CN109632786B - Method for accurately detecting content of phytosterol ester by utilizing microplate reader - Google Patents
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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Abstract
The invention discloses a method for accurately detecting the content of phytosterol ester by using an enzyme-labeling instrument, which comprises the following steps: adding a sample to be detected into ethyl acetate, and performing ultrasonic treatment until the sample is completely dissolved to obtain a phytosterol ester liquid to be detected; mixing and shaking the solution to be detected and a phosphorus-sulfur-iron color developing agent with the mass concentration of 0.0015-0.015%, and developing at room temperature to obtain a color developing solution; putting 50-300 mu L of developing solution into an enzyme-labeling instrument for detection, and measuring the light absorption luminosity value of a sample to be detected; and drawing a standard curve to obtain the content of the phytosterol ester in the sample to be detected. According to the method, ethyl acetate is used as a solvent of the phytosterol ester, a phosphorus-sulfur-iron solution with the mass concentration of 0.0015-0.015% is used as a color developing agent, and the color developing agent is matched with an enzyme labeling instrument for detection, so that the detection is realized under a micro condition, the aim of high flux detection is fulfilled, and the detection has the advantages of high accuracy, high precision, low detection limit and stable color development.
Description
Technical Field
The invention relates to the technical field of detection of phytosterol ester content, in particular to a method for accurately detecting the phytosterol ester content by using an enzyme-labeling instrument.
Background
The phytosterol has effects of reducing total cholesterol and low density lipoprotein cholesterol in blood plasma. Because the water solubility and the oil solubility of the phytosterol are not ideal, the solubility of the phytosterol in edible oil and fat can be obviously improved after the esterification, the sterol ester can be converted into the sterol and the fatty acid in a human body, and the hydrolysis rate of the phytostanol ester in intestinal tracts reaches 90 percent, so the phytostanol ester has the physiological functions of the phytosterol and the fatty acid. Plant stanol esters mainly comprise beta-sitosterol ester, plant sterol ester and campesterol ester, and at present, the plant sterol ester is mainly applied to spreads and salad dressings. Phytosterol ester preparations have gained FDA "Generally Recognized As Safe (GRAS)" approval and have become a development direction for functional foods.
The detection of phytosterol ester has few researches at home and abroad, and mainly focuses on the detection of phytosterol.
At present, methods for detecting phytosterol include spectrophotometry, high performance liquid chromatography, thin layer chromatography, gas chromatography, infrared spectroscopy and the like. The high performance liquid chromatography, the thin layer chromatography, the gas chromatography and the infrared spectroscopy are not suitable for detecting large-batch samples due to high determination cost and complex operation, and the spectrophotometry is simple and convenient to operate and low in cost, and mainly comprises a visible colorimetric method and an enzyme-linked immunosorbent assay.
An ultraviolet-visible spectrophotometer is common in the detection of phytosterol, and a pretreatment method of a sample mainly comprises the following steps: preparing a phytosterol solution with a certain concentration by using absolute ethyl alcohol as a solvent, adding a certain amount of 10% phosphorus-sulfur-iron color developing agent, shaking up by shaking, and developing for a certain time at room temperature. And detecting the content of the phytosterol by using an ultraviolet-visible spectrophotometer. However, the method has the problems of low detection flux, complex operation, generally only about three samples to be detected at one time, long operation time, inconvenient cleaning, poor color development stability of a large batch of samples, large amount of detected samples and the like in the detection process.
Aiming at the defects, the multifunctional microplate reader is adopted to replace an ultraviolet-visible spectrophotometer, so that the phytosterol ester content can be efficiently, accurately and quickly detected, and the time and labor are saved. However, for the detection of phytosterol ester, the sample pretreatment method adopted when the conventional ultraviolet-visible spectrophotometer is used for detecting phytosterol is not suitable for the detection of phytosterol ester on a multifunctional microplate reader.
Therefore, it is necessary to explore a method suitable for detecting phytosterol ester on a multifunctional microplate reader, so as to simultaneously achieve the purposes of high-throughput detection and accurate detection.
Disclosure of Invention
The invention provides a method for accurately detecting the content of phytosterol ester by using an enzyme-labeling instrument, which not only can detect the content under the trace condition to achieve the aim of high-flux detection, but also has the characteristics of high detection accuracy, high precision, low detection limit, stable color development and the like, and is particularly suitable for detecting the phytosterol ester.
The specific technical scheme is as follows:
a method for accurately detecting the content of phytosterol ester by using a microplate reader comprises the following steps:
(1) adding a to-be-detected sample containing phytosterol ester into ethyl acetate serving as a solvent, and performing ultrasonic treatment until the phytosterol ester is completely dissolved to obtain a to-be-detected phytosterol ester solution;
(2) mixing and shaking up the phytosterol ester solution to be detected and a phosphorus-sulfur-iron color developing agent with the mass concentration of 0.0015-0.015%, and developing at room temperature to obtain a color developing solution;
(3) putting 50-300 mu L of developing solution into an enzyme-labeling instrument for detection, and measuring the light absorption luminosity value of a sample to be detected;
(4) taking a phytosterol ester standard sample, obtaining the absorbance values of the standard samples with different concentrations according to the steps (1) - (3), drawing a standard curve, and obtaining a linear regression equation of the absorbance values and the phytosterol ester content;
(5) and (4) substituting the absorbance value of the sample to be detected in the step (3) into the linear regression equation in the step (4) to obtain the content of the phytosterol ester in the sample to be detected.
The sample to be tested containing the phytosterol ester can be functional protein beverage or artificial cream oil containing the phytosterol ester. The detection method can eliminate the interference of monosaccharide, protein and Tween series organic solvents, and is particularly suitable for determining the content of phytosterol ester in protein drinks.
According to the invention, a large number of solvent types are screened (in the embodiment, four solvents are taken as an example), and the finding is that only by adopting ethyl acetate as a solvent, the obtained phytosterol ester solution to be detected can be matched with the phosphorus-sulfur-iron color developing agent within a specific concentration range, so that a good linear relation is obtained, and the detection effects of high accuracy, high precision, low detection limit and stable color development are achieved on an enzyme-labeling instrument. Wherein, the ethyl acetate simultaneously has the following three conditions:
(1) the phytosterol ester can be quickly dissolved, and the solubility is high;
(2) the color developing agent can be mutually dissolved, and the layering phenomenon is avoided;
(3) does not have color development reaction with the color developing agent, and has stable color development.
The above conditions have great influence on the accuracy of the detection result of the microplate reader.
In addition, the color developing agent adopted by the invention is particularly suitable for detection of an enzyme-labeling instrument, and the dosage of the color developing agent is also a key factor for determining the detection accuracy of the enzyme-labeling instrument. In the method for measuring the content of the phytosterol by a spectrophotometry, the mass concentration of the phosphorus-sulfur-iron color developing agent is usually about 0.15%; however, the experiment of the invention shows that for an enzyme-linked immunosorbent assay, FeCl with the mass concentration of 0.0015-0.015 percent is selected3The phosphoric acid solution is used as a color developing agent, so that the light absorption value displayed by the enzyme-labeling instrument can be ensured to be between 0.2 and 0.7, the color development is good, the influence of a solvent cannot be caused at the concentration, and the color development is stable; and the accuracy of the detection result can be ensured.
Further, in the step (1), the ultrasonic treatment time is 5-60 min, and the power is 90-110W; further, the time of the ultrasonic treatment is 5 min. If the ultrasonic treatment time is too short, the dissolution is incomplete, and if the ultrasonic treatment time is too long, the ultrasonic treatment time is not necessary.
Further, in the step (1), the mass-to-volume ratio of the phytosterol ester to the ethyl acetate in the sample to be detected is 25-125 mug: 1 mL; in the actual detection process, the approximate content range of the phytosterol ester in the sample to be detected can be preliminarily estimated, and then the dosage of the ethyl acetate is determined; tests show that the detection effect is good under the concentration, and the concentration of the phytosterol ester and the absorbance value are in a linear relation.
Further, in the step (2), the color development time is 25 min-2 h; further, the developing time is 25 min; tests show that the color development effect is good after 25min of color development, and the color development effect is stable within two hours.
Further, in the step (2), the preparation method of the phosphorus-sulfur-iron color developer comprises the following steps: FeCl is added3﹒6H2Dissolving O in concentrated phosphoric acid, adding phosphoric acid to constant volume,obtaining FeCl3A solution; wherein the volume concentration of the concentrated phosphoric acid is 80-90%.
Further, in the step (3), the absorption wavelength of the microplate reader during detection is 400-700 nm; furthermore, the absorption wavelength is 480-500 nm; tests prove that the color development liquid has larger absorption wavelength at 492nm and larger absorbance value of color development reaction.
Further, in the step (3), the concentration range of the phytosterol ester in the standard curve is 25-125 mug/mL; namely, the concentration range of the phytosterol ester which can be detected by the invention is 25-125 mu g/mL, and if the concentration range is beyond the range, the standard curve R2Is less than 0.99, and is not in a linear relation.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the method, ethyl acetate is used as a solvent of the phytosterol ester, a phosphorus-sulfur-iron solution with the mass concentration of 0.0015-0.015% is used as a color developing agent, and the color developing agent is matched with an enzyme-labeling instrument for detection, so that the detection is realized under a micro condition, the aim of high flux detection is fulfilled, and the detection has the advantages of high accuracy, high precision, low detection limit and stable color development.
(2) The method of the invention detects the content of the phytosterol ester by the multifunctional microplate reader, is accurate, efficient and convenient, has low detection cost and high detection flux, and can detect in large batch.
(3) The ferric phosphate-sulfur solution is used as a color developing agent, the ethyl acetate is used as a solvent, the brown-yellow complex is formed with the phytosterol ester, the effect of dissolving the phytosterol ester by the solvent is good, the time is short, the color development is stable, the layering is avoided, and the mutual solubility of the water phase and the oil phase in a certain amount is realized.
(4) The method can also eliminate the interference of monosaccharides such as glucose and the like, proteins such as rice protein and the like, Tween series such as Tween 20 and the like on the detection sample.
Drawings
FIG. 1 is a standard graph of the content of different phytosterol esters prepared in example 1 in ethyl acetate versus the corresponding absorbance values.
FIG. 2 is a graph showing absorbance values corresponding to color reactions at different wavelengths (400 to 700) obtained in example 2.
FIG. 3 is a graph showing absorbance values corresponding to color reactions at different wavelengths (480 to 500) obtained in example 2.
FIG. 4 is a standard curve of the addition of different developers and the corresponding absorbance values obtained in example 2.
FIG. 5 is a standard graph of different development times versus corresponding absorbance values obtained in example 2.
Detailed Description
The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.
In the following examples, the preparation of the phosphorus-sulfur-iron color developer was as follows:
taking 10g of FeCl3﹒6H2Dissolving O (AR grade) in 85% concentrated phosphoric acid, diluting to 100mL with phosphoric acid, storing in brown bottle, refrigerating, and storing for one year to obtain FeCl with mass volume concentration of 100g/L3A solution;
taking 1mL of FeCl at 100g/L3Adding the solution into a 100mL volumetric flask, diluting to constant volume with phosphoric acid, storing in brown bottle, refrigerating, and storing for one year to obtain a volume concentration of 1g/LFeCl3A solution; take 1g/LFeCl3Solution, 10g/LFeCl3Solutions and 100g/LFeCl31.5mL of each solution is transferred into a 100mL brown volumetric flask, and concentrated sulfuric acid (AR grade) is used for metering volume to a scale mark, so that the phosphorus-sulfur-iron color developing agent with three concentrations of 0.0015%, 0.015% and 0.15% is obtained.
The phytosterol ester samples to be tested used in the following examples were prepared as self-made solutions by the following method: taking soybean protein isolate containing 10mg/mL, preparing a mixture of sterol ester and protein, wherein the ratio of sterol ester to protein is 1: 5 as the phytosterol ester sample to be tested.
Example 1
The embodiment provides a method for accurately detecting the content of phytosterol ester by using a microplate reader, which is obtained by searching a large number of tests (the test searching process is shown as an embodiment 2), and the method comprises the following specific steps:
(1) adding 1mL of a to-be-detected sample containing phytosterol ester into about 40mL of ethyl acetate solvent, carrying out ultrasonic treatment (100W) for 5min, then using ethyl acetate to fix the volume to 50mL, and completely dissolving phytosterol ester in the ethyl acetate to obtain a to-be-detected phytosterol ester solution (which is equivalent to 3.75mg/mL of phytosterol ester);
(2) mixing 200 mu L of phytosterol ester solution to be detected and 100 mu L of phosphorus-sulfur-iron color developing agent with the mass concentration of 0.0015%, shaking uniformly, and developing for 25min at room temperature to obtain color developing solution;
(3) putting 200 mu L of developing solution into an enzyme-linked immunosorbent assay for detection, and measuring the absorbance value of a sample to be detected;
(4) placing 100mg of phytosterol ester standard sample in a beaker, adding 80mL of ethyl acetate, carrying out ultrasonic treatment until the phytosterol ester standard sample is completely dissolved, transferring the mixture into a 100mL volumetric flask, carrying out constant volume with ethyl acetate to obtain a phytosterol ester standard sample solution to be detected, and carrying out ultrasonic treatment for 5min to completely dissolve the phytosterol ester standard sample;
when the phytosterol ester liquid A is used, 10mL of the phytosterol ester standard substance to-be-detected liquid is sucked, ethyl acetate is used for fixing the volume to 100mL, and the phytosterol ester liquid A is obtained, wherein the concentration of the phytosterol ester liquid A is 100 mu g/mL. Sucking 20mL of the solution to be detected, and using ethyl acetate to fix the volume to 100mL to obtain phytosterol ester using solution B, wherein the concentration of the phytosterol ester using solution B is 200 mu g/mL.
Phytosterol ester use solutions a or B were added separately as in table 1 and shaken. Then, the color was developed at room temperature for 25min, and the absorbance (A) was measured at 492nm with a microplate reader, and a standard curve was plotted with the phytosterol ester concentration as abscissa and the absorbance (A) as ordinate.
Table 1 addition of reagents made from phytosterol ester standard curves
The obtained standard curve is shown in fig. 1, and the relationship between the concentration of phytosterol ester and the absorbance value is as follows: y is 0.0051x +0.0667, R2The concentration is 0.9974, and the concentration range is 25-125 mu g/mL.
(5) And (4) substituting the absorbance light value of the sample to be detected in the step (3) into the linear regression equation in the step (4) to obtain the content of the phytosterol ester in the sample to be detected, and measuring the result to obtain the content of the phytosterol ester (76.4 +/-0.7) mu g/mL.
Example 2
This example was searched, optimized and verified for the conditions of the respective steps in the detection method, and in order to facilitate the search, optimization and verification of the detection method, this example employed the experiment directly using phytosterol esters which are conventional commercially available products, and the same as example 1 was followed except that the mentioned steps were changed during the search of the following conditions.
1. Solvent selection and sonication time
And (3) putting 100mg of phytosterol ester in a beaker, adding 80mL of organic solvent, carrying out ultrasonic treatment until the phytosterol ester is completely dissolved, transferring the mixture into a 100mL volumetric flask, and carrying out constant volume by using the same organic solvent to obtain the phytosterol ester solution to be detected.
When in use, 10mL of the solution to be detected is absorbed, and the volume is adjusted to 100mL by using an organic solvent. Four solvents of ethyl acetate, absolute ethyl alcohol, normal butanol and normal hexane are respectively added with 200 mu L of 50 mu g/mL plant sterol ester solution and 100 mu L of phosphorus-sulfur-iron color developing agent, and the color development reaction is observed, and the research shows that: ethyl acetate works well as a solvent for phytosterol esters, and the results are shown in table 2 below.
TABLE 2 color reaction and ultrasonic dissolution time of phytosterol esters in different solvents
2. Determination of absorption wavelength during detection
(1) Placing 100mg of phytosterol ester in a beaker, adding about 80mL of ethyl acetate, carrying out ultrasonic treatment until the phytosterol ester is completely dissolved, transferring the mixture into a 100mL volumetric flask, and carrying out constant volume with the ethyl acetate to obtain a phytosterol ester solution to be detected;
when in use, sucking 10mL of the solution to be detected, and fixing the volume to 100mL by using ethyl acetate, wherein the content of the phytosterol ester is 100 mug/mL; sucking 20mL of the solution to be detected, and using ethyl acetate to fix the volume to 100mL, wherein the content of the phytosterol ester is 200 mug/mL.
(2) Adding 100 mu L of phosphorus-sulfur-iron color developing agent into 200 mu L of phytosterol ester ethyl acetate solution with the concentration of 25 mu g/mL, 50 mu g/mL, 75 mu g/mL or 100 mu g/mL respectively; and then vibrating in an enzyme-linked immunosorbent assay, developing at room temperature for 25min, measuring the absorbance (A) of the enzyme-linked immunosorbent assay at the step length of 10nm under the condition of 400-700 nm, and drawing a standard curve by taking the absorption wavelength as a horizontal coordinate and the absorbance (A) as a vertical coordinate.
The research finds that: the four groups of concentrations all have larger absorbance at 480-500 nm and larger absorbance at 492nm, and the results are shown in FIG. 2 and FIG. 3. Therefore, 492nm was selected as the absorption wavelength of the color reaction.
3. Addition amount of phosphorus-sulfur-iron color developing agent
(1) Placing 100mg of phytosterol ester in a beaker, adding 80mL of ethyl acetate, carrying out ultrasonic treatment until the phytosterol ester is completely dissolved, transferring the mixture into a 100mL volumetric flask, and carrying out constant volume with the ethyl acetate to obtain a phytosterol ester solution to be detected; researches show that the ultrasonic time is about 60min to completely dissolve;
when in use, sucking 10mL of the solution to be detected, and fixing the volume to 100mL by using ethyl acetate, wherein the content of the phytosterol ester is 100 mug/mL; sucking 20mL of the solution to be detected, and using ethyl acetate to fix the volume to 100mL, wherein the content of the phytosterol ester is 200 mug/mL.
(2) 50 to 200. mu.L of a phosphothioferric developer was added to a 50. mu.g/mL-200. mu.L ethyl acetate solution of phytosterol ester, and the mixture was developed at room temperature for 25min, and the absorbance (A) was measured at 492nm with a microplate reader, and as a result, the amount of the developer added was selected to be 100. mu.L, as shown in FIG. 4.
4. Color reaction time and stabilization time
(1) Putting 100mg of phytosterol ester in a beaker, adding 80mL of ethyl acetate, carrying out ultrasonic treatment until the phytosterol ester is completely dissolved, transferring the phytosterol ester to a 100mL volumetric flask, carrying out constant volume with the ethyl acetate to obtain a phytosterol ester solution to be detected, and finding that the phytosterol ester solution can be completely dissolved within about 60min of ultrasonic time; when in use, sucking 10mL of the solution to be detected, and fixing the volume to 100mL by using ethyl acetate, wherein the content of the phytosterol ester is 100 mug/mL; sucking 20mL of the solution to be detected, and using ethyl acetate to fix the volume to 100mL, wherein the content of the phytosterol ester is 200 mug/mL.
(2) Adding 100 mu L of phosphorus-sulfur-iron color developing agent into 200 mu L of plant sterol ester ethyl acetate solution with the concentration of 50 mu g/mL, then developing for 5-120 min at room temperature, measuring the absorbance (A) of the solution by using an enzyme-labeling instrument at 492nm, and drawing a standard curve by using the absorption wavelength as a horizontal coordinate and the absorbance (A) as a vertical coordinate; the research finds that: the color development was stable after 25min, the results are shown in FIG. 5. Therefore, the color development time is 25min, and the color development is stable within 120 min.
5. Detection limit
And (3) detecting the sample by spectrophotometry according to the regulations of the International Union of theory and applied chemistry (I mu PAC), wherein the concentration corresponding to the absorbance of 0.01 after blank value is deducted is the detection limit.
Preparing a series of low-concentration phytosterol ester solutions, measuring the absorbance values according to the method of the standard curve, and determining the detection limit of the method according to the linear relation and the absorbance values, wherein the detection limit is 1.25 mu g/mL, and the result is shown in Table 3.
TABLE 3 detection limit test results
6. Experiment of interfering substances
(1) Putting 100mg of phytosterol ester in a beaker, adding 80mL of ethyl acetate, carrying out ultrasonic treatment until the phytosterol ester is completely dissolved, transferring the phytosterol ester to a 100mL volumetric flask, carrying out constant volume with the ethyl acetate to obtain a phytosterol ester solution to be detected, and finding that the phytosterol ester solution can be completely dissolved within about 60min of ultrasonic time; when in use, 10mL of the solution to be detected is sucked, and the volume is determined to be 100mL by ethyl acetate, wherein the content of the phytosterol ester is 100 mug/mL; sucking 20mL of the solution to be detected, and using ethyl acetate to fix the volume to 100mL, wherein the content of the phytosterol ester is 200 mug/mL.
(2) Respectively putting 0.1g of glucose, 0.1g of rice protein, 0.1g of konjac glucomannan and 0.1g of Tween 20 into four beakers, adding 100mL of ethyl acetate, and performing ultrasonic treatment until complete dissolution to obtain a glucose interference solution, a rice protein interference solution, a konjac glucomannan interference solution and a Tween 20 interference solution.
(3) 100. mu.L of a phytosterol ester ethyl acetate solution (100. mu.g/mL) was added, 100. mu.L of the above-mentioned interference solution was added to obtain a mixed solution of phytosterol ester and each component, 100. mu.L of a phospho-thioferric developer was added, color development was carried out at room temperature for 25min, and the absorbance (A) was measured at 492nm using a microplate reader, and it was found that glucose, rice protein, and Tween 20 did not cause an interference reaction in the color development system, but konjac glucomannan developed a color, and the results are shown in Table 4.
TABLE 4 interference substances experimental results
Example 3 precision and recovery
1) Standard curves were plotted for Δ a and different concentrations of phytosterol esters: adding 200 μ L of ethyl acetate solution of phytosterol ester with different concentrations and 100 μ L of phosphorus-sulfur-iron color developing agent into microporous enzyme label plate, placing into multifunctional enzyme label apparatus, oscillating, developing at room temperature for 25min, and measuring absorbance value at optimum wavelength of 492nm, wherein the blank of reagent is marked as A0The solution of phytosterol ester in ethyl acetate is denoted as A and the amount of Δ A ═ A-A is calculated0A standard curve of Δ a versus phytosterol ester concentration was established.
2) Preparing a phosphorus-sulfur-iron color developing agent: weighing 1g FeCl3﹒6H2Dissolving O (AR grade) in 85% concentrated phosphoric acid, diluting to 100mL with phosphoric acid, storing in brown bottle, refrigerating for one year to obtain 10% FeCl3A solution; imbibe 1mL 10% FeCl3Adding the solution into a 100mL volumetric flask, diluting to constant volume with phosphoric acid, storing in a brown bottle, refrigerating, and storing for one year to obtain 0.1% FeCl3A solution; taking 0.1% FeCl3The solution 1.5mL was placed in a 100mL brown flask and the volume was increased to the mark with concentrated sulfuric acid (AR grade).
3) Preparation of 100 μ g/mL phytosterol ester standard solution: accurately weighing 100.0mg of phytosterol ester standard sample, dissolving in ethyl acetate by using ultrasound, fixing the volume to 100mL, storing in a brown bottle, and storing at low temperature for later use, wherein the content of phytosterol ester is 1.00 mg/mL. When in use, 10mL of the solution to be detected is sucked, and the volume is determined to be 100mL by ethyl acetate, wherein the content of the phytosterol ester is 100 mug/mL; sucking 20mL of the solution to be detected, and using ethyl acetate to fix the volume to 100mL, wherein the content of the phytosterol ester is 200 mug/mL.
4) Determination of phytosterol ester content in the sample: demulsifying the phytosterol ester nanoemulsion, extracting with ethyl acetate, diluting to obtain 200 μ L solution, measuring according to the detection method in step 1, measuring the absorbance value A at 492nm, and calculating the value of delta A-A0Substituting the delta A into a linear regression equation to obtain the content of the phytosterol ester in the sample.
The standard curve obtained in the experiment is that y is 0.0051x +0.0667, R2The concentration is 0.9974, and the concentration range is 25-125 mu g/mL. The reaction selects ethyl acetate as a solvent of phytosterol ester, the color development reaction time is 25min, the absorption wavelength is 492nm, and meanwhile, interference of monosaccharides such as glucose and the like, proteins such as rice protein and the like, and Tween series such as Tween 20 and the like on a color development experiment can be eliminated. Six replicates of each of the three concentrations, 25.0. mu.g/mL, 50.0. mu.g/mL and 75.0. mu.g/mL, were selected for precision testing with RSD of 1.90%, 1.34% and 0.83%, respectively, and the results are shown in Table 5. The phytosterol ester nanoemulsion is subjected to demulsification, ethyl acetate extraction and dilution to obtain a solution, the concentration of nine groups of parallel samples is measured, standard samples of 12.5 mu g/mL, 25.0 mu g/mL and 37.5 mu g/mL are added for a standard recovery rate test, and the result is shown in Table 6.
TABLE 5 results of the precision test
TABLE 6 analysis of sample recovery by adding standard
Example 3
This example was tested directly with the commercial phytosterol ester, namely: and (3) putting 100mg of phytosterol ester in a beaker, adding 80mL of organic solvent, carrying out ultrasonic treatment until the phytosterol ester is completely dissolved, transferring the mixture into a 100mL volumetric flask, and carrying out constant volume by using the same organic solvent to obtain the phytosterol ester solution to be detected.
The rest of the steps were the same as those of example 1 except that the parameters shown in Table 7 below were changed.
TABLE 7
As can be seen from Table 7, the concentration of the developer is 0.0015% when ethyl acetate is used as the solvent, the standard curve fitting degree is the best when the enzyme-linked immunosorbent assay is used for detection, and the dissolution of phytosterol ester by ethyl acetate is much faster than that by absolute ethyl alcohol.
Claims (7)
1. A method for accurately detecting the content of phytosterol ester by using a microplate reader is characterized by comprising the following steps:
(1) adding a sample to be detected containing phytosterol ester into ethyl acetate solvent, performing ultrasonic treatment until the phytosterol ester is completely dissolved to obtain a phytosterol ester solution to be detected,
in the step (1), the mass-to-volume ratio of the phytosterol ester to the ethyl acetate in the sample to be detected is 25-125 mug: 1 mL;
(2) mixing and shaking up the phytosterol ester solution to be detected and a phosphorus-sulfur-iron color developing agent with the mass concentration of 0.0015-0.015%, and developing at room temperature to obtain a color developing solution;
(3) taking 50-300 mu L of developing solution, placing the developing solution into an enzyme-labeling instrument for detection, measuring the light absorption luminosity value of a sample to be detected,
in the step (3), the absorption wavelength of the enzyme-labeling instrument during detection is 480-500 nm;
(4) taking a phytosterol ester standard sample, obtaining the absorbance values of the standard samples with different concentrations according to the steps (1) - (3), drawing a standard curve, and obtaining a linear regression equation of the absorbance values and the phytosterol ester content;
(5) and (4) substituting the absorbance value of the sample to be detected in the step (3) into the linear regression equation in the step (4) to obtain the content of the phytosterol ester in the sample to be detected.
2. The method for accurately detecting the content of the phytosterol ester by using the microplate reader as claimed in claim 1, wherein in the step (1), the ultrasonic treatment time is 5-60 min, and the power is 90-110W.
3. The method for accurately detecting the content of the phytosterol ester by using the microplate reader as claimed in claim 1, wherein in the step (2), the mass concentration of the phospho-thioferric developer is 0.0015%.
4. The method for accurately detecting the content of the phytosterol ester by using the microplate reader as claimed in claim 1, wherein in the step (2), the developing time is 25 min-2 h.
5. The method for accurately detecting the content of phytosterol ester by using a microplate reader as claimed in claim 1, wherein the developing time is 25 min.
6. The method for accurately detecting the content of the phytosterol ester by using the microplate reader as recited in claim 1, wherein in the step (3), the absorption wavelength of the microplate reader during detection is 492 nm.
7. The method for accurately detecting the content of the phytosterol ester by using a microplate reader as claimed in claim 1, wherein in the step (4), the concentration range of the phytosterol ester in the standard curve is 25-125 μ g/mL.
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