichloromethane, so that lutein and zeaxanthin can be well dissolved, the lutein and zeaxanthin in the sample can be completely dissolved at one time only by short ultrasonic treatment, and the volume is determined to the scale by the dichloromethane, so that the sample treatment time is shortened;
3. the method can effectively separate the lutein and the zeaxanthin with various structures, and the separation degree is more than 1.5;
4. the sample analysis time is shortened, and the sample detection efficiency is improved. The traditional method needs about 30min for running the program once, and the method can complete the running of the program once only by 10 min.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.
The reagents used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1
A high performance liquid chromatography detection method for high content lutein and zeaxanthin comprises the following steps:
(1) preparing a reference substance solution:
preparing a standard stock solution: precisely weighing 125.0mg lutein oil secondary reference substance in a 50mL volumetric flask, adding 30mL dichloromethane, ultrasonically dissolving, diluting with dichloromethane to scale,
drawing a standard working curve: taking 0.2ml, 1.0ml, 2.0ml, 3.0ml and 4.0ml to 50ml volumetric flasks of the stock solution of the standard substance, diluting the flask by using 30ppm BHT ethyl acetate solution as a diluent to prepare 5 standard working solutions with different concentrations, filtering the solution by using a 0.45um filter membrane, and injecting the solution into HPLC;
(2) sample treatment:
weighing a sample equivalent to 1.2g, placing the sample in a 100ml volumetric flask, adding a proper amount of dichloromethane for ultrasonic dissolution, using dichloromethane for constant volume till the volume reaches the scale, putting the obtained solution in a 50ml centrifuge tube at 3000rpm/15 min; precisely placing 5.0ml of supernatant in a 50ml volumetric flask, diluting to scale with 30ppmBHT ethyl acetate solution, and mixing; filtering with 0.45um filter membrane, and injecting into HPLC;
(3) liquid chromatography detection:
respectively injecting the standard solution and the sample solution into a high performance liquid chromatography system to detect the measurement coefficient R of the standard curve2Not less than 0.999; the conditions of the liquid chromatography detection are as follows: a chromatographic column: YMCC30 column, 250X 4.6mm, 5 μm; flow rate: 1.5mL/min, detection wavelength: 450nm, sample size ((A)): 20 mul, column temperature: at 30 ℃, mobile phase: methanol: dichloromethane =80: 20;
(4) and (3) calculating a detection result:
calculating according to the standard curves of the lutein and the zeaxanthin to obtain the concentrations of the lutein and the zeaxanthin in the sample respectively, and calculating the concentrations of the lutein and the zeaxanthin in the sample by adopting the following formula:
X=C*V*K/M*m
in the formula: x: the content of lutein and zeaxanthin in the sample is mg/g;
c, calculating the concentration of lutein and zeaxanthin in the sample according to the corresponding standard curve, namely mg/ml;
v, the volume of the sample is determined;
k is sample dilution multiple;
m, weighing a sample, and g;
m: and (4) loading the weight on average.
As a result: the lutein content is 7.0mg/cap (required to be more than or equal to 5.0 mg/cap), and the zeaxanthin content is 1.4mg/cap (required to be more than or equal to 1.0 mg/cap).
Example 2
A high performance liquid chromatography detection method for low content lutein and zeaxanthin comprises the following steps:
(1) preparing a reference substance solution:
preparing a standard stock solution: accurately weighing 12.5mg lutein oil secondary reference substance in a 50mL volumetric flask, adding 30mL dichloromethane, ultrasonically dissolving, diluting with dichloromethane to scale,
drawing a standard working curve: taking 0.2ml, 1.0ml, 2.0ml, 3.0ml and 4.0ml to 50ml volumetric flasks of the stock solution of the standard substance, diluting the flask by using 30ppm BHT ethyl acetate solution as a diluent to prepare 5 standard working solutions with different concentrations, filtering the solution by using a 0.45um filter membrane, and injecting the solution into HPLC;
(2) sample treatment:
weighing a sample equivalent to 1.0g, placing the sample in a 100ml volumetric flask, adding a proper amount of dichloromethane for ultrasonic dissolution, using dichloromethane for constant volume till the volume reaches the scale, putting the obtained solution in a 50ml centrifugal tube, and keeping the speed at 3500rpm/12 min; precisely placing 5.0ml of supernatant in a 50ml volumetric flask, diluting to scale with 30ppmBHT ethyl acetate solution, and mixing; filtering with 0.45um filter membrane, and injecting into HPLC;
(3) liquid chromatography detection:
respectively injecting the standard solution and the sample solution into a high performance liquid chromatography system to detect the measurement coefficient R of the standard curve2Not less than 0.999; the conditions of the liquid chromatography detection are as follows: a chromatographic column: YMCC30 column, 250X 4.6mm, 5 μm; flow rate: 1.5mL/min, detection wavelength: 450nm, sample size ((A)): 20 mul, column temperature: at 30 ℃, mobile phase: methanol: dichloromethane =80: 20;
(4) and (3) calculating a detection result:
calculating according to the standard curves of the lutein and the zeaxanthin to obtain the concentrations of the lutein and the zeaxanthin in the sample respectively, and calculating the concentrations of the lutein and the zeaxanthin in the sample by adopting the following formula:
X=C*V*K/M*m
in the formula: x: the content of lutein and zeaxanthin in the sample is mg/g;
c, calculating the concentration of lutein and zeaxanthin in the sample according to the corresponding standard curve, namely mg/ml;
v, the volume of the sample is determined;
k is sample dilution multiple;
m, weighing a sample, and g;
m: and (4) loading the weight on average.
As a result: the lutein content is 0.6mg/cap (required to be more than or equal to 0.4 mg/cap), and the zeaxanthin content is 0.2mg/cap (required to be more than or equal to 0.1 mg/cap).
Example 3
A high performance liquid chromatography detection method for the content of lutein comprises the following steps:
(1) preparing a reference substance solution:
preparing a standard stock solution: precisely weighing 125.0mg lutein oil secondary reference substance in a 50mL volumetric flask, adding 30mL dichloromethane, ultrasonically dissolving, diluting with dichloromethane to scale,
drawing a standard working curve: taking 0.2ml, 1.0ml, 2.0ml, 3.0ml and 4.0ml to 50ml volumetric flasks of the stock solution of the standard substance, diluting the flask by using 30ppm BHT ethyl acetate solution as a diluent to prepare 5 standard working solutions with different concentrations, filtering the solution by using a 0.45um filter membrane, and injecting the solution into HPLC;
(2) sample treatment:
weighing a sample equivalent to 1.2g, placing the sample in a 100ml volumetric flask, adding a proper amount of dichloromethane for ultrasonic dissolution, using dichloromethane for constant volume till the volume reaches the scale, putting the obtained solution in a 50ml centrifuge tube at 2500rpm/20 min; precisely placing 5.0ml of supernatant in a 50ml volumetric flask, diluting to scale with 30ppmBHT ethyl acetate solution, and mixing; filtering with 0.45um filter membrane, and injecting into HPLC;
(3) liquid chromatography detection:
respectively injecting the standard solution and the sample solution into a high performance liquid chromatography system to detect the measurement coefficient R of the standard curve2Not less than 0.999; the conditions of the liquid chromatography detection are as follows: a chromatographic column: YMCC30 column, 250X 4.6mm, 5 μm; flow rate: 1.5mL/min, detection wavelength: 450nm, sample size ((A)): 20 mul, column temperature: at 30 ℃, mobile phase: methanol: dichloromethane =80: 20;
(4) and (3) calculating a detection result:
calculating to obtain the concentration of the lutein in the sample according to the standard curve of the lutein, and calculating the concentration of the lutein in the sample by adopting the following formula:
X=C*V*K/M*m
in the formula: x: the content of lutein in the sample, mg/g;
c, calculating the concentration of the lutein in the sample, mg/ml, according to the corresponding standard curve;
v, the volume of the sample is determined;
k is sample dilution multiple;
m, weighing a sample, and g;
m: and (4) loading the weight on average.
As a result: the lutein content is 5.9mg/cap (required to be more than or equal to 4.5 mg/cap).
Example 4
A high performance liquid chromatography detection method for zeaxanthin content comprises the following steps:
(1) preparing a reference substance solution:
preparing a standard stock solution: accurately weighing 12.5mg zeaxanthin reference substance in a 50mL volumetric flask, adding 30mL dichloromethane, dissolving with ultrasound, diluting with dichloromethane to scale,
drawing a standard working curve: taking 0.2ml, 1.0ml, 2.0ml, 3.0ml and 4.0ml to 50ml volumetric flasks of the stock solution of the standard substance, diluting the flask by using 30ppm BHT ethyl acetate solution as a diluent to prepare 5 standard working solutions with different concentrations, filtering the solution by using a 0.45um filter membrane, and injecting the solution into HPLC;
(2) sample treatment:
weighing 1.0g of sample, placing the sample in a 100ml volumetric flask, adding a proper amount of dichloromethane for ultrasonic dissolution, using dichloromethane for constant volume to scale, placing the obtained solution in a 50ml centrifugal tube at 3000rpm/15 min; precisely placing 5.0ml of supernatant in a 50ml volumetric flask, diluting to scale with 30ppmBHT ethyl acetate solution, and mixing; filtering with 0.45um filter membrane, and injecting into HPLC;
(3) liquid chromatography detection:
respectively injecting the standard solution and the sample solution into a high performance liquid chromatography system to detect the measurement coefficient R of the standard curve2Not less than 0.999; the conditions of the liquid chromatography detection are as follows: a chromatographic column: YMCC30 column, 250X 4.6mm, 5. mu.m; flow rate: 1.5mL/min, detection wavelength: 450nm, sample size ((A)): 20 mul, column temperature: at 30 ℃, mobile phase: methanol: dichloromethane =80: 20;
(4) and (3) calculating a detection result:
calculating the concentration of the zeaxanthin in the sample according to the standard curve of the zeaxanthin, and calculating the concentration of the zeaxanthin in the sample by adopting the following formula:
X=C*V*K/M*m
in the formula: x: the content of zeaxanthin in the sample, mg/g;
c, calculating the concentration of the zeaxanthin in the sample, mg/ml, according to the corresponding standard curve;
v, the volume of the sample is determined;
k is sample dilution multiple;
m, weighing a sample, and g;
m: and (4) loading the weight on average.
As a result: the zeaxanthin content is 0.8mg/cap (required to be more than or equal to 0.5 mg/cap).
The following results were obtained by comparing the HPLC detection methods of examples 1 to 4 with those of GB5009.248-2016, and are shown in Table 1.
TABLE 1
| Whether extraction filtration is required | Operating time min | |
| Method of examples 1 to 4 | Whether or not | 20-30min |
| GB5009.248-2016 method | Is that | 50-60min |
The following results were obtained from the above comparison: the high performance liquid chromatography detection method for determining the contents of lutein and zeaxanthin is simple in procedure and remarkably shortened in operation time.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (6)
1. A high performance liquid chromatography detection method for the contents of lutein and zeaxanthin is characterized by comprising the following steps:
(1) preparing a reference substance solution:
preparing a standard stock solution: precisely weighing 125.0mg of lutein or zeaxanthin reference substance in a 50mL volumetric flask, adding 30mL of dichloromethane, ultrasonically dissolving, and diluting with dichloromethane to scale;
drawing a standard working curve: taking 0.2ml, 1.0ml, 2.0ml, 3.0ml and 4.0ml to 50ml volumetric flasks of the stock solution of the standard substance, diluting the flask by using 30ppm of BHT ethyl acetate solution as a diluent to prepare 5 standard working solutions with different concentrations, filtering the solution by using a filter membrane, and injecting the solution into HPLC;
(2) sample treatment:
weighing a sample with the concentration equivalent to the middle point of the curve, placing the sample in a 100ml volumetric flask, adding a proper amount of dichloromethane for ultrasonic dissolution, using dichloromethane for constant volume till the volume is scaled, and taking the obtained solution to be centrifuged in a 50ml centrifugal tube; precisely placing 5.0ml of supernatant in a 50ml volumetric flask, diluting to scale with 30ppmBHT ethyl acetate solution, and mixing; filtering with filter membrane, and injecting into HPLC;
(3) liquid chromatography detection:
respectively injecting the standard solution and the sample solution into a high performance liquid chromatography system to detect the measurement coefficient R of the standard curve2≥0.999；
(4) And (3) calculating a detection result:
and respectively calculating according to the standard curves of the lutein and the zeaxanthin to respectively obtain the concentrations of the lutein and the zeaxanthin in the sample.
2. The HPLC method for detecting the xanthophyll and zeaxanthin content as claimed in claim 1, wherein in the step of processing the sample, the rotation speed of 2500-.
3. The HPLC method for detecting the contents of lutein and zeaxanthin in claim 2, wherein the rotation speed of 3000rpm is adopted during the centrifugation treatment, and the treatment time is 15 min.
4. The method for detecting the xanthophyll and zeaxanthin content according to claim 1, wherein in the step of drawing the standard working curve and processing the sample, the filter membrane is 0.45um filter membrane.
5. The high performance liquid chromatography detection method for lutein and zeaxanthin content as claimed in claim 1, wherein in the liquid chromatography detection process, the conditions of liquid chromatography detection are as follows: a chromatographic column: YMCC30 column, 250X 4.6mm, 5 μm; flow rate: 1.5mL/min, detection wavelength: 450nm, sample size ((A)): 20 μ L, column temperature: at 30 ℃, mobile phase: methanol: dichloromethane =80: 20.
6. the high performance liquid chromatography detection method for the contents of lutein and zeaxanthin of claim 1, characterized in that in the calculation process of the detection result, the following formula is adopted to calculate the concentrations of lutein and zeaxanthin in the sample:
X=C*V*K/M*m
in the formula: x: the content of lutein or zeaxanthin in the sample, mg/g;
c: calculating the concentration of lutein or zeaxanthin in the obtained sample according to the corresponding standard curve, mg/mL;
v: the volume of the sample is determined;
k: sample dilution factor;
m: sample weighing, g;
m: and (4) loading the weight on average.
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| CN114279992A (en) * | 2021-12-30 | 2022-04-05 | 广电计量检测（成都）有限公司 | Method for extracting and detecting yellow pigment in Huanggongfang pepper |
| CN114279992B (en) * | 2021-12-30 | 2024-03-22 | 广电计量检测（成都）有限公司 | Extraction and detection method of yellow pigment in yellow Gong Jiao |
| CN115078567A (en) * | 2022-05-19 | 2022-09-20 | 江苏艾兰得营养品有限公司 | Determination method of lutein ester and zeaxanthin ester |
| CN115078567B (en) * | 2022-05-19 | 2023-10-24 | 江苏艾兰得营养品有限公司 | Method for determining lutein ester and zeaxanthin ester |
| CN115078590A (en) * | 2022-07-16 | 2022-09-20 | 福建省产品质量检验研究院(福建省缺陷产品召回技术中心) | Detection method for determining 8 carotenoid species by two-dimensional liquid chromatography |
| CN115078590B (en) * | 2022-07-16 | 2023-08-04 | 福建省产品质量检验研究院(福建省缺陷产品召回技术中心) | Detection method for measuring 8 carotenoid types by two-dimensional liquid chromatography |
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