CN115792018A - Method for determining content of 13 carotenoids in sweet potatoes - Google Patents
Method for determining content of 13 carotenoids in sweet potatoes Download PDFInfo
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Abstract
The invention belongs to the technical field of food detection, and provides a method for determining the content of 13 carotenoids in sweet potatoes, which comprises the following steps: pretreating a sample to be detected to obtain a sample solution to be loaded on a computer; respectively preparing standard solutions of violaxanthin, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene and drawing a standard curve; and carrying out ultra-performance liquid chromatography detection on the solution of the sample to be tested, and substituting the peak areas of the obtained components into a standard curve to obtain the concentrations of the 13 components in the sample to be tested. Compared with other detection methods, the 13-carotenoid linear relation in the determination method is good, the precision, the repeatability, the stability and the recovery rate result all meet the requirements, and a foundation can be laid for the quality analysis of mass sweet potato germplasm resources and breeding materials.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a method for determining the content of 13 carotenoids in sweet potatoes.
Background
Sweet potatoes (Ipomoea batatas L.) also called sweet potatoes, sweet potatoes and the like are the sixth most important grain crops after rice, wheat, potatoes, corn and cassava. The sweet potato contains rich carotenoid, anthocyanin, polyphenol, flavonoid and other active substances, wherein the carotenoid is a main source substance for synthesizing human vitamin A, and has the effects of resisting oxidation, resisting cancer, improving cardiovascular diseases, improving eyesight and the like. With the increasing health consciousness of people, the demand of natural carotenoids has increased, and sweet potatoes have been recognized and favored as health foods and functional foods. The color of the sweet potato pulp is used as an important component of the commodity quality, the nutritional quality and the processing quality of the sweet potato, and directly determines the market value of the fresh sweet potato. Studies have shown that yellow and orange sweet potatoes are mainly determined by the composition and content distribution of carotenoids. Therefore, the determination of the composition and the content of the carotenoid in the sweet potatoes not only can screen high-quality sweet potato varieties, but also provides data reference for the development and the utilization of the sweet potatoes.
At present, the detection method of carotenoid content in sweet potatoes mainly comprises the following steps: spectrophotometer method, high Performance Liquid Chromatography (HPLC), and high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). Although the spectrophotometer method is simple to operate, the method can only measure the total carotenoid content in the sweet potatoes, cannot identify the carotenoid components, and has limitation. The HPLC method is one of the most commonly used methods, has the advantages of good stability, easy operation and the like, but has the defects of long time (generally more than 30 min), large solvent consumption and low separation degree. The LC-MS/MS technology combines the advantages of liquid chromatography and mass spectrum, has higher separation performance and identification characteristics, but is difficult to popularize and use due to the high price of the instrument. With the continuous development of chromatographic technology, the appearance of ultra-high performance liquid chromatography (UPLC) makes up for the defects of long detection period, slow analysis speed and low separation degree of HPLC, reduces analysis cost, has the characteristics of higher separation degree, higher sensitivity, higher detection speed and the like, and gradually becomes an effective tool for analysis and detection in the fields of food, traditional Chinese medicine and the like. However, no UPLC analysis method has been reported for sweet potato carotenoids.
Disclosure of Invention
The invention aims to provide a method for measuring the content of 13-carotenoid in sweet potatoes, compared with other detection methods, the method for measuring the content of 13-carotenoid in sweet potatoes has the advantages that the linear relation of 13-carotenoid is good, the precision, the repeatability, the stability and the recovery rate result all meet the requirements, and a foundation can be laid for the quality analysis of mass sweet potato germplasm resources and breeding materials.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for measuring the content of 13 carotenoids in sweet potatoes, which comprises the following steps:
(1) Extracting a sample to be detected by using a mixed solution of ethanol, acetone and normal hexane to obtain an extracting solution, drying the extracting solution to obtain carotenoid, re-dissolving the carotenoid by using a methyl tert-butyl ether solution, and filtering to obtain a sample solution to be detected;
(2) Preparing standard solutions of violaxanthin, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene respectively, and drawing standard curves of the 13 components respectively;
(3) And (2) carrying out ultra performance liquid chromatography detection on the solution of the to-be-detected computer sample obtained in the step (1), and substituting the peak areas of the obtained components into the standard curve in the step (2) to obtain the concentrations of the 13 components in the to-be-detected computer sample.
Preferably, the volume ratio of the ethanol, the acetone and the n-hexane in the step (1) is 0.5-1.5.
Preferably, in the step (1), the ratio of the sample to be detected to the mixed solution of ethanol, acetone and n-hexane is 0.2-0.8 g: 4-6 mL.
Preferably, the extraction times in the step (1) are 2-4 times, and the extraction time is 1-3 min.
Preferably, after the extraction in the step (1) is finished, centrifuging to obtain an extracting solution; the temperature of the centrifugation is 3-5 ℃; the rotating speed of the centrifugation is 3000-5000 r/min, and the time of the centrifugation is 2-6 min.
Preferably, the methyl tert-butyl ether solution in the step (1) contains 0.005-0.015% by mass of BHT.
Preferably, the redissolution process in step (1) is accompanied by ultrasonic oscillation, and the time of the ultrasonic oscillation is 8-12 min.
Preferably, step (1) is carried out under exclusion of light.
Preferably, the conditions of the ultra high performance liquid chromatography detection in the step (3) are as follows: adopting YMC Carotenoid C30 chromatographic column with flow rate of 0.7-0.9 mL min -1 The temperature of the chromatographic column is 20-23 ℃, the sample injection amount is 0.5-1.5 mu L, and the detection wavelength is 420-460 nm;
the mobile phase A adopted by the ultra-high performance liquid chromatography is a methanol-acetonitrile mixed solution containing 0.01 percent of BHT and 0.1 percent of formic acid; mobile phase B was methyl t-butyl ether containing 0.01% of BHT;
the gradient elution procedure was: 0-1min, 85% mobile phase A and 15% mobile phase B; 1-2min, 80% of mobile phase A and 20% of mobile phase B; 2-3min, 70% of mobile phase A and 30% of mobile phase B; 3-4min, 55% of mobile phase A and 45% of mobile phase B; 4-5min, 40% of mobile phase A and 60% of mobile phase B; 5-5.1min, 25% of mobile phase A and 75% of mobile phase B; 5.1-8min, 85% of mobile phase A and 15% of mobile phase B.
Preferably, the volume ratio of methanol to acetonitrile in the mobile phase a is 1:2 to 4.
Compared with the prior art, the invention has the beneficial effects that:
the invention establishes an analysis method for simultaneously determining purple yellow, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene 13 carotenoid in sweet potatoes by using an ultra-high performance liquid chromatography, and realizes the simultaneous detection of multiple components. The 13-carotenoid linear relation in the determination method is good, the precision, the repeatability, the stability and the recovery rate result meet the requirements, compared with the conventional sweet potato HPLC analysis method, the UPLC has more carotenoid detection components, shorter analysis time and less flow cancellation consumption, can better meet the rapid and accurate analysis of the components and the content of the carotenoid in mass sweet potatoes, is an effective method for detecting the carotenoid in the sweet potatoes, and is suitable for the evaluation of mass sweet potato germplasm resources and the selection of high-quality varieties.
Drawings
FIG. 1 is a chromatogram of carotenoids in different elution procedures, wherein 1 is lutein, 2 is zeaxanthin, 3 is alpha-cryptoxanthin, 4 is epsilon-carotene, 5 is beta-cryptoxanthin, 6 is alpha-carotene, 7 is beta-carotene, 8 is delta-carotene, 9 is gamma-carotene, 10 is lycopene, 11 is violaxanthin, 12 is neoxanthin, and 13 is epoxy zeaxanthin;
FIG. 2 is a chromatogram of carotenoids at different temperatures of a chromatographic column, wherein 1 is violaxanthin, 2 is neoxanthin, 3 is epoxyzeaxanthin, 4 is lutein, 5 is zeaxanthin, 6 is alpha-cryptoxanthin, 7 is epsilon-carotene, 8 is beta-cryptoxanthin, 9 is alpha-carotene, 10 is beta-carotene, 11 is delta-carotene, 12 is gamma-carotene, and 13 is lycopene;
FIG. 3 is a chromatogram of carotenoids at different flow rates, wherein 1 is violaxanthin, 2 is neoxanthin, 3 is epoxyzeaxanthin, 4 is lutein, 5 is zeaxanthin, 6 is alpha-cryptoxanthin, 7 is epsilon-carotene, 8 is beta-cryptoxanthin, 9 is alpha-carotene, 10 is beta-carotene, 11 is delta-carotene, 12 is gamma-carotene, and 13 is lycopene;
FIG. 4 is a chromatogram of carotenoids in different amounts, wherein 1 is violaxanthin, 2 is neoxanthin, 3 is epoxyzeaxanthin, 4 is lutein, 5 is zeaxanthin, 6 is alpha-cryptoxanthin, 7 is epsilon-carotene, 8 is beta-cryptoxanthin, 9 is alpha-carotene, 10 is beta-carotene, 11 is delta-carotene, 12 is gamma-carotene, and 13 is lycopene.
Detailed Description
The invention provides a method for measuring the content of 13 carotenoids in sweet potatoes, which comprises the following steps:
(1) Extracting a sample to be detected by using a mixed solution of ethanol, acetone and normal hexane to obtain an extracting solution, drying the extracting solution to obtain carotenoid, re-dissolving the carotenoid by using a BHT solution, and filtering to obtain a sample solution to be detected;
(2) Preparing standard solutions of violaxanthin, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene respectively, and drawing standard curves of the 13 components respectively;
(3) And (3) carrying out ultra-performance liquid chromatography detection on the to-be-tested machine sample solution obtained in the step (1), and substituting the obtained peak areas of all components into the standard curve in the step (2) to obtain the concentrations of the 13 components in the to-be-tested sample.
In the invention, firstly, a sample to be detected is extracted by using a mixed solution of ethanol, acetone and normal hexane to obtain an extracting solution, the extracting solution is dried to obtain carotenoid, the carotenoid is redissolved by using a methyl tert-butyl ether solution, and the solution is filtered to obtain a sample solution to be detected. The volume ratio of ethanol, acetone and n-hexane is 0.5 to 1.5. The proportion of the sample to be detected to the mixed liquid of ethanol, acetone and normal hexane is 0.2-0.8 g:4 to 6mL, preferably 0.3 to 0.7g:4.5 to 5.5mL, more preferably 0.4 to 0.6g:4.8 to 5.2mL. The extraction times are 2-4 times, preferably 3 times, and the time for each extraction is 1-3 min, preferably 1.5-2.5 min, and more preferably 1.8-2.2 min.
In the invention, after extraction is finished, centrifuging to obtain an extracting solution; the centrifugation temperature is 3-5 ℃, preferably 3.5-4.5 ℃, and more preferably 3.8-4.2 ℃; the centrifugal speed is 3000-5000 r/min, preferably 3500-4500 r/min, more preferably 3800-4200 r/min; the centrifugation time is 2 to 6min, preferably 3 to 5min, and more preferably 3.5 to 4.5min.
In the present invention, the drying is preferably performed by nitrogen-blowing using a nitrogen blower.
In the present invention, the methyl t-butyl ether solution contains 0.005 to 0.015% by mass of BHT, and the mass percentage of BHT is preferably 0.008 to 0.012%, and more preferably 0.009 to 0.011%.
In the present invention, the redissolution process is accompanied by ultrasonic oscillation, and the ultrasonic oscillation time is 8 to 12min, preferably 8.5 to 11min, and more preferably 9 to 10min.
In the present invention, the filtration is preferably performed using a 0.22 μm filter.
In the present invention, the above steps are all performed under a condition of being protected from light.
In the invention, the standard solutions of violaxanthin, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene are prepared respectively, and the standard curves of the 13 components are drawn respectively.
In the invention, the solution of the computer sample to be tested obtained in the step (1) is subjected to ultra high performance liquid chromatography detection, and the obtained peak areas of all components are substituted into the standard curve in the step (2) to obtain the concentrations of the 13 components in the sample to be tested.
In the invention, the conditions of the ultra-high performance liquid chromatography detection are as follows: adopting a YMC Carotenoid C30 chromatographic column, wherein the specification of the chromatographic column is (2 mm multiplied by 100mm,3 mu m); the flow rate is 0.7-0.9 mL min -1 Preferably 0.75 to 0.85 mL/min -1 More preferably 0.78 to 0.82mLmin -1 (ii) a The temperature of the chromatographic column is 20-23 ℃, preferably 20.5-22.5 ℃, and further preferably 21-22 ℃; the sample size is 0.5 to 1.5. Mu.L, preferably 0.6 to 1.4. Mu.L, and more preferably 0.8 to 1.2. Mu.L; the detection wavelength is 420 to 460nm, preferably 430 to 450nm, and more preferably 435 to 445nm.
In the present invention, the mobile phase A used in the ultra high performance liquid chromatography is a methanol-acetonitrile mixed solution containing 0.01% BHT and 0.1% formic acid; mobile phase B was methyl t-butyl ether containing 0.01% of BHT; the gradient elution procedure was: 0-1min, 85% of mobile phase A and 15% of mobile phase B; 1-2min, 80% of mobile phase A and 20% of mobile phase B; 2-3min, 70% of mobile phase A and 30% of mobile phase B; 3-4min, 55% of mobile phase A and 45% of mobile phase B; 4-5min, 40% of mobile phase A and 60% of mobile phase B; 5-5.1min, 25% of mobile phase A and 75% of mobile phase B; 5.1-8min, 85% mobile phase A and 15% mobile phase B.
In the invention, the volume ratio of methanol to acetonitrile in the mobile phase A is 1:2 to 4, preferably 1.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A method for determining the content of 13 carotenoids in Ipomoea batatas Lam.9 comprises the following steps:
(1) 0.500g of sweet potato powder is weighed into a 15mL centrifuge tube, 5mL of ethanol is added: acetone: a mixed solution of n-hexane = 1; repeating the above extraction steps twice, and mixing the centrifuged supernatants; and blowing nitrogen to dryness by using a nitrogen blower to obtain the carotenoid. Redissolving with 1mL of methyl tert-butyl ether containing 0.01% (v/v) BHT, ultrasonically shaking for 10min to dissolve completely, filtering with 0.22 μm filter membrane, storing in brown sample bottle, and processing the whole sample in dark. Obtaining the sample solution to be loaded on the computer.
(2) Dissolving violaxanthin, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene 13 carotenoid standard substances with methyl tert-butyl ether containing 0.01 percent BHT respectively, diluting the dissolved solution into standard solutions with different concentrations of 0.1, 0.5, 1, 5, 10, 100 and 200 mu g/mL, and passing through a 0.22 mu m filter membrane, and sequentially carrying out ultra high performance liquid chromatography detection on the obtained standard solutions, wherein the detection conditions are as follows:
a column of YMC Carotenoid C30 (2 mm. Times.100mm, 3 μm) was used, purified with methanol: acetonitrile (1, 3,v/v) was added to 0.01% BHT (gmL) -1 ) And 0.1% formic acid as mobile phase A, adding 0.01% BHT as mobile phase B, gradient eluting at flow rate of 0.8mL min -1 The detection wavelength is 450nm, the temperature of the chromatographic column is 23 ℃, and the sample injection amount is 1 mu L. The gradient elution procedure was: 0-1min, 85% mobile phase A and 15% mobile phase B; 1-2min, 80% of mobile phase A and 20% of mobile phase B; 2-3min, 70% of mobile phase A and 30% of mobile phase B; 3-4min, 55% of mobile phase A and 45% of mobile phase B; 4-5min, 40% of mobile phase A and 60% of mobile phase B; 5-5.1min, 25% of mobile phase A and 75% of mobile phase B; 5.1-8min, 85% of mobile phase A and 15% of mobile phase B.
And (4) taking the concentration of the carotenoid standard substance as a horizontal coordinate and taking the peak area as a vertical coordinate to draw standard curves of different carotenoids.
(3) And (3) carrying out ultra-high performance liquid chromatography detection on the to-be-tested machine sample solution obtained in the step (1) under the detection conditions of the step (2), and substituting the obtained peak areas of all components into the standard curve in the step (2) to obtain the concentrations of the 13 components in the to-be-tested sample.
Example 2
A method for measuring the content of 13 carotenoids in Guangdong potato 87 sweet potatoes comprises the following steps:
(1) 0.500g of sweet potato powder was weighed into a 15mL centrifuge tube, 4.5mL of ethanol: acetone: n-hexane =0.8, 1.2 (v/v/v), vortex at room temperature for 2.5min, centrifuge at 4500r/min for 4min at 4 ℃, and then transfer the supernatant to a new 15mL centrifuge tube; repeating the above extraction steps twice, and mixing the centrifuged supernatants; and blowing nitrogen to dryness by using a nitrogen blower to obtain the carotenoid. Redissolving with 1mL of methyl tert-butyl ether containing 0.012% BHT, ultrasonically shaking for 12min to dissolve completely, filtering with 0.22 μm filter membrane, storing in brown sample bottle, and processing the whole sample in dark. Obtaining the sample solution to be loaded on the machine.
(2) Dissolving violaxanthin, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene 13 carotenoid standard products by using methyl tert-butyl ether containing 0.01 percent BHT respectively, diluting the dissolved solution into standard solutions with different concentrations of 0.1, 0.5, 1, 5, 10, 100 and 200 mu g/mL, and carrying out ultra-high performance liquid chromatography detection on the obtained standard solutions sequentially after passing through a 0.22 mu m filter membrane, wherein the detection conditions are as follows:
a column of YMC Carotenoid C30 (2 mm. Times.100mm, 3 μm) was used, purified with methanol: acetonitrile (1, 3,v/v) was added with 0.01% BHT (gmL -1 ) And 0.1% formic acid as mobile phase A, methyl t-butyl ether was added with 0.01% BHT as mobile phase B, and gradient elution was carried out at a flow rate of 0.8mL min -1 The detection wavelength is 450nm, the temperature of the chromatographic column is 23 ℃, and the sample injection amount is 1 mu L. The gradient elution procedure was: 0-1min, 85% mobile phase A and 15% mobile phase B; 1-2min, 80% of mobile phase A and 20% of mobile phase B; 2-3min, 70% of mobile phase A and 30% of mobile phase B; 3-4min, 55% of mobile phase A and 45% of mobile phase B; 4-5min, 40% of mobile phase A and 60% of mobile phase B; 5-5.1min, 25% of mobile phase A and 75% of mobile phase B; 5.1-8min, 85% mobile phase A and 15% mobile phase B.
And drawing standard curves of different carotenoids by taking the concentration of the carotenoid standard substance as a horizontal coordinate and taking the peak area as a vertical coordinate.
(3) And (3) carrying out ultra-high performance liquid chromatography detection on the to-be-tested machine sample solution obtained in the step (1) under the detection conditions of the step (2), and substituting the obtained peak areas of all components into the standard curve in the step (2) to obtain the concentrations of the 13 components in the to-be-tested sample.
Example 3
A method for determining the content of 13 carotenoids in Pushu 32 sweet potatoes comprises the following steps:
(1) 0.500g of sweet potato powder was weighed into a 15mL centrifuge tube, and 5.5mL of ethanol: acetone: a mixed solution of n-hexane = 1.2 (v/v/v) and 1 (v/v/v), vortexed at room temperature for 3min, centrifuged at 3800r/min for 6min at 4.5 ℃, and then the supernatant is transferred to a new 15mL centrifuge tube; repeating the above extraction steps twice, and mixing the centrifuged supernatants; and blowing nitrogen to dryness by using a nitrogen blower to obtain the carotenoid. Redissolving with 1mL of methyl tert-butyl ether containing 0.008% (v/v) BHT, ultrasonically shaking for 9min to dissolve completely, filtering with 0.22 μm filter membrane, storing in brown sample bottle, and processing the whole sample in dark. Obtaining the sample solution to be loaded on the machine.
(2) Dissolving violaxanthin, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene 13 carotenoid standard products by using methyl tert-butyl ether containing 0.01 percent BHT respectively, diluting the dissolved solution into standard solutions with different concentrations of 0.1, 0.5, 1, 5, 10, 100 and 200 mu g/mL, and carrying out ultra-high performance liquid chromatography detection on the obtained standard solutions sequentially after passing through a 0.22 mu m filter membrane, wherein the detection conditions are as follows:
a column of YMC Carotenoid C30 (2 mm. Times.100mm, 3 μm) was used, purified with methanol: acetonitrile (1, 3,v/v) was added to 0.01% BHT (gmL) -1 ) And 0.1% formic acid as mobile phase A, methyl t-butyl ether was added with 0.01% BHT as mobile phase B, and gradient elution was carried out at a flow rate of 0.8mL min -1 The detection wavelength is 450nm, the temperature of the chromatographic column is 23 ℃, and the sample injection amount is 1 mu L. The gradient elution procedure was: 0-1min, 85% of mobile phase A and 15% of mobile phase B; 1-2min, 80% of mobile phase A and 20% of mobile phase B; 2-3min, 70% of mobile phase A and 30% of mobile phase B; 3-4min, 55% of mobile phase A and 45% of mobile phase B; 4-5min, 40% of mobile phase A and 60% of mobile phase B; 5-5.1min, 25% of mobile phase A and 75% of mobile phase B; 5.1-8min, 85% mobile phase A and 15% mobile phase B.
And drawing standard curves of different carotenoids by taking the concentration of the carotenoid standard substance as a horizontal coordinate and taking the peak area as a vertical coordinate.
(3) And (3) carrying out ultra-performance liquid chromatography detection on the solution of the to-be-detected computer sample obtained in the step (1) under the detection condition of the step (2), and substituting the obtained peak areas of all components into the standard curve in the step (2) to obtain the concentrations of the 13 components in the to-be-detected computer sample.
Example 4
A method for measuring the content of 13 carotenoids in Guangdong potato 79 sweet potato comprises the following steps:
(1) 0.500g of sweet potato powder was weighed into a 15mL centrifuge tube, and 4.5mL of ethanol: acetone: a mixed solution of n-hexane = 1; repeating the above extraction steps twice, and mixing the centrifuged supernatants; and blowing nitrogen to dryness by using a nitrogen blower to obtain the carotenoid. Redissolving with 1mL of methyl tert-butyl ether containing 0.008% (v/v) BHT, ultrasonically shaking for 12min to dissolve completely, filtering with 0.22 μm filter membrane, storing in brown sample bottle, and processing the whole sample in dark. Obtaining the sample solution to be loaded on the machine.
(2) Dissolving violaxanthin, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene 13 carotenoid standard products by using methyl tert-butyl ether containing 0.01 percent BHT respectively, diluting the dissolved solution into standard solutions with different concentrations of 0.1, 0.5, 1, 5, 10, 100 and 200 mu g/mL, and carrying out ultra-high performance liquid chromatography detection on the obtained standard solutions sequentially after passing through a 0.22 mu m filter membrane, wherein the detection conditions are as follows:
a column of YMC Carotenoid C30 (2 mm. Times.100mm, 3 μm) was used, purified with methanol: acetonitrile (1, 3,v/v) was added to 0.01% BHT (gmL) -1 ) And 0.1% formic acid as mobile phase A, methyl t-butyl ether was added with 0.01% BHT as mobile phase B, and gradient elution was carried out at a flow rate of 0.8mL min -1 The detection wavelength is 450nm, the temperature of the chromatographic column is 23 ℃, and the sample injection amount is 1 mu L. The gradient elution procedure was: 0-1min, 85% mobile phase A and 15% mobile phase B; 1-2min, 80% of mobile phase A and 20% of mobile phase B;2 to 3min,70% of mobile phase A and30% mobile phase B; 3-4min, 55% of mobile phase A and 45% of mobile phase B; 4-5min, 40% of mobile phase A and 60% of mobile phase B; 5-5.1min, 25% of mobile phase A and 75% of mobile phase B; 5.1-8min, 85% of mobile phase A and 15% of mobile phase B.
And drawing standard curves of different carotenoids by taking the concentration of the carotenoid standard substance as a horizontal coordinate and taking the peak area as a vertical coordinate.
(3) And (3) carrying out ultra-high performance liquid chromatography detection on the to-be-tested machine sample solution obtained in the step (1) under the detection conditions of the step (2), and substituting the obtained peak areas of all components into the standard curve in the step (2) to obtain the concentrations of the 13 components in the to-be-tested sample.
Example 5
A method for determining the content of 13 carotenoids in Jishu 26 sweet potatoes comprises the following steps:
(1) 0.500g of sweet potato powder was weighed into a 15mL centrifuge tube, and 4mL of ethanol: acetone: n-hexane =0.8, 1 (v/v/v), vortexed at room temperature for 3min, centrifuged at 4200r/min for 4min at 3.5 ℃, and then the supernatant is transferred to a new 15mL centrifuge tube; repeating the above extraction steps twice, and mixing the centrifuged supernatants; and blowing nitrogen to dryness by using a nitrogen blower to obtain the carotenoid. Redissolving with 1mL of methyl tert-butyl ether containing 0.01% (v/v) BHT, ultrasonically shaking for 10min to dissolve completely, filtering with 0.22 μm filter membrane, storing in brown sample bottle, and processing the whole sample in dark. Obtaining the sample solution to be loaded on the machine.
(2) Dissolving violaxanthin, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene 13 carotenoid standard products by using methyl tert-butyl ether containing 0.01 percent BHT respectively, diluting the dissolved solution into standard solutions with different concentrations of 0.1, 0.5, 1, 5, 10, 100 and 200 mu g/mL, and carrying out ultra-high performance liquid chromatography detection on the obtained standard solutions sequentially after passing through a 0.22 mu m filter membrane, wherein the detection conditions are as follows:
a column of YMC Carotenoid C30 (2 mm. Times.100mm, 3 μm) was used, purified with methanol: acetonitrile (1V/v) 0.01% of BHT (gmL) -1 ) And 0.1% formic acid as mobile phase A, methyl t-butyl ether was added with 0.01% BHT as mobile phase B, and gradient elution was carried out at a flow rate of 0.8mL min -1 The detection wavelength is 450nm, the temperature of the chromatographic column is 23 ℃, and the sample injection amount is 1 mu L. The gradient elution procedure was: 0-1min, 85% mobile phase A and 15% mobile phase B; 1-2min, 80% of mobile phase A and 20% of mobile phase B; 2-3min, 70% of mobile phase A and 30% of mobile phase B; 3-4min, 55% of mobile phase A and 45% of mobile phase B; 4-5min, 40% of mobile phase A and 60% of mobile phase B; 5-5.1min, 25% of mobile phase A and 75% of mobile phase B; 5.1-8min, 85% of mobile phase A and 15% of mobile phase B.
And (4) taking the concentration of the carotenoid standard substance as a horizontal coordinate and taking the peak area as a vertical coordinate to draw standard curves of different carotenoids.
(3) And (3) carrying out ultra-high performance liquid chromatography detection on the to-be-tested machine sample solution obtained in the step (1) under the detection conditions of the step (2), and substituting the obtained peak areas of all components into the standard curve in the step (2) to obtain the concentrations of the 13 components in the to-be-tested sample.
Example 6
A method for determining the content of 13 carotenoids in 25 sweet potatoes of nicotiana tabacum comprises the following steps:
(1) 0.500g of sweet potato powder was weighed into a 15mL centrifuge tube, and 5mL of ethanol: acetone: a mixed solution of n-hexane = 1.2 (v/v/v), 1.2, vortex at room temperature for 2min, centrifuge at 4500r/min at 4 ℃ for 4min, and then transfer the supernatant to a new 15mL centrifuge tube; repeating the above extraction steps twice, and mixing the centrifuged supernatants; and blowing nitrogen to dryness by using a nitrogen blower to obtain the carotenoid. Redissolving with 1mL of methyl tert-butyl ether containing 0.01% (v/v) BHT, ultrasonic vibrating for 12min to dissolve completely, filtering with 0.22 μm filter membrane, storing in brown sample bottle, and processing the whole sample in dark place. Obtaining the sample solution to be loaded on the machine.
(2) Dissolving violaxanthin, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene 13 carotenoid standard substances with methyl tert-butyl ether containing 0.01 percent BHT respectively, diluting the dissolved solution into standard solutions with different concentrations of 0.1, 0.5, 1, 5, 10, 100 and 200 mu g/mL, and passing through a 0.22 mu m filter membrane, and sequentially carrying out ultra high performance liquid chromatography detection on the obtained standard solutions, wherein the detection conditions are as follows:
a column of YMC Carotenoid C30 (2 mm. Times.100mm, 3 μm) was used, purified with methanol: acetonitrile (1, 3,v/v) was added with 0.01% BHT (g mL -1 ) And 0.1% formic acid as mobile phase A, methyl t-butyl ether was added with 0.01% BHT as mobile phase B, and gradient elution was carried out at a flow rate of 0.8mL min -1 The detection wavelength is 450nm, the temperature of the chromatographic column is 23 ℃, and the sample injection amount is 1 mu L. The gradient elution procedure was: 0-1min, 85% mobile phase A and 15% mobile phase B; 1-2min, 80% of mobile phase A and 20% of mobile phase B; 2-3min, 70% of mobile phase A and 30% of mobile phase B; 3-4min, 55% of mobile phase A and 45% of mobile phase B; 4-5min, 40% of mobile phase A and 60% of mobile phase B; 5-5.1min, 25% of mobile phase A and 75% of mobile phase B; 5.1-8min, 85% of mobile phase A and 15% of mobile phase B.
And (4) taking the concentration of the carotenoid standard substance as a horizontal coordinate and taking the peak area as a vertical coordinate to draw standard curves of different carotenoids.
(3) And (3) carrying out ultra-high performance liquid chromatography detection on the to-be-tested machine sample solution obtained in the step (1) under the detection conditions of the step (2), and substituting the obtained peak areas of all components into the standard curve in the step (2) to obtain the concentrations of the 13 components in the to-be-tested sample.
Comparative example 1
Compared to example 1, the elution procedure for this comparative example was: 0-5min, 100% of mobile phase A and 0% of mobile phase B; 5-5.01min, 30% of mobile phase A and 70% of mobile phase B; 5.01-6 min,5% of mobile phase A and 95% of mobile phase B; 6-7min, 5% mobile phase A and 95% mobile phase B; 7-8min, 100% of mobile phase A and 0% of mobile phase B. The rest of the detection procedure was the same as in example 1.
Comparative example 2
The elution procedure for this comparative example compared to example 1 was: 0-2min, 85% mobile phase A and 15% mobile phase B; 2-2.1min, 75% of mobile phase A and 25% of mobile phase B; 2.1-2.5 min,60% of mobile phase A and 40% of mobile phase B; 2.5-3min, 40% of mobile phase A and 60% of mobile phase B; 3-3.5min, 5% of mobile phase A and 95% of mobile phase B; 3.5-4min, 60% of mobile phase A and 40% of mobile phase B; 4-4.10 min,85% of mobile phase A and 15% of mobile phase B; 4.10-6 min,85% of mobile phase A and 15% of mobile phase B; 6-8min, 85% mobile phase A and 15% mobile phase B. The remaining test procedures were the same as in example 1.
Comparative example 3
The elution procedure for this comparative example compared to example 1 was: 0-2min, 85% mobile phase A and 15% mobile phase B; 2-2.05min, 75% of mobile phase A and 25% of mobile phase B; 2.05-2.5 min,60% of mobile phase A and 40% of mobile phase B; 2.5-3min, 40% of mobile phase A and 60% of mobile phase B; 3-3.01min, 5% of mobile phase A and 95% of mobile phase B; 3.01-4min, 85% of mobile phase A and 15% of mobile phase B; 4-4.1min, 85% of mobile phase A and 15% of mobile phase B; 4.1-6 min,85% of mobile phase A and 15% of mobile phase B; 6-8min, 85% mobile phase A and 15% mobile phase B. The remaining test procedures were the same as in example 1.
Comparative example 4
The column temperature of this comparative example was 25 ℃ compared to example 1, and the rest of the detection procedure was the same as in example 1.
Comparative example 5
The temperature of the column of this comparative example was 28 ℃ as compared with example 1, and the remaining detection procedure was the same as in example 1.
Comparative example 6
The temperature of the column of this comparative example was 30 ℃ as compared with example 1, and the remaining detection procedure was the same as in example 1.
Comparative example 7
Compared with example 1, the flow rate of the sample injection in the comparative example is 0.6mL min -1 The rest of the detection procedure was the same as in example 1.
Comparative example 8
Compared with example 1, the flow rate of the sample injection in the comparative example is 1.0mL min -1 The rest of the detection procedure was the same as in example 1.
Comparative example 9
The sample amount of this comparative example was 2. Mu.L as compared with example 1, and the remaining testing procedure was the same as in example 1.
Comparative example 10
The sample amount of this comparative example was 3. Mu.L compared with example 1, and the remaining testing procedure was the same as in example 1.
Experimental example 1
The measurement method of example 1 was used to examine the linear relationship, detection limit and quantification limit of carotenoids, and the concentration of the standard solution corresponding to the signal-to-noise ratio S/N =3 was calculated to determine the detection Limit (LOD) and the concentration of the standard solution corresponding to the signal-to-noise ratio S/N =10 was calculated to determine the quantification Limit (LOQ). The linear regression equation of 13 carotenoids is shown in Table 1. As can be seen from Table 1, the linear range of the standard substance is wide, and the linear correlation coefficient r is>0.997, indicating that the carotenoid is in good linear relation in the linear range, and is suitable for the determination of the carotenoid of sweet potato 13. The detection limit of 13 carotenoid is 0.003 to 0.018 mu g mL -1 The quantitative limit is 0.009-0.06 mug mL -1 Thus, the method is high in sensitivity.
TABLE 1.13 Linear regression equation, correlation coefficient, detection limit and quantitation limit for carotenoids
The precision, the repeatability and the stability were examined by the measuring method of example 1, the peak area and the retention time of each component were obtained by continuously measuring 6 times using the carotenoid mixed standard solution, the Relative Standard Deviation (RSD) was calculated, and it was judged whether the precision of the instrument was good, and the relative standard deviation RSD was between 0.46 to 8.66% and 0.45 to 2.02%, respectively, indicating that the precision of the instrument was good. Taking 6 parts of mixed standard solution with a certain concentration, carrying out sample injection analysis, recording the peak area and the retention time, calculating RSD, and judging whether the repeatability is good or not. Taking a mixed standard solution with a certain concentration, placing the mixed standard solution at room temperature for 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours, carrying out sample injection analysis, recording the peak area and retention time of each component, selecting 6 groups of data from the peak area and retention time, calculating RSD, and judging the stability of the instrument. The results are shown in Table 2, and it is understood from Table 2 that the relative standard deviations of the peak areas and retention times of carotenoids were between 0.35 to 3.14% and 1.07 to 2.76%, respectively, indicating good reproducibility of the chromatographic analysis method; stability test 13 the carotenoid peak area and retention time relative standard deviation were between 1.80-14.05% and 4.38-8.16%, respectively, indicating that the solution was stable over 24 h.
TABLE 2 relative standard deviations for precision, reproducibility and stability
The recovery rate was examined by the method of example 1, and 3 carotenoid standards with different concentration levels were added to a mixed standard solution of known concentration as a background, and after sample analysis, the peak areas of the carotenoid components were recorded to calculate the recovery rates of the components. The results are shown in Table 3, the average recovery rates of the 13 carotenoids at different levels are 84.61-122.38%, which indicates that the detection method has good accuracy.
TABLE 3.13 recovery of carotenoids
Experimental example 2
According to the detection results of the embodiments 1 to 6, the carotenoid content in the sample to be detected is calculated and obtained, and the formula is as follows:
carotenoid content (μ g) -1 )y＝c×v/1000/m
Wherein c represents the carotenoid concentration (μ g mL) obtained by substituting the peak area of each carotenoid component in the sample into the standard curve -1 ) V represents the volume (. Mu.L) of the solution used in the reconstitution, and m represents the mass (g) of the sweet potato tuber sample measured.
The carotenoid content in the different sweet potato varieties is shown in Table 4, and it can be seen from Table 4 that 8 carotenoid components were detected in total in 6 sweet potato varieties, among which violaxanthin, epoxyzeaxanthin and beta-carotene were detected in all varieties. The carotenoid components of different sweet potato varieties are different, 8 kinds of carotenoids are detected in the tobacco potato 25, 7 kinds of carotenoids are detected in the dragon potato No. 9, 6 kinds of carotenoids are detected in the Guanhua potato 87 and the Guanhua potato 79 respectively, 5 kinds of carotenoids are detected in the Puhua potato 32, and 4 kinds of carotenoids are detected in the Jihua potato 26. The major carotenoid in the other 5 varieties was β -carotene, except that the major carotenoid in jima 26 was epoxy zeaxanthin.
TABLE 4. Carotenoid content of different sweet potato products
ND: it was not detected.
Experimental example 3
This experimental example examined the chromatograms obtained by the measurement methods of example 1 and comparative examples 1 to 10, and the results are shown in fig. 1 to 4.
Fig. 1 shows chromatograms obtained by different elution procedures, wherein a in fig. 1 shows that a chromatogram is obtained by an elution procedure of comparative example 1, B in fig. 1 shows that a chromatogram is obtained by an elution procedure of comparative example 2, C in fig. 1 shows that a chromatogram is obtained by an elution procedure of comparative example 3, and D in fig. 1 shows that a chromatogram is obtained by an elution procedure of example 1. As can be seen from FIG. 1, under the elution conditions of comparative example 1, the carotenoid was eluted after 3min, and the peak widths of the different components were greatly different; comparative example 2 and comparative example 3 elution procedures, which failed to provide better separation between the different components due to the relatively short elution times; example 1 elution procedure, 13 carotenoids were more effective than the first three elution conditions, and were completely eluted within 6min.
Fig. 2 is a carotenoid chromatogram obtained by using different column temperatures, wherein a in fig. 2 is a chromatogram obtained by using comparative example 4, B in fig. 2 is a chromatogram obtained by using comparative example 5, C in fig. 2 is a chromatogram obtained by using comparative example 6, and D in fig. 2 is a chromatogram obtained by using example 1. As can be seen from FIG. 2, the carotenoid separation effect becomes better and better as the temperature is gradually decreased.
Fig. 3 is a carotenoid chromatogram obtained using different flow rates, wherein a in fig. 3 is a chromatogram obtained using comparative example 7, B in fig. 3 is a chromatogram obtained using example 1, and C in fig. 3 is a chromatogram obtained using comparative example 8. As can be seen from FIG. 3, the flow rate has an effect on the time to peak carotenoid production, when the flow rate is 0.6mL min -1 When the peak of carotenoid is late, the separation degree between a solvent peak and a lycopene chromatographic peak is 1.25, which indicates that lycopene cannot be completely separated within 8 min; flow rate 1.0mL min -1 Although 13 carotenoids can be separated, the separation degree between the epsilon-carotene and the beta-cryptoxanthin chromatographic peak is only 0.89, and the two substances cannot be completely separated; flow rate 0.8mLmin -1 Under the conditions of (1), 13 carotenoids were completely separated within 6min.
Fig. 4 is a carotenoid chromatogram obtained by using different sample amounts, wherein a in fig. 4 is a chromatogram obtained by using example 1, B in fig. 4 is a chromatogram obtained by using comparative example 9, and C in fig. 4 is a chromatogram obtained by using comparative example 10. As can be seen from fig. 4, as the sample injection volume gradually increased, the separation degrees of the other carotenoid chromatographic peaks except lycopene all showed a decreasing trend, indicating that the separation effect of carotenoid was decreased; when the sample amount is 1 mu L, the separation degree of two chromatographic peaks of the violaxanthin and the neoxanthin is 1.12 and is more than 1, which indicates that the two chromatographic peaks are basically separated, and the separation degree of other chromatographic peaks is more than 1.5, which indicates that the two chromatographic peaks are completely separated.
The embodiment shows that the invention provides the method for measuring the content of 13 carotenoids in sweet potatoes, compared with other detection methods, the method for measuring the content of 13 carotenoids in sweet potatoes has the advantages that the linear relation of 13 carotenoids in the method for measuring the content of 13 carotenoids in sweet potatoes is good, the precision, the repeatability, the stability and the recovery rate result meet the requirements, and a foundation can be laid for the quality analysis of mass sweet potato germplasm resources and breeding materials.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (10)
1. A method for measuring the content of 13 carotenoids in sweet potatoes is characterized by comprising the following steps:
(1) Extracting a sample to be detected by using a mixed solution of ethanol, acetone and normal hexane to obtain an extracting solution, drying the extracting solution to obtain carotenoid, redissolving the carotenoid by using a methyl tert-butyl ether solution, and filtering to obtain a sample solution to be detected;
(2) Respectively preparing standard solutions of violaxanthin, neoxanthin, epoxy zeaxanthin, lutein, zeaxanthin, alpha-cryptoxanthin, epsilon-carotene, beta-cryptoxanthin, alpha-carotene, beta-carotene, delta-carotene, gamma-carotene and lycopene, and respectively drawing standard curves of the 13 components;
(3) And (3) carrying out ultra-performance liquid chromatography detection on the to-be-tested machine sample solution obtained in the step (1), and substituting the obtained peak areas of all components into the standard curve in the step (2) to obtain the concentrations of the 13 components in the to-be-tested sample.
2. The method for determining the content of 13 carotenoids in sweetpotato according to claim 1, wherein the volume ratio of ethanol, acetone and n-hexane in step (1) is 0.5-1.5.
3. The method for determining the content of 13 carotenoids in sweet potatoes as claimed in claim 1, wherein the ratio of the sample to be tested to the mixed solution of ethanol, acetone and n-hexane in step (1) is 0.2-0.8 g: 4-6 mL.
4. The method for determining the content of 13 carotenoids in sweetpotato according to claim 1, wherein the number of times of extraction in step (1) is 2-4, and the time for each extraction is 1-3 min.
5. The method for determining the content of 13 carotenoids in sweetpotato according to claim 1, wherein after the extraction in step (1) is completed, an extract is obtained by centrifugation; the temperature of the centrifugation is 3-5 ℃; the rotating speed of the centrifugation is 3000-5000 r/min, and the time of the centrifugation is 2-6 min.
6. The method for determining the content of 13-carotenoid in sweet potatoes as claimed in claim 1, wherein the methyl tert-butyl ether solution in the step (1) contains 0.005-0.015% by mass of BHT.
7. The method for determining the content of 13 carotenoids in sweetpotato according to claim 1, wherein the reconstitution process is accompanied by ultrasonic vibration for 8-12 min.
8. The method for determining the content of 13 carotenoids in sweet potatoes according to any one of claims 1 to 7, wherein step (1) is carried out under a dark condition.
9. The method for determining the content of 13 carotenoids in sweet potatoes according to claim 1,the conditions of the ultra-high performance liquid chromatography detection in the step (3) are as follows: adopting YMC Carotenoid C30 chromatographic column with flow rate of 0.7-0.9 mL min -1 The temperature of the chromatographic column is 20-23 ℃, the sample injection amount is 0.5-1.5 mu L, and the detection wavelength is 420-460 nm;
the mobile phase A adopted by the ultra-high performance liquid chromatography is a methanol-acetonitrile mixed solution containing 0.01 percent of BHT and 0.1 percent of formic acid; mobile phase B was methyl t-butyl ether containing 0.01% of BHT;
the gradient elution procedure was: 0-1min, 85% mobile phase A and 15% mobile phase B; 1-2min, 80% of mobile phase A and 20% of mobile phase B; 2-3min, 70% of mobile phase A and 30% of mobile phase B; 3-4min, 55% of mobile phase A and 45% of mobile phase B; 4-5min, 40% of mobile phase A and 60% of mobile phase B; 5-5.1min, 25% of mobile phase A and 75% of mobile phase B; 5.1-8min, 85% mobile phase A and 15% mobile phase B.
10. The method for determining the content of 13 carotenoids in sweet potatoes as claimed in claim 9, wherein the volume ratio of methanol to acetonitrile in the mobile phase A is 1:2 to 4.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116499832A (en) * | 2023-05-16 | 2023-07-28 | 佛山市沃特测试技术服务有限公司 | Method and device for detecting carotene content of agricultural products |
| CN117664689A (en) * | 2024-01-31 | 2024-03-08 | 南京市食品药品监督检验院 | Method for analyzing or identifying sweet potato/tapioca starch based on nitrogen stable isotope |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116499832A (en) * | 2023-05-16 | 2023-07-28 | 佛山市沃特测试技术服务有限公司 | Method and device for detecting carotene content of agricultural products |
| CN116499832B (en) * | 2023-05-16 | 2023-09-22 | 佛山市沃特测试技术服务有限公司 | Method and device for detecting carotene content of agricultural products |
| CN117664689A (en) * | 2024-01-31 | 2024-03-08 | 南京市食品药品监督检验院 | Method for analyzing or identifying sweet potato/tapioca starch based on nitrogen stable isotope |
| CN117664689B (en) * | 2024-01-31 | 2024-04-16 | 南京市食品药品监督检验院 | Method for analyzing or identifying sweet potato/tapioca starch based on nitrogen stable isotope |
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