CN109632786A - A method of accurately detecting phytosterol ester content using microplate reader - Google Patents
A method of accurately detecting phytosterol ester content using microplate reader Download PDFInfo
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Abstract
The invention discloses a kind of methods that phytosterol ester content is accurately detected using microplate reader, and this method: taking sample to be tested, are added in ethyl acetate, and ultrasonication obtains phytosterin ester prepare liquid to after being completely dissolved；The phosphorus sulphur iron color developing agent that prepare liquid and mass concentration are 0.0015~0.015% is mixed after shaking up, develops the color at room temperature, obtain developing solution；It takes the developing solution of 50~300 μ L as being detected in microplate reader, measures the absorption photometric value of sample to be tested；Standard curve is drawn, the content of phytosterin ester in sample to be tested is obtained.Solvent of the method for the present invention by using ethyl acetate as phytosterin ester, using mass concentration is 0.0015~0.015% phosphorus sulphur ferrous solution as color developing agent, cooperated with microplate reader detection, it not only realizes and is detected under the conditions of micro, achieve the purpose that flux high detection, and the accuracy detected is high, precision is high, detection limit is low, color stability.
Description
Technical field
The present invention relates to the detection technique fields of phytosterol ester content more particularly to a kind of utilization microplate reader accurately to detect
The method of phytosterol ester content.
Background technique
Phytosterol has the effect of reducing total plasma cholesterol and low density lipoprotein cholesterol.Due to phytosterol
Water-soluble and oil-soluble is undesirable, and the solubility in edible oil & fat can be significantly improved after being esterified, and sterol ester can
Sterol and fatty acid are converted in human body, percent hydrolysis of the plant stanol ester in enteron aisle reaches 90%, plants so it has
Object sterol and the two-part physiological function of fatty acid.Plant stanol ester mainly has cupreol ester, phytosterin ester, rape oil
Sterol ester three classes, currently, phytosterin ester is mainly used in tablespread and salad dressing.Phytosterol ester articles have obtained
The approval for obtaining FDA " it is generally acknowledged that safety (GRAS) ", becomes a kind of developing direction of functional food.
The research of the detection of phytosterin ester at home and abroad is less, focuses primarily upon the detection of phytosterol.
Currently, the method for detection phytosterol has spectrophotometry, high performance liquid chromatography, thin-layered chromatography, gas phase color
Spectrometry and infra-red sepectrometry etc..Wherein, high performance liquid chromatography, thin-layered chromatography, gas chromatography and infra-red sepectrometry by
It is high, complicated for operation in cost of determination, be not all suitable for the detection of batch samples, and spectrophotometry is easy to operate, cost compared with
It is low, mainly there are visible photolorimetry and microtitration assay in spectrophotometry.
Ultraviolet visible light spectrophotometer is more universal in the detection of phytosterol, the pre-treating method master of sample
It wants are as follows: prepare certain density phytosterol solution using dehydrated alcohol as solvent, a certain amount of 10% phosphorus sulphur iron colour developing is added
Agent concussion shakes up, and develop the color certain time at room temperature.Content of phytosterol is detected using ultraviolet visible light spectrophotometer.But
It is that this method is low in the presence of detection flux in the detection process, it is complicated for operation, three or so samples generally can only be once detected,
The problems such as, cleaning inconvenience long for the large batch of sample operation time, color stability are poor, and the sample size detected is big.
Against the above deficiency, ultraviolet visible light spectrophotometer is replaced using multi-function microplate reader, can efficiently, it is accurate,
Quickly detection phytosterol ester content is more time saving and energy saving.However, Conventional UV-can for the detection of phytosterin ester
Used sample-pretreating method, is not particularly suited for phytosterin ester in more function when light-exposed spectrophotometer detection phytosterol
Detection in energy microplate reader.
Therefore, it is necessary to a kind of method for being suitable for phytosterin ester and being detected on multi-function microplate reader is probed into, from
And the purpose realizing high-throughput detection simultaneously and precisely detecting.
Summary of the invention
The present invention provides a kind of method for accurately detecting phytosterol ester content using microplate reader, this method not only can be with
Content detection is carried out under the conditions of micro, achievees the purpose that high-throughput detection, but also there is accuracy in detection height, precision
High, the features such as detection limit is low, color stability, the detection of particularly suitable phytosterin ester.
Specific technical solution is as follows:
A method of accurately detecting phytosterol ester content using microplate reader, comprising the following steps:
(1) sample to be tested containing phytosterin ester is taken, is added in solvent ethyl acetate, ultrasonication to phytosterol
After ester is completely dissolved, phytosterin ester prepare liquid is obtained；
(2) the phosphorus sulphur iron color developing agent that phytosterin ester prepare liquid and mass concentration are 0.0015~0.015% is mixed and is shaken
It after even, develops the color at room temperature, obtains developing solution；
(3) it takes the developing solution of 50~300 μ L as being detected in microplate reader, measures the absorption photometric value of sample to be tested；
(4) phytosterin ester standard sample is taken, according to step (1)~(3), obtains the extinction light of various concentration standard sample
Angle value obtains the equation of linear regression of absorption photometric value Yu phytosterol ester content after drawing standard curve；
(5) will in step (3) sample to be tested absorption photometric value substitute into step (4) equation of linear regression in, obtain to
The content of phytosterin ester in sample.
The heretofore described sample to be tested containing phytosterin ester can be the functional protein drink containing phytosterin ester
Material or margarine etc..Wherein, since detection method can exclude monosaccharide, protein and TWEEN Series organic solvent
Interference, so it is particularly suitable measurement protein drink in phytosterol ester content.
The present invention has carried out a large amount of screenings (in case study on implementation by taking four kinds of solvents as an example) type of solvent, and discovery only uses
For ethyl acetate as solvent, the phytosterin ester prepare liquid of acquisition can be with the phosphorus sulphur iron color developing agent phase in certain concentration range
Mutually cooperation, obtains good linear relationship, and reaches in microplate reader that accuracy is high, precision is high, detection limit is low and colour developing is steady
Fixed detection effect.Wherein, ethyl acetate is provided simultaneously with following three conditions:
(1) phytosterin ester can be quickly dissolved, and solubility is high；
(2) it can dissolve each other with color developing agent, lamination do not occur；
(3) chromogenic reaction, and color stability do not occur with color developing agent.
Above-mentioned condition can have an immense impact on to the accuracy of microplate reader testing result.
In addition, the detection for the particularly suitable microplate reader of color developing agent that the present invention uses, and the dosage of color developing agent is also to determine enzyme
Mark the key factor of instrument detection accuracy.In Plants with Spectrophotometry sterol content, the quality of phosphorus sulphur iron color developing agent is dense
Degree usually all 0.15% or so；But the present invention tests discovery, for microplate reader, select mass concentration for 0.0015%~
0.015% FeCl3Phosphoric acid solution as color developing agent, can not only guarantee light absorption value that microplate reader is shown 0.2~0.7 it
Between, colour developing is good, will not be affected by the solvent under the concentration, color stability；And it can guarantee the accuracy of testing result.
Further, in step (1), the time of the ultrasonication is 5~60min, and power is 90~110W；More into
One step, the time of the ultrasonication is 5min.Not exclusively, ultrasonic time is too long, does not have for the too short then dissolution of ultrasonic time
It is necessary.
Further, in step (1), the mass volume ratio of phytosterin ester and ethyl acetate in sample to be tested is 25~
125μg:1mL；During atual detection, can first according to a preliminary estimate in sample to be tested phytosterin ester substantially content range,
The dosage of ethyl acetate is determined again；Test discovery, detection effect is good under the concentration, the concentration and absorbance of phytosterin ester
It is worth in a linear relationship.
Further, in step (2), the time of the colour developing is 25min~2h；Further, the colour developing when
Between be 25min；Test finds that color developing effect is good after the 25min that develops the color, and keeps stablizing in two hours.
Further, in step (2), the phosphorus sulphur iron color developing agent the preparation method comprises the following steps: by FeCl3﹒ 6H2O is dissolved in concentrated phosphoric acid
In, after phosphoric acid constant volume, obtain FeCl3Solution；Wherein, the volumetric concentration of concentrated phosphoric acid is 80~90%.
Further, in step (3), the absorbing wavelength when microplate reader detects is 400~700nm；Further,
The absorbing wavelength is 480~500nm；Tests prove that above-mentioned developing solution has larger absorbing wavelength at 492nm, colour developing is anti-
Answer absorbance value larger.
Further, in step (3), the concentration range of phytosterin ester is 25~125 μ g/mL in the standard curve；
The concentration range of phytosterin ester that can be detected i.e. of the invention is 25~125 μ g/mL, if exceeding the range, standard curve
R2< 0.99, non-linear relationship.
Compared with prior art, the invention has the following advantages:
(1) solvent of the method for the present invention by using ethyl acetate as phytosterin ester, use mass concentration for
0.0015%~0.015% phosphorus sulphur ferrous solution is cooperated with microplate reader detection, is not only realized micro as color developing agent
Under the conditions of detected, achieve the purpose that flux high detection, and the accuracy detected is high, precision is high, detection limit is low, colour developing
Stablize.
(2) the method for the present invention by multi-function microplate reader detect phytosterin ester content, not only accurately, efficiently, it is convenient,
And testing cost is low, detection flux is high, and it can be with mass detection.
(3) using phosphorus sulphur ferrous solution as color developing agent, ethyl acetate is solvent, forms brown complex with phytosterin ester,
Solvent dissolves that phytosterin ester effect is good, the time is short, color stability, not stratified, realizes that water phase and oil phase are mutual in a certain amount of
It is molten.
(4) the method for the present invention can also exclude the protein such as the monosaccharide such as glucose, rice protein and Tween 20 etc. and spit
Interference of the warm series to test sample.
Detailed description of the invention
Fig. 1 is the mark of different phytosterin ester contents and corresponding absorbance value in ethyl acetate obtained in embodiment 1
Directrix curve figure.
Fig. 2 is absorbance value figure corresponding to chromogenic reaction under different wave length (400~700) obtained in embodiment 2.
Fig. 3 is absorbance value figure corresponding to chromogenic reaction under different wave length (480~500) obtained in embodiment 2.
Fig. 4 is the canonical plotting of different color developing agent additive amounts and corresponding absorbance value obtained in embodiment 2.
Fig. 5 is the canonical plotting of different developing times and corresponding absorbance value obtained in embodiment 2.
Specific embodiment
The invention will be further described combined with specific embodiments below, and what is be exemplified below is only specific implementation of the invention
Example, but protection scope of the present invention is not limited only to this.
In the following example, phosphorus sulphur iron color developing agent the preparation method is as follows:
Take 10gFeCl3﹒ 6H2O (AR grades) is dissolved in the concentrated phosphoric acid that volume fraction is 85%, is settled to 100mL with phosphoric acid, is store
In brown bottle, refrigeration be can be reserved for 1 year, obtain the FeCl that mass-volume concentration is 100g/L3Solution；
Take the FeCl of 1mL, 100g/L3Solution is added in 100mL volumetric flask, with phosphoric acid constant volume, is stored in brown bottle,
Refrigeration can be reserved for 1 year, and obtaining volumetric concentration is 1g/LFeCl3Solution；Take 1g/LFeCl3Solution, 10g/LFeCl3Solution and
100g/LFeCl3Each 1.5mL of solution is transferred to respectively in 100mL brown volumetric flask, is settled to graduation mark with the concentrated sulfuric acid (AR grades),
Obtain 0.0015%, 0.015%, the phosphorus sulphur iron color developing agent of 0.15% 3 kind of concentration.
The phytosterin ester sample to be tested used in following Examples is makes liquid by oneself, the preparation method comprises the following steps: taking soybean containing 10mg/mL
Protein isolate prepares the phytosterin ester nanoemulsions that the ratio between sterol ester and albumen are 1:5, waits for test sample as phytosterin ester
Product.
Embodiment 1
The present embodiment provides a kind of method for accurately detecting phytosterol ester content using microplate reader, this method is through excessive
(it is as described in Example 2 that process is groped in test) that amount test obtains after groping, the specific steps are as follows:
(1) the sample to be tested 1mL containing phytosterin ester is taken, is added in about 40mL solvent ethyl acetate, ultrasonic wave
After (100W) handles 5min, it is settled to 50mL with ethyl acetate, phytosterin ester is dissolved completely in ethyl acetate, obtains plant
Sterol ester prepare liquid (phytosterin ester for being equivalent to 3.75mg/mL)；
(2) the phosphorus sulphur iron color developing agent that 200 μ L phytosterin ester prepare liquids and 100 μ L mass concentrations are 0.0015% is mixed
After shaking up, develop the color 25min at room temperature, obtains developing solution；
(3) it takes the developing solution of 200 μ L as being detected in microplate reader, measures the absorption photometric value of sample to be tested；
(4) it takes 100mg phytosterin ester standard sample to be placed in a beaker, 80mL ethyl acetate is added, be ultrasonically treated to molten
Solution completely, is transferred in 100mL volumetric flask and uses ethyl acetate constant volume, obtain phytosterin ester standard items prepare liquid, ultrasonic 5min
It can be completely dissolved；
In use, drawing above-mentioned phytosterin ester standard items prepare liquid 10mL, it is settled to 100mL with ethyl acetate, is obtained
Phytosterin ester uses liquid A, and concentration is 100 μ g/mL.Above-mentioned prepare liquid 20mL is drawn, is settled to 100mL with ethyl acetate,
Phytosterin ester is obtained using liquid B, concentration is 200 μ g/mL.
Phytosterin ester is added respectively by table 1 using liquid A or B, and is vibrated.Then, develop the color 25min at room temperature,
Its absorbance (A) is measured with microplate reader under 492nm, using phytosterol ester concentration as abscissa, absorbance (A) is ordinate, is drawn
Standard curve processed.
The reagent of 1 phytosterin ester standard curve making of table adds situation
Gained standard curve as shown in Figure 1, phytosterol ester concentration and absorbance value relationship are as follows: y=0.0051x+
0.0667, R2=0.9974, concentration range is 25~125 μ g/mL.
(5) will in step (3) sample to be tested absorption photometric value substitute into step (4) equation of linear regression in, obtain to
The content of phytosterin ester in sample, as a result measuring sterol ester content is (76.4 ± 0.7) μ g/mL.
Embodiment 2
The present embodiment is directed to each step condition in detection method and grope, optimize and verify, for the ease of inspection
Survey method is groped, optimizes and is verified, and the present embodiment use directlys adopt phytosterin ester and tested, which is
Conventional commercial product, and in following condition during groping, in addition to the step of referring to is varied, remaining content and implementation
Example 1 is identical.
1, solvent selection and ultrasonic time
It takes 100mg phytosterin ester to be placed in a beaker, 80mL organic solvent is added, ultrasonic treatment is complete to dissolving, transfer
Into 100mL volumetric flask, with organic solvent constant volume of the same race, phytosterin ester prepare liquid is obtained.
In use, drawing above-mentioned prepare liquid 10mL, 100mL is settled to organic solvent.It is quasi- to select ethyl acetate, anhydrous second
Four kinds of alcohol, n-butanol, n-hexane solvents add 50 μ g/mL phytosterol ester solution, 200 μ L, 100 μ of phosphorus sulphur iron color developing agent respectively
L observes chromogenic reaction, research shows that: ethyl acetate is preferable as the solvent effect of phytosterin ester, as a result as shown in table 2 below.
Chromogenic reaction and ultrasonic dissolution time of 2 phytosterin ester of table under different solvents
2, the determination of absorbing wavelength when detecting
(1) it taking 100mg phytosterin ester to be placed in a beaker, about 80mL ethyl acetate is added, ultrasonic treatment is complete to dissolving,
It is transferred in 100mL volumetric flask and uses ethyl acetate constant volume, obtain phytosterin ester prepare liquid；
In use, drawing above-mentioned prepare liquid 10mL, it is settled to 100mL with ethyl acetate, phytosterol ester content is at this time
100μg/mL；Above-mentioned prepare liquid 20mL is drawn, is settled to 100mL with ethyl acetate, phytosterol ester content is 200 μ g/ at this time
mL。
It (2) is 25 μ g/mL, the 200 μ L phytosterin ester ethyl acetate of 50 μ g/mL, 75 μ g/mL or 100 μ g/mL in concentration
In solution, 100 μ L of phosphorus sulphur iron color developing agent is added respectively；Then it is vibrated in microplate reader, and the 25min that develops the color at room temperature, 400
Under~700nm, step-length 10nm measures its absorbance (A) with microplate reader, and using absorbing wavelength as abscissa, absorbance (A) is vertical
Coordinate draws standard curve.
Research is found: four groups of concentration have larger absorbance in 480~500nm, and have larger absorbance at 492nm
Value, as a result as shown in Figures 2 and 3.Therefore select absorbing wavelength of the 492nm as chromogenic reaction.
3, phosphorus sulphur iron color developing agent additive amount
(1) it takes 100mg phytosterin ester to be placed in a beaker, 80mL ethyl acetate is added, ultrasonic treatment turns to dissolving completely
It moves in 100mL volumetric flask with ethyl acetate constant volume, obtains phytosterin ester prepare liquid；Research finds ultrasonic time about 60min
It can be completely dissolved；
In use, drawing above-mentioned prepare liquid 10mL, it is settled to 100mL with ethyl acetate, phytosterol ester content is at this time
100μg/mL；Above-mentioned prepare liquid 20mL is drawn, is settled to 100mL with ethyl acetate, phytosterol ester content is 200 μ g/ at this time
mL。
(2) 50 μ g/mL, 200 μ L phytosterin ester ethyl acetate solution in, add 50~200 μ of phosphorus sulphur iron color developing agent
L, develop the color 25min at room temperature, measures its absorbance (A) with microplate reader at 492nm, as a result as shown in figure 4, therefore selection colour developing
Agent additive amount is 100 μ L.
4, chromogenic reaction time and stable time
(1) it takes 100mg phytosterin ester to be placed in a beaker, 80mL ethyl acetate is added, ultrasonic treatment turns to dissolving completely
It moves in 100mL volumetric flask with ethyl acetate constant volume, obtains phytosterin ester prepare liquid, research finds ultrasonic time about 60min
It can be completely dissolved；In use, drawing above-mentioned prepare liquid 10mL, it is settled to 100mL with ethyl acetate, at this time phytosterol ester content
For 100 μ g/mL；Above-mentioned prepare liquid 20mL is drawn, is settled to 100mL with ethyl acetate, phytosterol ester content is 200 μ at this time
g/mL。
(2) in the 200 μ L phytosterin ester ethyl acetate solutions that concentration is 50 μ g/mL, phosphorus sulphur iron color developing agent 100 is added
μ L, then develop the color 5~120min at room temperature, measures its absorbance (A) with microplate reader at 492nm, is cross with absorbing wavelength
Coordinate, absorbance (A) are ordinate, draw standard curve；Research is found: the color stability after 25min, shown in result figure 5.
Therefore select developing time for 25min, and the color stability in 120min.
5, detection limit
According to the regulation of International Federation of Theoretical and Applied Chemistry (I μ PAC), spectrophotometry sample, to deduct sky
It is detection limit that absorbance after white value, which is 0.01 corresponding concentration,.
A series of phytosterol ester solution for preparing low concentrations measures its absorbance according to the method for above-mentioned standard curve
Value determines that the detection limit of method, detection limit value are 1.25 μ g/mL according to linear relationship and absorbance value, and the results are shown in Table 3.
3 detection limit test result of table
6, interference substance is tested
(1) it takes 100mg phytosterin ester to be placed in a beaker, 80mL ethyl acetate is added, ultrasonic treatment turns to dissolving completely
It moves in 100mL volumetric flask with ethyl acetate constant volume, obtains phytosterin ester prepare liquid, research finds ultrasonic time about 60min
It can be completely dissolved；Above-mentioned prepare liquid 10mL is drawn when use, is settled to 100mL with ethyl acetate, at this time phytosterol ester content
For 100 μ g/mL；Above-mentioned prepare liquid 20mL is drawn, is settled to 100mL with ethyl acetate, phytosterol ester content is 200 μ at this time
g/mL。
(2) 0.1g glucose, 0.1g rice protein, 0.1g konjac glucomannan, 0.1gTween 20 is taken to be placed in four beakers respectively
In, be added 100mL ethyl acetate, ultrasonic treatment to dissolve completely, obtain glucose interference solution, rice protein interference solution,
Konjac glucomannan interferes solution, Tween 20 to interfere solution.
(3) 100 μ g/mL phytosterin ester ethyl acetate solution, 100 μ L is added, adds above-mentioned 100 μ L of interference solution respectively
The mixed solution of phytosterin ester and each component is obtained, 100 μ L of phosphorus sulphur iron color developing agent is added, develop the color 25min at room temperature,
Its absorbance (A) is measured with microplate reader under 492nm, discovery glucose, rice protein, Tween 20 are not produced in the color development system
Raw disturbing reaction, and konjac glucomannan has colour developing phenomenon, as a result shown in table 4.
4 interference substance experimental result of table
3 precision of embodiment and the rate of recovery
1) standard curve of Δ A and the phytosterin ester of various concentration are drawn: it is different that 200 μ L being added in Xiang Weikong ELISA Plate
The ethyl acetate solution of the phytosterin ester of concentration, 100 μ L phosphorus sulphur iron color developing agents, is put into multi-function microplate reader and vibrates, in room
The lower 25min that develops the color of temperature, measures absorbance value, wherein reagent blank is denoted as A at optimal wavelength 492nm0, containing phytosterin ester
Ethyl acetate solution is denoted as A, and calculates Δ A=A-A0, establish the standard curve of Δ A Yu phytosterol ester concentration.
2) preparation of phosphorus sulphur iron color developing agent: 1gFeCl is weighed3﹒ 6H2O (AR grades) is dissolved in 85% concentrated phosphoric acid, with phosphoric acid constant volume
It to 100mL, stores in brown bottle, refrigerates, can be reserved for 1 year, obtain 10%FeCl3Solution；Draw 1mL10%FeCl3Solution adds
Enter into 100mL volumetric flask, with phosphoric acid constant volume, store in brown bottle, refrigerate, can be reserved for 1 year, obtain 0.1%FeCl3Solution；
Take 0.1%FeCl3Solution 1.5mL is settled to graduation mark in 100mL brown volumetric flask, with the concentrated sulfuric acid (AR grades).
3) preparation of 100 μ g/mL phytosterin ester standard solution: accurately weighing phytosterin ester standard specimen 100.0mg, utilizes
Ultrasound is dissolved in ethyl acetate, is settled to 100mL, and phytosterol ester content is 1.00mg/mL, stores in brown bottle, cryo-conservation
It is spare.Above-mentioned prepare liquid 10mL is drawn when use, is settled to 100mL with ethyl acetate, and phytosterol ester content is 100 μ at this time
g/mL；Above-mentioned prepare liquid 20mL is drawn, is settled to 100mL with ethyl acetate, phytosterol ester content is 200 μ g/mL at this time.
4) phytosterol ester content determines in sample: phytosterin ester nanoemulsions being taken to extract through demulsification, ethyl acetate, is dilute
The 200 μ L of solution obtained after releasing, is measured by step 1 detection method, its absorbance value A is measured at 492nm, and calculate Δ
A=A-A0, Δ A substitution equation of linear regression is acquired to the content of phytosterin ester in sample.
Standard curve obtained by this experiment is y=0.0051x+0.0667, R2=0.9974, concentration range is 25~125 μ
g/mL.The solvent of the selecting response ethyl acetate as phytosterin ester, chromogenic reaction time are 25min, and absorbing wavelength is
492nm, while the TWEEN Series such as the protein such as the monosaccharide such as glucose, rice protein, Tween 20 can be excluded, colour developing is tested
Interference.25.0 μ g/mL, 50.0 μ g/mL are selected, 75.0 each six parallel samples of every group of concentration of tri- kinds of μ g/mL do precision reality
It tests, RSD is respectively 1.90%, 1.34%, 0.83%, and the results are shown in Table 5.Phytosterin ester nanoemulsions are through demulsification, second
Obtained solution after acetoacetic ester extraction, dilution, and measure the concentration of nine groups of parallel samples, be added 12.5 μ g/mL, 25.0 μ g/mL,
The standard sample of 37.5 μ g/mL does recovery of standard addition experiment, and the results are shown in Table 6.
5 precision experiment result of table
6 sample recovery of standard addition of table analyzes result
Embodiment 3
The present embodiment is directly tested with commercially available phytosterin ester, it may be assumed that 100mg phytosterin ester is taken to be placed in beaker
In, 80mL organic solvent is added, ultrasonic treatment is transferred in 100mL volumetric flask to dissolving completely, fixed with organic solvent of the same race
Hold, obtains phytosterin ester prepare liquid.
In remaining step, in addition to the parameter shown in the following table 7 is changed, remaining content is same as Example 1.
Table 7
As shown in Table 7, for ethyl acetate as solvent, selecting chromogenic agent is 0.0015%, when being detected using microplate reader,
Standard curve fit degree is best, and ethyl acetate dissolution phytosterin ester is more faster than dehydrated alcohol.
Claims (9)
1. a kind of method for accurately detecting phytosterol ester content using microplate reader, which comprises the following steps:
(1) sample to be tested containing phytosterin ester is taken, is added in solvent ethyl acetate, ultrasonication is complete to phytosterin ester
After fully dissolved, phytosterin ester prepare liquid is obtained；
(2) the phosphorus sulphur iron color developing agent that phytosterin ester prepare liquid and mass concentration are 0.0015%~0.015% is mixed and is shaken up
Afterwards, it develops the color at room temperature, obtains developing solution；
(3) it takes the developing solution of 50~300 μ L as being detected in microplate reader, measures the absorption photometric value of sample to be tested；
(4) phytosterin ester standard sample is taken, according to step (1)~(3), obtains the absorption photometric of various concentration standard sample
Value obtains the equation of linear regression of absorption photometric value Yu phytosterol ester content after drawing standard curve；
(5) absorption photometric value of sample to be tested in step (3) is substituted into the equation of linear regression of step (4), is obtained to test sample
The content of phytosterin ester in product.
2. the method for accurately detecting phytosterol ester content using microplate reader as described in claim 1, which is characterized in that step
(1) in, the time of the ultrasonication is 5~60min, and power is 90~110W.
3. the method for accurately detecting phytosterol ester content using microplate reader as described in claim 1, which is characterized in that step
(1) in, the mass volume ratio of phytosterin ester and ethyl acetate in sample to be tested is 25~125 μ g:1mL.
4. the method for accurately detecting phytosterol ester content using microplate reader as described in claim 1, which is characterized in that step
(2) in, the mass concentration of the phosphorus sulphur iron color developing agent is 0.0015%.
5. the method for accurately detecting phytosterol ester content using microplate reader as described in claim 1, which is characterized in that step
(2) in, the time of the colour developing is 25min~2h.
6. the method for accurately detecting phytosterol ester content using microplate reader as described in claim 1, which is characterized in that described
The time of colour developing is 25min.
7. the method for accurately detecting phytosterol ester content using microplate reader as described in claim 1, which is characterized in that step
(3) in, the absorbing wavelength when microplate reader detects is 400~700nm.
8. the method for accurately detecting phytosterol ester content using microplate reader as described in claim 1, which is characterized in that step
(3) in, the absorbing wavelength when microplate reader detects is 492nm.
9. the method for accurately detecting phytosterol ester content using microplate reader as described in claim 1, which is characterized in that step
(3) in, the concentration range of phytosterin ester is 25~125 μ g/mL in standard curve.
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| CN115128020B (en) * | 2022-04-15 | 2024-05-03 | 盐城师范学院 | C in fermentation liquor21System for measuring steroid content, construction method and application thereof |
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