CN116203179A - A detection method for the determination of various free sterols and sterol esters in phytosterol esters - Google Patents

Abstract

The invention belongs to the technical field of quality standards of health-care foods and detection thereof, and particularly relates to a detection method for measuring total amounts of various free sterols and sterol esters in plant sterol esters. The invention utilizes a C18 solid phase extraction column purification technology to separate and remove other components and enrich target components, and utilizes a reversed phase liquid chromatography to quantitatively detect the original free sterol content in four raw materials of stigmasterol, campesterol, beta-sitosterol and brassicasterol; and then the sample is weighed for saponification to convert sterol ester into sterol, the methanol-water-phosphoric acid mixed solvent is taken as a mobile phase A, the methanol-isopropanol mixed solvent is taken as a mobile phase B, the methanol is taken as a mobile phase C, gradient elution is carried out through a reversed-phase C18 analytical column, and the total amount of sterol (including free sterol originally existing and sterol obtained by saponification and hydrolysis) in the sample is measured, so that the content of plant sterol ester in the raw material is converted.

Description

A method for determining the total amount of free sterols and sterol esters in plant sterol esters

Technical Field

The invention belongs to the technical field of quality standards and detection of health foods, and specifically relates to a detection method for determining the total amount of multiple free sterols and sterol esters in plant sterol esters.

Background Art

Phytosterol esters are obtained by saponification, extraction, crystallization and other processes using vegetable oil fractions such as soybean oil or taro oil as raw materials. They are a natural ingredient in plants. Phytosterols and sunflower oil fatty acids are then esterified to produce phytosterol esters, which were approved in 2010 to be included in the new resource food catalog as a raw material for health foods. Phytosterol esters mainly contain stigmasterol, campesterol, β-sitosterol, and a small amount of rapeseed sterol. During the production process, the various free phytosterols are not completely esterified to form phytosterol esters, which coexist in the product with phytosterol esters, accounting for about 2% to 6%. Phytosterol esters can inhibit the absorption of cholesterol in the intestines of humans and animals, and show that they can effectively reduce total cholesterol and low-density lipoprotein cholesterol in the blood. Based on its auxiliary effect of lowering blood lipids, it is favored by health product developers as a raw material for health foods.

At present, there is no authoritative detection method for the phytosterol ester components in health foods, and only literature on the detection of multiple sterols in vegetable oils can be consulted. Manufacturers that provide phytosterol ester raw materials use GC methods to detect free phytosterols and total phytosterols respectively. The gas chromatograph needs to be equipped with a cold column head to complete the detection of all indicators. The amount of free phytosterols is first deducted, and the sterols involved in the esterification reaction are converted into sterol esters by coefficient calculation according to the production process. The approved new resource food phytosterol esters use free phytosterols, total phytosterols, phytosterol esters, phytosterol esters and phytosterols (total) to control the quality of raw materials; when determining free phytosterols, a cold column head is required to inject and determine free phytosterols, and the sample is saponified and hydrolyzed, and the extract is extracted after derivatization. The total phytosterols (including the original free phytosterols and the hydrolyzed phytosterols) are determined by GC, and the process is cumbersome and complicated. Most of the other public literature reports use HPLC to determine various phytosterols in vegetable oils, but do not describe the specific data of the separation between the peaks of each sterol. There is only a textual description that the separation between the peaks of each sterol can reach baseline separation. In fact, when the HPLC method is used to detect various sterols in vegetable oils such as soybean oil, it is found that the separation of stigmasterol, campesterol, β-sitosterol, and rapeseed sterol has poor separation, or overlapping peaks, and the content of various phytosterols cannot be accurately determined. Some researchers also use GC to determine various phytosterols in vegetable oils. The sample pretreatment method is basically the same as that of the manufacturer providing the phytosterol ester raw material. All of them are saponified and hydrolyzed samples, and the extracts are extracted after derivatization for sterol determination. The results express the total amount of phytosterols in the sample, and the free sterols contained in the sample itself cannot be detected. Due to the limitation of the detection equipment (cold column head device), this method is not fully applicable to the detection of phytosterol ester raw materials, because phytosterol esters contain 2% to 6% of free phytosterols in addition to sterol esters.

Summary of the invention

The invention aims to solve the problem that the separation effect of various phytosterols in the prior art is poor, the accuracy of the determination result is low, and the operation process is cumbersome and complicated. Under the condition that a cold column head of a gas chromatograph is not provided, the invention provides a detection method for determining the total amount of various free sterols and sterol esters in phytosterol esters. The method uses a C18 solid phase extraction column purification technology to separate and remove other components, enrich the target components, and use a reversed phase liquid chromatography method to quantitatively detect the content of the free sterols originally existing in four raw materials, namely, stigmasterol, campesterol, β-sitosterol, and rapeseed sterol; then weigh the sample for saponification to convert the sterol esters into sterols, use a methanol-water-phosphoric acid mixed solvent as a mobile phase A, a methanol-isopropanol mixed solvent as a mobile phase B, and methanol as a mobile phase C, and perform gradient elution through a reversed phase C18 analytical column to determine the total amount of sterols in the sample (including the free sterols originally existing and the sterols obtained by saponification and hydrolysis), thereby converting the content of the phytosterol esters in the raw materials. In the detection method provided by the present invention, phosphoric acid is used as an ion inhibitor in the mobile phase system to affect the dissociation properties of the substance being measured, and gradient elution is used to make the peak shape of each component good, and the separation degree is greater than 1.4, thereby solving the problem that multiple sterol components in the prior art cannot be effectively separated, providing reference data for the formulation of quality standards and quality inspection of plant sterol ester raw materials and corresponding terminal health foods, as well as providing data support for raw material screening and determining process parameters for new product development.

In order to solve the above technical problems, the present invention adopts the following technical solutions:

A detection method for determining the total amount of multiple free sterols and sterol esters in plant sterol esters, using a methanol-water-phosphoric acid mixed solvent as mobile phase A, a methanol-isopropanol mixed solvent as mobile phase B, and methanol as mobile phase C, and using a reversed phase analytical column to perform gradient elution on different test solutions to respectively determine the total amount of multiple free sterols and sterol esters in the plant sterol esters.

Preferably, the gradient elution is performed using a methanol-water-phosphoric acid mixed solvent with a volume ratio of 385:115:0.4 as the mobile phase A, a methanol-isopropanol mixed solvent with a volume ratio of 7:3 as the mobile phase B, and methanol as the mobile phase C; the reverse phase analytical column is a _C18 analytical column.

Preferably, the specific operation of the gradient elution is: at 0 to 5 minutes, the initial volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is 90:10:0; at 5.1 to 8 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C changes linearly and uniformly from 90:10:0 to 8:10:82; at 8.1 to 28 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 8:10:82; at 28.1 to 38 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 90:10:0.

In the initial elution period, the properties of the target components are very similar, and the mobile phase needs to be changed gradually and precisely to successfully separate the target components. And because the target components have completely peaked before 28 minutes, the column returns to the starting gradient after 28 minutes for column equilibrium and preparation before the next injection. Therefore, there is no need to set a gradient in the last two periods, and the appropriate mobile phase can be used directly.

Preferably, the detection wavelength of the gradient elution is 210 nm; the flow rate of the gradient elution is 1.0 mL/min, the injection volume is 20 μL, and the column temperature is 38-42° C.

Preferably, the reverse phase analytical column is a Hypersil ODS2 C ₁₈ analytical column.

Preferably, the test solution is prepared before the gradient elution, and the test solution includes test solution I and test solution II;

The preparation of the test solution I is specifically as follows: weigh the sample, place it in a volumetric flask, add an organic solvent, shake and extract, take it out and put it at room temperature, make it up to volume with the organic solvent, shake it well, let it stand, take the supernatant as the sample extract, draw the sample extract for elution, after receiving the eluent, evaporate it to dryness, leave a residue, add a dissolving solvent to dissolve the residue, and obtain the test solution I for the determination of a plurality of free sterols;

The preparation of the test solution II is specifically as follows: weigh the sample, perform saponification reflux, transfer and make up the volume with the organic solvent, draw the supernatant into a separatory funnel, extract with an extractant, combine the extracts, wash with pure water, dehydrate the organic layer with a dehydrating agent, and evaporate to dryness to leave a residue, add the dissolving solvent to dissolve the residue, and obtain the test solution II for the determination of total sterols.

Preferably, the organic solvent includes one or more of water, methanol, ethanol, and isopropanol; the dissolving solvent includes one or more of methanol, ethanol, and isopropanol;

In the preparation of the test solution I, the shaking extraction includes ultrasonic extraction; the elution is carried out in a _C18 solid phase extraction column (2g/6mL) and eluted with methanol;

The type of solid phase extraction cartridge can also be Waters tC18 Cartridges (1 g/6 mL) or AgelaCleanert S C18 (1 g/6 mL). The cartridge pretreatment method is to wash the cartridge with 3 column volumes of methanol.

In the preparation of the test solution II, the saponification reflux is carried out in a BHT potassium hydroxide ethanol solution; the heating reflux method of the saponification reflux is water bath heating; the extractant is n-hexane; and the dehydrating agent is anhydrous sodium sulfate.

Preferably, the organic solvent is a 95% ethanol aqueous solution; the dissolving solvent is a mixture of methanol and isopropanol in a volume ratio of 1:4;

In the preparation of the test solution I, the mass volume ratio of the sample to the organic solvent is 0.7 g:100 mL; the power of the ultrasonic extraction is 500 W, and the time is 60 min; the volume ratio of the sample amount to methanol in the elution is 2 mL:25 mL; the volume ratio of the sample amount to the dissolving solvent is 2 mL:5 mL;

In the preparation of the test solution II, the mass volume ratio of the sample to the BHT potassium hydroxide ethanol solution is 0.25g:20mL; the temperature of the saponification reflux is 95°C, and the reflux time is 90min; the number of extractions with the extractant is 3 times; the number of pure water washings is 3 times; the mass volume ratio of the sample to the dissolving solvent is 0.05g:50mL.

Preferably, after the gradient elution, the chromatogram is recorded, and qualitative analysis is performed according to the retention time of the reference substances of rapeseed sterol, stigmasterol, campesterol, and β-sitosterol. The concentrations of rapeseed sterol, stigmasterol, campesterol, and β-sitosterol in the test solution are respectively obtained from the standard curve, and the corresponding content of each sterol is calculated respectively, and the total amount of free sterols and the total amount of sterols are calculated, and the total amount of phytosterol esters is calculated according to the following formula:

Z＝(Y-W)×1.62

Among them, the sterol ester coefficient is provided by the raw material manufacturer.

Preferably, the standard curve is obtained by gradient elution of a standard curve solution under the same conditions, and the preparation of the standard curve solution comprises:

A. Accurately weigh 10 mg of rapeseed sterol reference substance, dissolve it in isopropanol and make up to 10 mL, to obtain a 1 mg/mL rapeseed sterol stock solution; accurately weigh 20 mg of stigmasterol reference substance, dissolve it in isopropanol and make up to 20 mL, to obtain a 1 mg/mL stigmasterol stock solution; accurately weigh 10 mg of campesterol reference substance, dissolve it in isopropanol and make up to 10 mL, to obtain a 1 mg/mL campesterol stock solution; accurately weigh 20 mg of β-sitosterol reference substance, dissolve it in isopropanol and make up to 10 mL, to obtain a 2 mg/mL β-sitosterol stock solution;

B. Accurately pipette 1.0 mL of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, respectively, mix them, and dilute them to 5 mL with a dilution solution to prepare control solutions with a rapeseed sterol concentration of 0.2 mg/mL, a stigmasterol concentration of 0.2 mg/mL, a campesterol concentration of 0.2 mg/mL, and a β-sitosterol concentration of 0.4 mg/mL, as standard curve solution V;

C. Accurately pipette 1.0 mL of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, respectively, mix them, and dilute them to 10 mL with the dilution solution to prepare control solutions with a rapeseed sterol concentration of 0.1 mg/mL, a stigmasterol concentration of 0.1 mg/mL, a campesterol concentration of 0.1 mg/mL, and a β-sitosterol concentration of 0.2 mg/mL, as standard curve IV;

D. Accurately pipette 1.0 mL of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, respectively, mix them, and dilute them to 25 mL with the dilution solution to prepare control solutions with a rapeseed sterol concentration of 0.04 mg/mL, a stigmasterol concentration of 0.04 mg/mL, a campesterol concentration of 0.04 mg/mL, and a β-sitosterol concentration of 0.08 mg/mL, as standard curve III;

E. Accurately pipette 1.0 mL of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, respectively, mix them, and dilute them to 50 mL with the dilution solution to prepare control solutions with a rapeseed sterol concentration of 0.02 mg/mL, a stigmasterol concentration of 0.02 mg/mL, a campesterol concentration of 0.02 mg/mL, and a β-sitosterol concentration of 0.04 mg/mL, as standard curve II;

F. Accurately pipette 1.0 mL of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, respectively, mix them, and dilute them to 100 mL with the dilution solution to prepare control solutions with a rapeseed sterol concentration of 0.01 mg/mL, a stigmasterol concentration of 0.01 mg/mL, a campesterol concentration of 0.01 mg/mL, and a β-sitosterol concentration of 0.02 mg/mL, as standard curve I;

The dilution solution is a mixture of methanol and isopropanol in a volume ratio of 1:4.

Compared with the prior art, the present invention has the following beneficial effects:

(1) C18 solid phase extraction column purification technology was used to separate and remove other components, enrich the target components, and eliminate the interference of other components on the detection of free phytosterols.

(2) Using methanol-water-phosphoric acid mixed solvent as A, methanol-isopropanol mixed solvent as mobile phase B, methanol as mobile phase C, gradient elution, phosphoric acid is used as an ion inhibitor to affect the dissociation properties of the substance being measured, and in combination with gradient elution, it has a good adsorption and desorption effect, improves the separation ability of reversed-phase chromatography, enables a variety of sterols to be well separated, and the detection results are accurate and reliable.

(3) The problem of poor separation of various plant sterols and low accuracy of measurement results in the prior art is solved, the cumbersome sample pretreatment operation process is simplified, the amount of solvent used is reduced, costs are saved, and the pressure on environmental protection is reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a chromatogram of the specificity test of the present invention.

DETAILED DESCRIPTION

In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Example 1

A method for determining the total amount of free sterols and sterol esters in phytosterol esters comprises the following steps:

(I) Preparation of test solution I: Weigh 0.7 g of plant sterol ester sample accurately, place in a 100 mL volumetric flask, add 60 mL of 95% ethanol, perform ultrasonic extraction (500 W, 60 min), shake occasionally, take out and place at room temperature, dilute with 95% ethanol, shake well, let stand for 10 min, and take the supernatant as the sample extract. Take an Agela Cleanert S C18 column (1 g/6 mL), rinse the column with methanol 3 times, 6 mL each time. Accurately pipette 2.0 mL of sample extract onto the sample, open the valve, and use a stoppered test tube at the bottom of the device to receive the wash solution. When the extract flows to the same level as the stationary phase, rinse the column with methanol 5 times, 5 mL each time. After receiving all the wash solution, place it at 60°C and recycle it to dryness. Accurately pipette 5 mL of methanol to dissolve the residue, filter it, and obtain the free sterol determination solution.

(II) Preparation of test solution II: Accurately weigh 0.25 g of phytosterol ester sample, place in a 150 mL conical flask, add 20 mL of potassium hydroxide ethanol solution (weigh 12 g of potassium hydroxide, 10-15 mg of BHT, place in a beaker, add 10 mL of pure water, after the solid is dissolved, add 90 mL of anhydrous ethanol, mix well, and let it cool to room temperature), place in a 95°C water bath for reflux extraction for 90 min, gently shake from time to time during the reflux process, take out and let it cool to room temperature, completely transfer the saponified solution to a 100 mL volumetric flask, make up to volume with 95% ethanol, shake well, and let it stand for 10 min , accurately pipette 20.0 mL of supernatant into a separatory funnel, add 20 mL of n-hexane, extract for 30 seconds, let stand and separate the layers, take the upper n-hexane liquid into another clean separatory funnel, extract the water layer of the extract twice with n-hexane in the same way, combine the three n-hexane extracts, wash with pure water three times, 100 mL each time, dehydrate the n-hexane with anhydrous sodium sulfate, collect it in a round-bottom flask, recover it by rotary evaporation at 40°C to dryness, and obtain the residue, accurately add 50.0 mL of methanol-isopropanol (1:4) to dissolve the residue, filter it, and obtain the total sterol determination solution.

(III) HPLC conditions

Mobile phase: using a methanol-water-phosphoric acid mixed solvent with a volume ratio of 385:115:0.4 as mobile phase A, a methanol-isopropanol mixed solvent with a volume ratio of 7:3 as mobile phase B, and methanol as mobile phase C, for the gradient elution.

Gradient elution: at 0 to 5 minutes, the initial volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is 90:10:0; at 5.1 to 8 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C changes linearly and uniformly from 90:10:0 to 8:10:82; at 8.1 to 28 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 8:10:82; at 28.1 to 38 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 90:10:0.

Chromatographic column: Hypersil ODS2 C18 analytical column (Elite, specification 4.6×250mm, 5μm)

(IV) Preparation of standard solution

A. Preparation of reference substance stock solution:

Accurately weigh 10 mg of rapeseed sterol reference substance, dissolve it in isopropanol and make up to 10 mL, to obtain a 1 mg/mL rapeseed sterol stock solution; accurately weigh 20 mg of stigmasterol reference substance, dissolve it in isopropanol and make up to 20 mL, to obtain a 1 mg/mL stigmasterol stock solution; accurately weigh 10 mg of campesterol reference substance, dissolve it in isopropanol and make up to 10 mL, to obtain a 1 mg/mL campesterol stock solution; accurately weigh 20 mg of β-sitosterol reference substance, dissolve it in isopropanol and make up to 10 mL, to obtain a 2 mg/mL β-sitosterol stock solution;

B. Accurately pipette 1.0 mL of each of the rapeseed sterol stock solution, stigmasterol stock solution, campesterol stock solution, and β-sitosterol stock solution, mix them, and dilute to 5 mL with methanol-isopropanol (1:4). The control solution with a rapeseed sterol concentration of 0.2 mg/mL, a stigmasterol concentration of 0.2 mg/mL, a campesterol concentration of 0.2 mg/mL, and a β-sitosterol concentration of 0.4 mg/mL is used as the standard curve solution V.

C. Accurately pipette 1.0 mL of each of the rapeseed sterol stock solution, stigmasterol stock solution, campesterol stock solution, and β-sitosterol stock solution, mix them, and dilute to 10 mL with methanol-isopropanol (1:4). The control solution with a rapeseed sterol concentration of 0.1 mg/mL, a stigmasterol concentration of 0.1 mg/mL, a campesterol concentration of 0.1 mg/mL, and a β-sitosterol concentration of 0.2 mg/mL is used as the standard curve solution IV.

D. Accurately pipette 1.0 mL of each of the rapeseed sterol stock solution, stigmasterol stock solution, campesterol stock solution, and β-sitosterol stock solution, mix them, and dilute them to 25 mL with methanol-isopropanol (1:4). The control solution with a rapeseed sterol concentration of 0.04 mg/mL, a stigmasterol concentration of 0.04 mg/mL, a campesterol concentration of 0.04 mg/mL, and a β-sitosterol concentration of 0.08 mg/mL is used as the standard curve solution III.

E. Accurately pipette 1.0 mL of each of the rapeseed sterol stock solution, stigmasterol stock solution, campesterol stock solution, and β-sitosterol stock solution, mix them, and dilute to 50 mL with methanol-isopropanol (1:4). The control solution with a rapeseed sterol concentration of 0.02 mg/mL, a stigmasterol concentration of 0.02 mg/mL, a campesterol concentration of 0.02 mg/mL, and a β-sitosterol concentration of 0.04 mg/mL is used as the standard curve solution II.

F. Accurately pipette 1.0 mL of each of the rapeseed sterol stock solution, stigmasterol stock solution, campesterol stock solution, and β-sitosterol stock solution, mix them, and dilute to 100 mL with methanol-isopropanol (1:4). The control solution with a rapeseed sterol concentration of 0.01 mg/mL, a stigmasterol concentration of 0.01 mg/mL, a campesterol concentration of 0.01 mg/mL, and a β-sitosterol concentration of 0.02 mg/mL is used as the standard curve solution I.

(V) Preparation of test solution II spiked solution: Take the above test solution II and standard curve solution III and mix them in a volume ratio of 1:1.

(VI) Reference substance mixture: Take the above-mentioned standard curve solution II as the reference substance mixture.

(VII) Take 20 μL of methanol-isopropanol (1:4), standard curve solution I to standard curve solution V, test solution I, and test solution II, respectively, and inject them into the chromatograph to record the chromatogram according to the chromatographic conditions of (III) in the technical scheme, as shown in Figure 1. According to the retention time of the reference substances of rapeseed sterol, stigmasterol, campesterol, and β-sitosterol, the concentrations of rapeseed sterol, stigmasterol, campesterol, and β-sitosterol in test solution I and test solution II are obtained from the standard curve, and the corresponding content of each sterol is calculated, and the total amount of free sterols and the total amount of sterols are calculated, and the total amount of phytosterol esters is calculated according to the following formula.

Z＝(Y-W)×1.62

Example 2

Selection of chromatographic conditions

There is no authoritative detection method for the detection of plant sterol ester components in health foods. The published literature mainly involves liquid chromatography, gas chromatography, and gas chromatography-mass spectrometry to detect various sterols in vegetable oils. From the perspective of detection cost, gas chromatography-mass spectrometry requires the use of multiple gases, and its detection cost is relatively high, which is not the best choice. The published detection of various sterols in vegetable oils (liquid chromatography, gas chromatography) is not suitable for the detection of sterol esters in plant sterol ester raw materials and their corresponding preparations.

After a lot of trial and error, the inventors used a mixed solvent of methanol, isopropanol, and phosphoric acid solution, combined with a gradient elution system, to make the separation between rapeseed sterol, stigmasterol, campesterol, and β-sitosterol greater than 1.4. The developed chromatographic conditions are shown in (iii) of the technical scheme of Example 1. If only a mixed solvent of methanol and isopropanol is used for elution, the separation effect between the sterols is worse than that described in (iii) of the technical scheme of Example 1. If a mixed solvent of methanol, isopropanol, and phosphoric acid solution is used for isocratic elution, the separation effect is also worse than that described in (iii) of the technical scheme of Example 1, as shown in Table 1.

Table 1 Chromatographic conditions investigation

Example 3

Determination of extraction solvent for pretreatment of test solution I

The same batch of plant sterol ester samples were taken, and after weighing the samples, 75% ethanol, 95% ethanol, methanol, and methanol-isopropanol (4:1) were used as extraction solvents, respectively. Other conditions were the same as those in the technical scheme (I) of Example 1. The contents of free rapeseed sterol, stigmasterol, campesterol, and β-sitosterol in the samples were determined. The results are shown in Table 2.

Table 2 Results of the extraction solvent investigation of test solution I (unit: mg/g)

Sample No. Ultrasonic extraction solvent Brassicasterol Stigmasterol Campesterol β-Sitosterol Sample 1 75% ethanol 0.018 1.921 0.19 2.05 Sample 2 95% ethanol 0.086 2.96 1.77 6.39 Sample 3 Methanol 0.026 1.52 1.24 4.25 Sample 4 Methanol-isopropanol (4:1) 0.085 2.87 1.78 6.53

Conclusion: The extraction effects of 95% ethanol and methanol-isopropanol (4:1) are comparable. It is better to choose 95% ethanol because it is cheaper and less toxic.

Example 4

Determination of saponification conditions of test solution Ⅱ

Take the same batch of plant sterol ester samples, weigh the samples, place them in 150mL conical flasks, add 20mL of potassium hydroxide ethanol solution (weigh 12g of potassium hydroxide and 15mg of BHT, place them in a beaker, add 10mL of pure water, and after the solid is dissolved, add 90mL of anhydrous ethanol, mix well, and let it cool to room temperature), and carry out saponification reaction under the following conditions: reflux at 85°C for 1.5h; reflux at 95°C for 1.5h; reflux at 98°C for 1.5h; the remaining conditions are the same as the technical scheme of Example 1, and the contents of rapeseed sterol, stigmasterol, campesterol, and β-sitosterol are determined. The results are shown in Table 3.

Table 3 Results of investigation on saponification reaction conditions in the determination of total phytosterols (unit: mg/g)

Sample No. Saponification reaction conditions Brassicasterol Stigmasterol Campesterol β-Sitosterol Total sterol content Sample 1 Reflux at 85℃ for 90min 7.92 152.66 106.01 259.25 525.84 Sample 2 Reflux at 95℃ for 90min 8.24 163.43 110.29 262.37 544.34 Sample 3 Reflux at 98℃ for 90min 8.26 163.41 110.65 262.33 544.65

Conclusion: The conversion of sterol esters into sterols is related to temperature. The extraction effects of 95℃ and 98℃ reflux are equivalent. Since the maximum operating temperature of the water bath is 98℃, in order to make the temperature conditions more stable, 95℃ reflux is the best choice.

Example 5

Column selection

There is no description of the separation data of each sterol in the existing literature. The inventors have conducted a large number of exploratory experiments and took a mixed standard solution containing rapeseed sterol, stigmasterol, campesterol, and β-sitosterol to investigate most of the chromatographic columns available on the market. Only the chromatographic column described in the technical solution (iii) of Example 1 can achieve good separation of multiple sterols under the gradient elution described in the solution.

Example 6

Specificity and system suitability experiments

According to the technical scheme of Example 1, 20 μL of each of the blank solvent methanol-isopropanol (1:4), test solution I, test solution II, reference substance mixed solution, and test solution II spiked solution were accurately taken for measurement and the chromatogram was recorded as shown in Figure 1.

The determination results show that no obvious chromatographic peaks were detected at the main peak positions of the blank solvents rapeseed sterol, stigmasterol, campesterol, and β-sitosterol, and there was no interference, indicating that the specificity of the method met the requirements; based on the chromatogram and peak area of the reference substance mixture obtained by continuous injection of 6 times, the RSD and separation were calculated, and the separation of each component peak was greater than 1.4. The experimental results are shown in Table 5.

Table 5 System suitability experimental data

Example 7

Limit of Detection and Limit of Quantitation

Rapeseed sterol, stigmasterol, campesterol, and β-sitosterol reference substances were prepared into quantitative limit reference solutions and detection limit reference solutions, and the samples were injected and measured according to the chromatographic conditions of "Technical Scheme of Example 1", and the chromatograms were recorded. The quantitative limit of rapeseed sterol was 0.028 μg, and the detection limit was 0.014 μg; the quantitative limit of stigmasterol was 0.032 μg, and the detection limit was 0.016 μg; the quantitative limit of campesterol was 0.024 μg, and the detection limit was 0.012 μg; the quantitative limit of β-sitosterol was 0.066 μg, and the detection limit was 0.033 μg.

Example 8

Linear relationship test

According to the chromatographic conditions of "Technical Scheme of Example 1", 20 μL of each of standard solution I to standard solution VI was accurately drawn and injected into the liquid chromatograph, and the chromatogram was recorded. The peak area of each component was used as the ordinate and the concentration was used as the abscissa to calculate the regression equation. From the results, it can be seen that the standard linear equation of rapeseed sterol is y=103.2337x-0.0219, and the correlation coefficient r ² =0.9992; the standard linear equation of stigmasterol is y=99.8980x+0.9949, and the correlation coefficient r ² =0.9988; the standard linear equation of campesterol is y=91.8408x+0.2588, and the correlation coefficient r ² =0.9993; the standard linear equation of β-sitosterol is y=83.7421x+0.5470, and the correlation coefficient r ² =0.9992; it shows that the method presents a good linear relationship.

Example 9

Precision test

The same batch of phytosterol ester samples were taken to prepare test solution I and test solution II according to the "Technical Scheme of Example 1", and the chromatographic conditions were respectively measured, and the chromatograms were recorded. The concentrations of rapeseed sterol, stigmasterol, campesterol, and β-sitosterol in test solution I and test solution II were respectively obtained according to the measured standard curves, and the corresponding content of each sterol was calculated respectively, and then the total amount of free sterols and the total amount of sterols were calculated and converted into the amount of sterol esters. The average and RSD of the total phytosterol ester content of the 6 samples were calculated. The results are shown in Table 6.

Table 6 Precision test results

Example 10

Investigate the effect of different column temperatures (38°C and 42°C) on sample determination

Two portions of the same batch of samples were taken respectively, and the test solution I and the test solution II were prepared according to the operation described in "Technical Scheme of Example 1". According to the chromatographic conditions in "Technical Scheme of Example 1 (III)", the column temperatures were 38°C and 42°C, respectively. The separation results between the four sterols at 38°C to 42°C are shown in Table 8. The results show that the separation between the four sterols is not greater than 1.4, and is relatively excellent when the column temperature is 40°C.

Table 8 Separation results between four sterols at different temperatures

Separation Brassicasterol and stigmasterol Stigmasterol and Campesterol Campesterol and β-sitosterol 38℃ 3.71 1.41 1.50 40℃ 3.75 1.43 1.54 42℃ 3.81 1.42 1.55

Embodiment 11

Determination of free sterols and total sterol esters in several batches of phytosterol esters

Three batches of plant sterol ester samples were taken and operated according to the technical scheme of Example 1 to measure the total amount of various free sterols and sterol esters in the samples. The measurement results are shown in Table 9.

Table 9 Determination results of total amount of free sterols and sterol esters in three batches of phytosterol esters

batch number Total sterols Free sterols Total sterol esters A1 54.43% 1.12% 86.37% A2 55.25% 1.13% 87.69% A3 54.65% 1.10% 86.76%

From the determination results, it can be seen that the free sterol and total sterol ester contents in the plant sterol ester samples can be quickly detected by the method of the present invention, and the determination results of the three batches of plant sterol ester samples are in line with the internal control standards of the enterprise.

The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims (10)

1. the detection method of multiple free sterols and sterol ester total amount determination in a kind of phytosterol ester, it is characterized in that, be mobile phase A with methanol-water-phosphoric acid mixed solvent, methanol-isopropanol mixed solvent is mobile phase B , Methanol was the mobile phase C, and the reversed-phase analytical column was used to carry out gradient elution for different test solutions, and the various free sterols and the total amount of sterol esters in phytosterol esters were determined respectively. 2. the detection method that multiple free sterols and sterol ester total amount are measured in phytosterol ester as claimed in claim 1 is characterized in that, be the methanol-water-phosphoric acid mixed solvent that is 385:115:0.4 with volume ratio as described Mobile phase A, the methanol-isopropanol mixed solvent with a volume ratio of 7:3 is the mobile phase B, methanol is the mobile phase C, and the gradient elution is carried out; the reversed-phase analytical column is _C18 Analytical column. 3. the detection method of multiple free sterols and sterol ester total amount determination in the phytosterol ester as claimed in claim 1, is characterized in that, the specific operation of described gradient elution is: at 0～5min, described mobile phase A , the initial volume ratio of the mobile phase B and the mobile phase C is 90:10:0; 5.1～8min, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is from 90: 10:0, change linearly and uniformly to 8:10:82; at 8.1~28min; the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C remains at 8:10:82; at 28.1~38min , the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 90:10:0. 4. the detection method that multiple free sterols and sterol ester total amount are measured in phytosterol ester as claimed in claim 1, is characterized in that, the detection wavelength of described gradient elution is 210nm; The flow velocity of described gradient elution is 1.0 mL/min, the injection volume is 20 μL, and the column temperature is 38-42°C. 5. the detection method of multiple free sterols and sterol ester total amount determination in the phytosterol ester as claimed in claim 1, is characterized in that, described reverse-phase analysis post is Hypersil ODS2 C ₁₈ analysis post. 6. the detection method that multiple free sterols and sterol ester total amount are measured in the phytosterol ester as claimed in claim 1, is characterized in that, before described gradient elution, carries out the preparation of test solution, and described test solution comprises for Test solution Ⅰ and test solution Ⅱ; The preparation of the test solution I is as follows: weigh the sample, put it in a volumetric flask, add an organic solvent, shake and extract, take it out and put it at room temperature, use the organic solvent to make up the volume, shake it up, let it stand, take the The supernatant, as the sample extract, was drawn to elute the sample extract, and after receiving the eluate, it was rotary evaporated to dryness, and the residue was left, and a dissolving solvent was added to dissolve the residue to obtain the test solution I, which was used for determination Various free sterols; The preparation of the test solution II specifically includes: weighing the sample, carrying out saponification and reflux, transferring and constant volume with the organic solvent, drawing the supernatant into a separatory funnel, extracting with an extractant, and combining the extracts, After washing with pure water, the organic layer was dehydrated with a dehydrating agent, and then rotary evaporated to dryness to leave a residue, which was dissolved by adding the dissolving solvent to obtain the test solution II for the determination of total sterols. 7. the detection method that multiple free sterols and sterol ester total amount are measured in the phytosterol ester as claimed in claim 6, is characterized in that, described organic solvent comprises one or more in water, methyl alcohol, ethanol, Virahol A kind; The dissolving solvent comprises one or more in methanol, ethanol, isopropanol; In the preparation of the test solution I, the oscillating extraction includes ultrasonic extraction; the elution is carried out in a _C18 solid-phase extraction cartridge, and eluted with methanol; In the preparation of the test solution II, the saponification reflux was carried out in BHT potassium hydroxide ethanol solution; the heating and reflux mode of the saponification reflux was heating in a water bath; the extractant was n-hexane; the dehydrating agent was anhydrous sodium sulfate. 8. the detection method that multiple free sterols and sterol ester total amount are measured in phytosterol ester as claimed in claim 7, is characterized in that, described organic solvent is 95% ethanol aqueous solution; Described dissolving solvent is methyl alcohol, Virahol Mix by volume ratio 1:4; In the preparation of the test solution I, the mass volume ratio of the sample to the organic solvent was 0.7g:100mL; the power of the ultrasonic extraction was 500w, and the time was 60min; The volume ratio of the sample is 2mL: 25mL; the volume ratio of the loading amount to the dissolving solvent is 2mL: 5mL; In the preparation of the test solution II, the mass volume ratio of the sample to the BHT potassium hydroxide ethanol solution was 0.25g: 20mL; the saponification reflux temperature was 95°C, and the reflux time was 90min; the extractant The number of extractions is 3 times; the number of times of pure water washing is 3 times; the mass volume ratio of the sample to the dissolving solvent is 0.05g:50mL. 9. the detection method that multiple free sterols and sterol ester total amount are measured in phytosterol ester as claimed in claim 1, is characterized in that, after described gradient elution, record chromatogram, according to brassicasterol, stigmasterol, rapeseed oil The retention time of sterol and β-sitosterol reference substance is qualitatively determined, and the concentrations of brassicasterol, stigmasterol, campesterol and β-sitosterol in the test solution are obtained respectively from the standard curve, and the corresponding contents of each sterol are calculated respectively, and calculated The total amount of free sterols and the total amount of sterols, the total amount of phytosterol esters is calculated according to the following formula: Z=(Y-W)×1.62 Z—the total amount of phytosterol esters, in %; Y—the total amount of phytosterols, in %; W - the total amount of free sterols, in %; 1.62—the conversion coefficient of sterol to sterol ester. 10. the detection method that multiple free sterols and sterol ester total amount are measured in phytosterol ester as claimed in claim 9, is characterized in that, described standard curve obtains by standard curve liquid gradient elution under identical conditions, and described standard The preparation of curve liquid includes: A. Accurately weigh 10mg of brassicasterol reference substance, dissolve it with isopropanol and set the volume to 10mL, and obtain a brassicasterol stock solution with a concentration of 1mg/mL; accurately weigh 20mg of stigmasterol reference substance, dissolve it with isopropanol to volume to 20mL to obtain a stock solution of stigmasterol with a concentration of 1mg/mL; accurately weigh 10mg of the campesterol reference substance, dissolve it in isopropanol to 10mL, and obtain a stock solution of campesterol with a concentration of 1mg/mL; precisely weigh 20mg of β-sitosterol reference substance was dissolved in isopropanol to 10mL to obtain a β-sitosterol stock solution with a concentration of 2mg/mL; B. Accurately draw respectively 1.0mL of the brassicasterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix and release to 5mL with a diluted solution to obtain The brassicasterol concentration is 0.2mg/mL, the stigmasterol concentration is 0.2mg/mL, the campesterol concentration is 0.2mg/mL, and the β-sitosterol concentration is the control solution of 0.4mg/mL, as the standard curve solution V; C. Accurately draw respectively 1.0 mL of the brassicasterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix and release to 10 mL with the diluted solution, Prepare a control solution with a brassicasterol concentration of 0.1 mg/mL, a stigmasterol concentration of 0.1 mg/mL, a campesterol concentration of 0.1 mg/mL, and a β-sitosterol concentration of 0.2 mg/mL as a standard curve IV; D. Accurately draw respectively 1.0 mL of the brassicasterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix and release to 25 mL with the diluted solution, Prepare a control solution with a brassicasterol concentration of 0.04 mg/mL, a stigmasterol concentration of 0.04 mg/mL, a campesterol concentration of 0.04 mg/mL, and a β-sitosterol concentration of 0.08 mg/mL as a standard curve III; E. Accurately draw 1.0 mL each of the brassicasterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution respectively, mix and release to 50 mL with the diluted solution, Prepare a control solution with a brassicasterol concentration of 0.02 mg/mL, a stigmasterol concentration of 0.02 mg/mL, a campesterol concentration of 0.02 mg/mL, and a β-sitosterol concentration of 0.04 mg/mL as a standard curve II; F. Accurately draw respectively 1.0 mL of the brassicasterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix and release to 100 mL with the diluted solution, A control solution with a brassicasterol concentration of 0.01 mg/mL, a stigmasterol concentration of 0.01 mg/mL, a campesterol concentration of 0.01 mg/mL and a β-sitosterol concentration of 0.02 mg/mL was prepared as standard curve I; The diluted solution is formed by mixing methanol and isopropanol at a volume ratio of 1:4.

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