CN102175789A - HILC (high performance liquid chromatography) measuring method of beta-carotene in spiral seaweed - Google Patents
HILC (high performance liquid chromatography) measuring method of beta-carotene in spiral seaweed Download PDFInfo
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- CN102175789A CN102175789A CN2011100019076A CN201110001907A CN102175789A CN 102175789 A CN102175789 A CN 102175789A CN 2011100019076 A CN2011100019076 A CN 2011100019076A CN 201110001907 A CN201110001907 A CN 201110001907A CN 102175789 A CN102175789 A CN 102175789A
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Abstract
The invention discloses a measuring method of beta-carotene in spiral seaweed, comprising the following steps: (1) preparing a test article solution by dissolving the beta-carotene in spiral seaweed by a saponification extracting method; (2) preparing a standard curve; and (3) calculating the content of beta-carotene in the liquid to be measured by using the concentration of the standard applying liquid determined in the step (2) as an external standard. The measuring method disclosed by the invention has good separability, high sensitivity, good selectivity and specificity to the beta-carotene in spiral seaweed, can effectively remove interferences of matrix, and uses saponification extraction during the processing procedure of samples, so that the method is suitable for spiral seaweed products. The method disclosed by the invention simplifies the experimental steps for measuring the beta-carotene in spiral seaweed, reduces the error generated for a standard reserving solution and the instrumental sensitivity are not calibrated, and improves the repeatability and accuracy of the measuring result; and the measuring method also can be used for other powder products.
Description
Technical field
The present invention relates to a kind of assay method of beta carotene, particularly relate to the assay method of content beta-carotene in a kind of spirulina.
Background technology
Beta carotene (C40H56) is one of carotenoid, it also is the crocus fat-soluble compound, beta carotene can be converted to vitamin A by human body, 6 microgram beta carotenes are equivalent to 1 microgram vitamin A, beta carotene is a kind of antioxidant, has detoxication, is to safeguard the indispensable nutrient of health, anticancer, angiocardiopathy preventing, cataract and anti-oxidant on significant function is arranged, and and then prevent the aging and old and feeble multiple degenerative disorders that causes.
The assay method of beta carotene is sherwood oil-acetone extraction, alundum (Al column chromatography, high performance liquid chromatography and paper chromatography, sherwood oil-acetone extraction, this method complex operation of alundum (Al column chromatography, simultaneously because do not demarcate standard solution, easily detected value is surveyed high, paper chromatography is because pre-treatment is loaded down with trivial details, accurate with spectrophotometry not as high performance liquid chromatography, all influence accuracy as a result.
Summary of the invention
In order to solve problems of the prior art, the invention provides the assay method of content beta-carotene in a kind of spirulina, overcome in the prior art a. result is detected higher, the emulsion in the b. sample preparation, the problem of c. beta carotene degraded.
The present invention is achieved by the following technical solutions: the assay method of beta carotene in a kind of spirulina comprises the steps:
(1) preparation of need testing solution: adopt the beta carotene in the saponification extraction method dissolving spirulina, be specially: take by weighing the spirulina sample, adding ethanolic solution, ascorbic acid and potassium hydroxide solution refluxes in thermostat water bath, gained solution is transferred to the beta carotene in 30-60 ℃ of petroleum ether extraction saponification extract of separating funnel usefulness, get the upper strata petroleum ether layer, wash with water 3-5 time, discard water layer, petroleum ether layer is carried out drying by anhydrous sodium sulfate, be transferred to the volumetric flask constant volume then promptly;
(2) preparation of typical curve:
A, standard reserving solution: accurately take by weighing the beta carotene standard items in volumetric flask, add chloroform dissolving, use the sherwood oil constant volume, be mixed with the carrotene storing solution, keep in Dark Place in-18 ℃, face and use preceding demarcation;
The demarcation of standard solution: get standard reserving solution, use the normal hexane constant volume, mixing is measured its light absorption value, is blank with the normal hexane, lambda1-wavelength 450nm, and average is got in three parts of replicate determinations.Calculate solution concentration by following formula:
X: carrot concentration of standard solution, unit are every milliliter of microgram
A: light absorption value
E: beta carotene in hexane solution, lambda1-wavelength 450nm, cuvette thickness 1cm, solution concentration is the absorptivity of 1ug/ml, is 0.2368
B, get the beta carotene solution of carrotene storing solution preparation variable concentrations, go up HPLC mensuration respectively, try to achieve peak area drawing standard curve;
C, standard are used liquid: the beta carotene standard reserving solution preparation standard of measuring existing demarcation is used liquid;
(3) utilize the standard of determining among step (2) C to use the concentration of liquid to calculate the content of beta carotene in the liquid to be measured as external standard:
Prepare test sample liquid to be measured according to the described method of step (1),
The content of beta carotene in the X--sample, unit are milligram every hectogram (mg/100g)
The C-sample is measured the concentration of beta carotene in the liquid, and unit is every milliliter of microgram (ng/mL)
M-sample mass or volume, unit is gram or milliliter (g or mL).
Described step (1) spirulina sample is 0.1-1g, the volume that adds ethanol is 25-50ml, ascorbic acid concentrations is 5%-15%, volume is 3-10ml, potassium hydroxide solution concentration is 40-60%, volume is 8-15ml, and described bath temperature is 80-100 ℃, and described saponification time is 0.5-1.5h.
Beta carotene volume in described step (1) the PetroChina Company Limited. ether extraction saponification extract is 40-70ml, jolts 1-5min; The volume of institute's water is 5ml, anhydrous sodium sulfate 6-10g.
The HPLC chromatographic condition is:
Moving phase: methyl alcohol 85-95 parts by volume
Acetonitrile 5-15 parts by volume
Flow velocity is 1.2mL/min; Sample size: 10 μ L, column temperature: 30 ℃.
Beneficial effect of the present invention: assay method of the present invention is good to the beta carotene separation property in the spirulina, highly sensitive, selectivity and specificity are good, can effectively get rid of matrix interference, and in the sample preparation process, adopt saponification to extract, be applicable to the spirulina goods.
The assay method of the beta carotene of bibliographical information is sherwood oil-acetone extraction, alundum (Al column chromatography, high performance liquid chromatography and paper chromatography, sherwood oil-acetone extraction, this method complex operation of alundum (Al column chromatography, simultaneously because do not demarcate standard solution, easily testing result is surveyed high, paper chromatography is because pre-treatment is loaded down with trivial details, accurate with spectrophotometry not as high performance liquid chromatography, all influence accuracy as a result.The present invention has simplified the experimental procedure of measuring beta carotene in the spirulina, reduced because of not demarcating the accidental error that standard reserving solution and instrumental sensitivity thereof produce, improved the repeatability and the accuracy of measurement result, and this detection method can be used also in other powder series products.
Description of drawings
Fig. 1 is a content beta-carotene bioassay standard curve map in the spirulina.
Embodiment
Below will the invention will be further described by example, these descriptions will help those skilled in the art further to understand the present invention, but not limit the present invention in any form.
(1) preparation of need testing solution:
Beta carotene in A, the employing saponification extraction method dissolving spirulina, beta carotene in the spirulina is fully extracted, specific practice is: the spirulina sample that takes by weighing 0.5g, place conical flask, the ethanol that adds 30ml, adding concentration is 10% ascorbic acid 5ml, and adding concentration is 50% potassium hydroxide solution 10ml, is placed in the thermostat water bath and refluxes.Described bath temperature is 90 ℃, and described saponification time is 0.5h; From water-bath, take out cooling immediately, cessation reaction.
B, the solution that A is gone on foot gained are transferred in the separating funnel; Be the beta carotene in sherwood oil (boiling range is 30-60 ℃) the extraction saponification extract of 60ml with volume, jolt 2min, standing demix.
C, discard lower floor, wash petroleum ether layer 4 times, discard water layer with the moisture of 5ml;
D, in glass funnel, fill the 8g anhydrous sodium sulfate, petroleum ether layer is carried out drying by anhydrous sodium sulfate;
E, dried petroleum ether layer is transferred in the volumetric flask of 50ml, shifts constant volume, promptly obtain test solution with sherwood oil flushing separating funnel and anhydrous sodium sulfate;
(2) chromatographic condition:
Moving phase: methyl alcohol 90 parts by volume, acetonitrile 10 parts by volume flow velocitys are 1.2mL/min; Sample size: 10 μ L, column temperature: 30 ℃.
(3) preparation of typical curve:
A, standard reserving solution: accurately take by weighing 12.5mg beta carotene standard items in the 50ml volumetric flask, add 3-6ml chloroform dissolving, use the sherwood oil constant volume, being mixed with concentration is the carrotene storing solution of 250ug/ml, keeps in Dark Place in-18 ℃, faces and uses preceding demarcation.
The demarcation of standard solution: get standard reserving solution 50ul, be settled to 10ml with normal hexane, mixing is measured its light absorption value, and cuvette thickness is 1cm, is blank with the normal hexane, lambda1-wavelength 450nm, and average is got in three parts of replicate determinations.Calculate solution concentration by following formula:
X: carrot concentration of standard solution, unit are every milliliter of microgram
A: light absorption value
E: beta carotene in hexane solution, lambda1-wavelength 450nm, cuvette thickness 1cm, solution concentration is the absorptivity of 1ug/ml, is 0.2368
B, draw carrotene storing solution 0 μ l, 200 μ l, 400 μ l, 600 μ l, 800 μ l, the 1000 μ l of 250ug/ml in the 10ml volumetric flask respectively, add sherwood oil and be settled to scale, shake up standby, this solution content beta-carotene is respectively 0ug/mL, 5ug/mL, 10ug/mL, 15ug/mL, 20ug/mL, 25ug/mL, the machine of going up is respectively measured, and tries to achieve peak area drawing standard song.
C, standard use liquid: measure beta carotene standard reserving solution that 50 μ L now demarcate in the 10mL volumetric flask, use the sherwood oil constant volume, be mixed with the standard use liquid about 5ug/mL.
(3) utilize the standard of determining among step (2) C to use the concentration of liquid to calculate the content of beta carotene in the liquid to be measured as external standard:
Computing formula:
Prepare test sample liquid to be measured according to the described method of step (1),
The content of beta carotene in the X--sample, unit are milligram every hectogram (mg/100g)
The C-sample is measured the concentration of beta carotene in the liquid, and unit is every milliliter of microgram (μ g/mL)
M-sample mass or volume, unit is gram or milliliter (g or mL)
The measurement result of content beta-carotene in table 1 spirulina
Claims (4)
1. the HPLC assay method of beta carotene in the spirulina is characterized in that, comprises the steps:
(1) preparation of need testing solution: adopt the beta carotene in the saponification extraction method dissolving spirulina, be specially: take by weighing the spirulina sample, adding ethanolic solution, ascorbic acid and potassium hydroxide solution refluxes in thermostat water bath, it is the 30-60 ℃ of beta carotene in the petroleum ether extraction saponification extract that gained solution is transferred to the separating funnel boiling range, get the upper strata petroleum ether layer, wash with water 3-5 time, discard water layer, petroleum ether layer is carried out drying by anhydrous sodium sulfate, be transferred to the volumetric flask constant volume then promptly;
(2) preparation of typical curve:
A, standard reserving solution: accurately take by weighing the beta carotene standard items in volumetric flask, add chloroform dissolving, use the sherwood oil constant volume, be mixed with the carrotene storing solution, keep in Dark Place in-18 ℃, face and use preceding demarcation;
The demarcation of standard solution: get standard reserving solution, use the normal hexane constant volume, mixing is measured its light absorption value, is blank with the normal hexane, lambda1-wavelength 450nm, and average is got in three parts of replicate determinations.Calculate solution concentration by following formula:
X: carrot concentration of standard solution, unit are every milliliter of microgram
A: light absorption value
E: beta carotene in hexane solution, lambda1-wavelength 450nm, cuvette thickness 1cm, solution concentration is the absorptivity of 1ug/ml, is 0.2368
B, get the beta carotene solution of carrotene storing solution preparation variable concentrations, go up HPLC mensuration respectively, try to achieve peak area drawing standard curve;
C, standard are used liquid: the beta carotene standard reserving solution preparation standard of measuring existing demarcation is used liquid;
(3) utilize the standard of determining among step (2) C to use the concentration of liquid to calculate the content of beta carotene in the liquid to be measured as external standard:
Prepare test sample liquid to be measured according to the described method of step (1),
The content of beta carotene in the X--sample, unit are milligram every hectogram (mg/100g)
The C-sample is measured the concentration of beta carotene in the liquid, and unit is every milliliter of microgram (ng/mL)
M-sample mass or volume, unit is gram or milliliter (g or mL).
2. according to the HPLC assay method of beta carotene in the described spirulina of claim 1, it is characterized in that, described step (1) spirulina sample is 0.1-1g, the volume that adds ethanol is 25-50ml, and ascorbic acid concentrations is 5%-15%, and volume is 3-10ml, potassium hydroxide solution concentration is 40-60%, volume is 8-15ml, and described bath temperature is 80-100 ℃, and described saponification time is 0.5-1.5h.
3. according to the HPLC assay method of beta carotene in the described spirulina of claim 1, it is characterized in that the beta carotene volume in described step (1) the PetroChina Company Limited. ether extraction saponification extract is 40-70ml, jolts 1-5min; The volume of institute's water is 5ml, anhydrous sodium sulfate 6-10g.
4. according to the HPLC assay method of beta carotene in the described spirulina of claim 1, it is characterized in that described step (1) HPLC chromatographic condition is:
Moving phase: methyl alcohol 85-95 parts by volume
Acetonitrile 5-15 parts by volume
Flow velocity is 1.2mL/min; Sample size: 10 μ L, column temperature: 30 ℃.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102645512A (en) * | 2012-04-17 | 2012-08-22 | 东华大学 | Method for quickly determining content of hydrophilic organic solvent by utilizing dual-aqueous phase system |
| CN102702059A (en) * | 2012-06-13 | 2012-10-03 | 北京林业大学 | Spirulina beta-renieratene extract and preparation method thereof |
| CN102735767A (en) * | 2012-06-04 | 2012-10-17 | 华中科技大学 | Method for detecting content of carotenoids having cis-trans isomers through high-polarity mobile phase |
| CN103175934A (en) * | 2013-03-05 | 2013-06-26 | 福建省农业科学院作物研究所 | Strawberry carotenoid component content determination method |
| CN107561171A (en) * | 2017-07-06 | 2018-01-09 | 北京林业大学 | The high efficiency extraction and assay method of carotenoid in Chinese rose petal |
| CN111812235A (en) * | 2020-07-06 | 2020-10-23 | 南京中科药业有限公司 | Method for measuring content of beta-carotene |
-
2011
- 2011-01-06 CN CN2011100019076A patent/CN102175789A/en active Pending
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102645512A (en) * | 2012-04-17 | 2012-08-22 | 东华大学 | Method for quickly determining content of hydrophilic organic solvent by utilizing dual-aqueous phase system |
| CN102735767A (en) * | 2012-06-04 | 2012-10-17 | 华中科技大学 | Method for detecting content of carotenoids having cis-trans isomers through high-polarity mobile phase |
| CN102702059A (en) * | 2012-06-13 | 2012-10-03 | 北京林业大学 | Spirulina beta-renieratene extract and preparation method thereof |
| CN103175934A (en) * | 2013-03-05 | 2013-06-26 | 福建省农业科学院作物研究所 | Strawberry carotenoid component content determination method |
| CN107561171A (en) * | 2017-07-06 | 2018-01-09 | 北京林业大学 | The high efficiency extraction and assay method of carotenoid in Chinese rose petal |
| CN111812235A (en) * | 2020-07-06 | 2020-10-23 | 南京中科药业有限公司 | Method for measuring content of beta-carotene |
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Application publication date: 20110907 |