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

Abstract

The invention belongs to the quality standard and detection technology field of health food, and is specifically related to a detection method for measuring the total amount of multiple free sterols and sterol esters in a kind of plant sterol ester. The present invention utilizes C18 solid phase extraction cartridge purification technology, separates and removes other components, enriches target component, utilizes reversed-phase liquid chromatography, first quantitatively detects the free sterol content originally present in four kinds of raw materials of stigmasterol, campesterol, β-sitosterol, and rapeseed sterol; Then weigh the sample and carry out saponification to convert sterol ester into sterol, with methanol-water-phosphoric acid mixed solvent as mobile phase A, methanol-isopropanol mixed solvent as mobile phase B, methanol as mobile phase C, gradient elution is carried out through reversed-phase C18 analytical column, the total amount of sterols in the determination sample (including the free sterols originally present and the sterols obtained by saponification and hydrolysis) is determined, so as to convert the content of plant sterol ester in raw material.

Description

Technical Field

[0001] The invention belongs to the technical field of quality standards and detection of health foods, and particularly relates to a detection method for determining the total amount of multiple free sterols and sterol esters in plant sterol esters. Background Art

[0002] Phytosterol esters are derived from vegetable oil fractions such as soybean oil or tall oil through saponification, extraction, and crystallization. These phytosterols are naturally occurring plant components. These phytosterols are then esterified with sunflower oil fatty acids to produce phytosterol esters. In 2010, these esters were approved for inclusion in the New Resource Food Catalogue as a health food ingredient. Phytosterol esters primarily contain stigmasterol, campesterol, β-sitosterol, and a small amount of rapeseed sterol. During the production process, incomplete esterification of various free phytosterols may occur, resulting in phytosterol esters. These esters, along with the phytosterol esters, contribute approximately 2% to 6% of the product. Phytosterol esters can effectively lower total cholesterol and LDL cholesterol in the blood by inhibiting cholesterol absorption in the intestines of humans and animals. Due to their lipid-lowering properties, they are favored by health product developers as a health food ingredient.

[0003] Currently, there is no authoritative detection method for the phytosterol ester component in health foods, and only literature on the detection of various sterols in vegetable oils can be found. Manufacturers that provide phytosterol ester raw materials use GC methods to detect the free phytosterol and total phytosterol content respectively. The gas chromatograph must be equipped with a cold column chromatograph 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 ester uses four indicators to control the quality of raw materials: free phytosterols, total phytosterols, phytosterol esters, phytosterol esters and phytosterols (total); when determining free phytosterols, a cold column chromatograph is required to inject the sample to determine the free phytosterols, and the sample is saponified and hydrolyzed, and then extracted after derivatization to obtain the extract. The total phytosterols (including the original free phytosterols and the hydrolyzed phytosterols) are determined by GC. The process is tedious and complicated. Most other published reports use HPLC to determine various phytosterols in vegetable oils. Specific data on the resolution between individual sterol peaks are not provided, with only a textual statement stating that baseline resolution is achieved. In practice, when using HPLC to determine various sterols in vegetable oils such as soybean oil, poor resolution or overlapping peaks were observed for stigmasterol, campesterol, β-sitosterol, and rapeseed sterol, preventing accurate determination of the various phytosterol contents. Other researchers have used GC to determine various phytosterols in vegetable oils. Sample pretreatment methods are essentially the same as those used by the manufacturer of the phytosterol ester raw materials: saponification and hydrolysis of the samples, followed by derivatization and extraction, to obtain extracts for sterol determination. The results represent the total amount of phytosterols in the sample and cannot measure free sterols present in the sample. Due to limitations in the testing equipment (cold on-column analyzer), this method is not fully applicable to the determination of phytosterol ester raw materials, as phytosterol esters contain 2% to 6% free phytosterols in addition to sterol esters. Summary of the Invention

[0004] The present invention aims to address the current situation in the prior art where separation effects of various phytosterols are poor, the accuracy of determination results is low, and the operation process is cumbersome and complicated. In the absence of a cold on-column gas chromatograph, the present invention provides a detection method for determining the total amount of various free sterols and sterol esters in phytosterol esters. The method utilizes a C18 solid-phase extraction cartridge purification technique to separate and remove other components and enrich target components. Reverse-phase liquid chromatography is used to first quantitatively detect the content of free sterols originally present in four raw materials, namely, stigmasterol, campesterol, β-sitosterol, and rapeseed sterol. The sample is then weighed and saponified to convert the sterol esters into sterols. Gradient elution is performed on a reverse-phase C18 analytical column 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. The total amount of sterols in the sample (including the originally present free sterols and the sterols obtained by saponification and hydrolysis) is determined, 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. In combination with gradient elution, the peak shape of each component is good, and the separation degree is greater than 1.4. This solves the problem of the inability to effectively separate multiple sterol components in the prior art, provides reference data for the formulation of quality standards and quality testing of plant sterol ester raw materials and corresponding terminal health foods, and provides data support for raw material screening and determining process parameters for new product development.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A detection method for determining the total amount of multiple free sterols and sterol esters in phytosterol esters, comprising: 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 employing 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 phytosterol esters.

[0007] 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 C 18 Analytical column.

[0008] Preferably, the specific operation of the gradient elution is: from 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; from 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; from 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; from 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.

[0009] During the initial elution period, the properties of the target components are extremely similar, requiring precise, controlled, and gradual changes in the mobile phase to successfully separate them. Furthermore, since the target components have completely eluted before 28 minutes, the column returns to the starting gradient after 28 minutes to equilibrate and prepare for the next injection. Therefore, a gradual change is not required during the last two periods; the appropriate mobile phase can be used directly.

[0010] 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.

[0011] Preferably, the reverse phase analytical column is Hypersil ODS2 C 18 Analytical column.

[0012] Preferably, the test solution is prepared before the gradient elution, and the test solution includes test solution I and test solution II;

[0013] The test solution I is prepared as follows: weighing a sample, placing it in a volumetric flask, adding an organic solvent, extracting by shaking, taking it out and letting it cool to room temperature, making up the volume with the organic solvent, shaking it well, letting it stand, taking the supernatant as the sample extract, drawing the sample extract for elution, receiving the eluate, and rotary evaporating it to dryness to leave a residue, adding a dissolving solvent to dissolve the residue to obtain the test solution I for the determination of multiple free sterols;

[0014] The preparation of the test solution II is specifically as follows: weighing the sample, performing saponification reflux, transferring and fixing the volume with the organic solvent, absorbing the supernatant into a separatory funnel, extracting with an extractant, combining the extracts, washing with pure water, dehydrating the organic layer with a dehydrating agent, and rotary evaporating to dryness to leave a residue, adding the dissolving solvent to dissolve the residue to obtain the test solution II for the determination of total sterols.

[0015] 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;

[0016] In the preparation of the test solution I, the shaking extraction includes ultrasonic extraction; the elution is carried out at C 18 The solid phase extraction was performed in a small column (2 g / 6 mL) and eluted with methanol;

[0017] 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 flush the cartridge with 3 column volumes of methanol.

[0018] 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.

[0019] 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;

[0020] 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 volume to methanol in the elution is 2 mL:25 mL; the volume ratio of the sample volume to the dissolving solvent is 2 mL:5 mL;

[0021] In the preparation of the test solution II, the mass volume ratio of the sample to the BHT potassium hydroxide ethanol solution is 0.25 g:20 mL; the saponification reflux temperature is 95° C., and the reflux time is 90 min; the extraction number of the extractant is 3 times; the pure water washing number is 3 times; and the mass volume ratio of the sample to the dissolving solvent is 0.05 g:50 mL.

[0022] 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. The total amount of phytosterol esters is calculated according to the following formula:

[0023] Z=(YW)×1.62

[0024]

[0025] Among them, the sterol ester coefficient is provided by the raw material manufacturer.

[0026] 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:

[0027] A. Accurately weigh 10 mg of rapeseed sterol reference substance, dissolve it in isopropanol and make the volume 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 the volume 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 the volume 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 the volume to 10 mL to obtain a 2 mg / mL β-sitosterol stock solution;

[0028] B. Accurately pipette 1.0 mL each of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix them, and dilute to 5 mL with the 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, which serve as standard curve solution V;

[0029] C. Accurately pipette 1.0 mL each of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix them, and dilute 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;

[0030] D. Accurately pipette 1.0 mL each of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix them, and dilute 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, which serve as standard curve III;

[0031] E. Accurately pipette 1.0 mL each of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix them, and dilute 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;

[0032] F. Accurately pipette 1.0 mL each of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix them, and dilute to 100 mL with the diluted 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;

[0033] The dilution solution is a mixture of methanol and isopropanol in a volume ratio of 1:4.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) Using C18 solid phase extraction cartridge purification technology, other components were separated and removed, the target component was enriched, and the interference of other components on the detection of free phytosterols was eliminated.

[0036] (2) Using methanol-water-phosphoric acid mixed solvent as A, methanol-isopropanol mixed solvent as mobile phase B, methanol as mobile phase C, and gradient elution, phosphoric acid was used as an ion suppressant to affect the dissociation properties of the substance being measured. In combination with gradient elution, it exerted a good adsorption and desorption effect, improved the separation ability of reversed-phase chromatography, and achieved good separation of various sterols, resulting in accurate and reliable detection results.

[0037] (3) It solves the problems of poor separation of various plant sterols and low accuracy of measurement results in the existing technology, simplifies the tedious sample pretreatment operation process, reduces the use of solvents, saves costs, and reduces environmental pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG1 is a chromatogram of the specificity test of the present invention. DETAILED DESCRIPTION

[0039] To make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings so that those skilled in the art can better understand the present invention and implement it, but the embodiments 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 methods, steps or conditions of the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0040] Example 1

[0041] A method for determining the total amount of multiple free sterols and sterol esters in plant sterol esters comprises the following steps:

[0042] (I) Preparation of Test Solution I: Accurately weigh 0.7 g of the phytosterol ester sample into a 100 mL volumetric flask. Add 60 mL of 95% ethanol and perform ultrasonic extraction (500 W, 60 min) with occasional shaking. Remove and bring to room temperature. Dose to volume with 95% ethanol, shake well, and let stand for 10 min. The supernatant is used as the sample extract. An Agela Cleanert S C18 cartridge (1 g / 6 mL) is placed and rinsed three times with 6 mL of methanol. Accurately pipette 2.0 mL of the sample extract onto the cartridge. Open the valve and collect the wash solution in a stoppered test tube at the bottom of the device. Wait until the extract flows to the same level as the stationary phase. Rinse the cartridge five times with 5 mL of methanol. After collecting all the wash solution, recycle it at 60°C and evaporate to dryness. Accurately pipette 5 mL of methanol to dissolve the residue. Filter to obtain the free sterol assay solution.

[0043] (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 and 10-15 mg of BHT, place in a beaker, add 10 mL of pure water, wait for the solid to dissolve, add 90 mL of anhydrous ethanol, mix well, and let it cool to room temperature), place in a 95°C water bath and reflux 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 draw 20.0mL of supernatant into a separatory funnel, add 20mL of n-hexane, extract for 30s, let stand and separate the layers, take the upper n-hexane liquid into another clean separatory funnel, and extract the aqueous layer of the extract twice with n-hexane in the same way. Combine the three n-hexane extracts and wash with pure water three times, 100mL each time. Dehydrate the n-hexane through anhydrous sodium sulfate and collect it in a round-bottom flask. Recover it by rotary evaporation at 40℃ to dryness to obtain the residue. Accurately add 50.0mL of methanol-isopropanol (1:4) to dissolve the residue and filter to obtain the total sterol determination solution.

[0044] (3) HPLC conditions

[0045] 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.

[0046] Gradient elution: from 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; from 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; from 8.1 to 28 minutes, the volume ratio of the mobile phase A, the mobile phase B, and the mobile phase C remains at 8:10:82; from 28.1 to 38 minutes, the volume ratio of the mobile phase A, the mobile phase B, and the mobile phase C remains at 90:10:0.

[0047] Chromatographic column: Hypersil ODS2 C18 analytical column (Elite, size 4.6×250mm, 5μm)

[0048]

[0049] (IV) Preparation of standard solution

[0050] A. Preparation of reference substance stock solution:

[0051] 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;

[0052] B. Accurately pipette 1.0 mL 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, stigmasterol concentration of 0.2 mg / mL, campesterol concentration of 0.2 mg / mL, and β-sitosterol concentration of 0.4 mg / mL is used as the standard curve solution V.

[0053] C. Accurately pipette 1.0 mL 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, stigmasterol concentration of 0.1 mg / mL, campesterol concentration of 0.1 mg / mL, and β-sitosterol concentration of 0.2 mg / mL is used as the standard curve solution IV.

[0054] D. Accurately pipette 1.0 mL each of the rapeseed sterol stock solution, stigmasterol stock solution, campesterol stock solution, and β-sitosterol stock solution, mix them, and dilute to 25 mL with methanol-isopropanol (1:4). The control solution with a rapeseed sterol concentration of 0.04 mg / mL, stigmasterol concentration of 0.04 mg / mL, campesterol concentration of 0.04 mg / mL, and β-sitosterol concentration of 0.08 mg / mL will serve as standard curve solution III.

[0055] E. Accurately pipette 1.0 mL 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 will serve as standard curve solution II.

[0056] F. Accurately pipette 1.0 mL 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.

[0057] (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.

[0058] (6) Reference substance mixture: Take the above-mentioned standard curve solution II as the reference substance mixture.

[0059] (VII) Take 20 μL each of methanol-isopropanol (1:4), standard curve solutions I to V, test solution I, and test solution II, and inject them into the chromatograph according to the chromatographic conditions in (III) of the technical plan to record the chromatogram, as shown in Figure 1. Qualitative analysis is performed based on the retention time of the reference substances for 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 determined from the standard curves, and the corresponding content of each sterol is calculated. The total amount of free sterols and the total amount of sterols are also calculated. The total amount of phytosterol esters is calculated according to the following formula.

[0060] Z=(YW)×1.62

[0061]

[0062] Example 2

[0063] Selection of chromatographic conditions

[0064] Currently, there is no authoritative method for detecting phytosterol esters in health foods. Published literature primarily involves liquid chromatography, gas chromatography, and gas chromatography-mass spectrometry for detecting various sterols in vegetable oils. Gas chromatography-mass spectrometry requires the use of multiple gases, which is costly and not optimal. Published methods for detecting individual sterols in vegetable oils (liquid chromatography and gas chromatography) are not suitable for detecting sterol esters in phytosterol ester raw materials and their corresponding preparations.

[0065] After extensive trial and error, the inventors have discovered that using a mixed solvent of methanol, isopropanol, and phosphoric acid solution, combined with a gradient elution system, they have achieved separations greater than 1.4 between rapeseed sterol, stigmasterol, campesterol, and β-sitosterol. The chromatographic conditions developed are shown in (iii) of the technical solution in Example 1. When elution was performed using only a mixed solvent of methanol and isopropanol, the separation effect between the sterols was inferior to that described in (iii) of the technical solution in Example 1. When isocratic elution was performed using a mixed solvent of methanol, isopropanol, and phosphoric acid solution, the separation effect was also inferior to that described in (iii) of the technical solution in Example 1, as shown in Table 1.

[0066] Table 1 Chromatographic conditions

[0067]

[0068]

[0069] Example 3

[0070] Determination of extraction solvent for pretreatment of test solution I

[0071] The same batch of plant sterol ester samples were taken and weighed. 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 solution (I) of Example 1. The contents of free brassicasterol, stigmasterol, campesterol, and β-sitosterol in the samples were determined. The results are shown in Table 2.

[0072] Table 2 Results of the extraction solvent investigation of test solution I (unit: mg / g)

[0073] Sample number 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

[0074] Conclusion: The extraction effects of 95% ethanol and methanol-isopropanol (4:1) are comparable. 95% ethanol is a better choice due to its lower price and lower toxicity.

[0075] Example 4

[0076] Determination of saponification conditions for test solution Ⅱ

[0077] The same batch of plant sterol ester samples were taken, weighed, and placed in 150 mL conical flasks, and 20 mL of potassium hydroxide ethanol solution was added (weighing 12 g of potassium hydroxide and 15 mg of BHT, placed in a beaker, and 10 mL of pure water was added. After the solid was dissolved, 90 mL of anhydrous ethanol was added, mixed, and allowed to cool to room temperature). Saponification reaction was carried out under the following conditions: reflux at 85° C. for 1.5 h; reflux at 95° C. for 1.5 h; and reflux at 98° C. for 1.5 h. The remaining conditions were the same as those in the technical scheme of Example 1. The contents of rapeseed sterol, stigmasterol, campesterol, and β-sitosterol were determined. The results are shown in Table 3.

[0078] Table 3 Results of saponification reaction conditions in the determination of total phytosterols (unit: mg / g)

[0079] Sample number Saponification reaction conditions Brassicasterol Stigmasterol Campesterol β-Sitosterol Total sterol content Sample 1 Reflux at 85°C for 90 minutes 7.92 152.66 106.01 259.25 525.84 Sample 2 Reflux at 95°C for 90 minutes 8.24 163.43 110.29 262.37 544.34 Sample 3 Reflux at 98°C for 90 minutes 8.26 163.41 110.65 262.33 544.65

[0080] 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℃, 95℃ reflux is more preferred to make the temperature condition more stable.

[0081] Example 5

[0082] Column selection

[0083] Existing literature does not describe the separation data of each sterol. After a large number of exploratory experiments, the inventors took a mixed standard solution containing rapeseed sterol, stigmasterol, campesterol, and β-sitosterol and examined most commercially available chromatographic columns. Only the chromatographic column described in the technical solution (iii) of Example 1 was able to achieve good separation of multiple sterols under the gradient elution described in this solution.

[0084]

[0085]

[0086] Example 6

[0087] Specificity and system suitability experiments

[0088] 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 mixture, and test solution II spiked solution were accurately taken for measurement and the chromatogram was recorded, as shown in Figure 1.

[0089] 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, indicating no interference, indicating that the specificity of the method meets the requirements; based on the chromatograms and peak areas of the reference sample mixture obtained by continuous injection for 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.

[0090] Table 5 System suitability experimental data

[0091]

[0092] Example 7

[0093] Limit of detection and limit of quantification

[0094] Reference substances for rapeseed sterol, stigmasterol, campesterol, and β-sitosterol were prepared into limit of quantification control solutions and limit of detection control solutions, respectively. The samples were injected and measured according to the chromatographic conditions of "Technical Scheme of Example 1," and the chromatograms were recorded. The limit of quantification for rapeseed sterol was 0.028 μg, and the limit of detection was 0.014 μg; the limit of quantification for stigmasterol was 0.032 μg, and the limit of detection was 0.016 μg; the limit of quantification for campesterol was 0.024 μg, and the limit of detection was 0.012 μg; and the limit of quantification for β-sitosterol was 0.066 μg, and the limit of detection was 0.033 μg.

[0095] Example 8

[0096] Linear relationship test

[0097] According to the chromatographic conditions of "Technical Scheme of Example 1", 20 μL of each of standard solutions I to VI was accurately drawn and injected into the liquid chromatograph. 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, indicating that the method presents a good linear relationship.

[0098] Example 9

[0099] Precision test

[0100] The same batch of phytosterol ester samples were taken and test solution I and test solution II were prepared according to the "Technical Scheme of Example 1". They were measured according to the chromatographic conditions 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. The corresponding content of each sterol was calculated, 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 value and RSD of the total phytosterol ester content of the 6 samples were calculated. The results are shown in Table 6.

[0101] Table 6 Precision test results

[0102]

[0103]

[0104] Example 10

[0105] Investigate the effects of different column temperatures (38°C and 42°C) on sample determination

[0106] Two portions of the same sample were taken, and the test solution I and the test solution II were prepared according to the "Technical Scheme of Example 1". According to the chromatographic conditions in "Technical Scheme (III) of Example 1", 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 better when the column temperature is 40°C.

[0107] Table 8 Separation results between four sterols at different temperatures

[0108] 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

[0109] Example 11

[0110] Determination of free sterols and total sterol esters in multiple batches of phytosterol esters

[0111] Three batches of plant sterol ester samples were taken and operated according to the technical scheme of Example 1. The total amount of various free sterols and sterol esters in the samples was determined respectively. The determination results are shown in Table 9.

[0112] Table 9 Determination results of total amount of free sterols and sterol esters in three batches of phytosterol esters

[0113] 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％

[0114] From the determination results, it can be seen that the method of the present invention can be used to quickly detect the contents of free sterols and total sterol esters in plant sterol ester samples, and the determination results of the three batches of plant sterol ester samples all meet the company's internal control standards.

[0115] The above disclosure is merely a preferred embodiment of the present invention and 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

1. A method for determining the total amount of free sterols and sterol esters in plant sterol esters, characterized in that: A methanol-water-phosphoric acid mixed solvent was used as mobile phase A, a methanol-isopropanol mixed solvent was used as mobile phase B, and methanol was used as mobile phase C. A reversed-phase analytical column was used to perform gradient elution on different test solutions to respectively determine the total amount of various free sterols and sterol esters in plant sterol esters; a methanol-water-phosphoric acid mixed solvent with a volume ratio of 385:115:0.4 was used as the mobile phase A, a methanol-isopropanol mixed solvent with a volume ratio of 7:3 was used as the mobile phase B, and methanol was used as the mobile phase C to perform the gradient elution; the reversed-phase analytical column was C 18 Analytical column; the free sterols include brassicasterol, stigmasterol, campesterol, and β-sitosterol; The specific operation of the gradient elution is: from 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; from 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; from 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; from 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; the detection wavelength of the gradient elution is 210 nm.

2. The method for determining the total amount of free sterols and sterol esters in plant sterol esters according to claim 1, wherein: The flow rate of the gradient elution was 1.0 mL / min, the injection volume was 20 μL, and the column temperature was 38-42° C.

3. The method for determining the total amount of free sterols and sterol esters in plant sterol esters according to claim 1, wherein: The reverse phase analytical column is Hypersil ODS2 C 18 Analytical column.

4. The method for determining the total amount of free sterols and sterol esters in plant sterol esters according to claim 1, wherein: Before the gradient elution, a test solution is prepared, wherein the test solution includes a test solution I and a test solution II; The test solution I is prepared as follows: weighing a sample, placing it in a volumetric flask, adding an organic solvent, extracting by shaking, taking it out and letting it cool to room temperature, making up the volume with the organic solvent, shaking it well, letting it stand, taking the supernatant as the sample extract, drawing the sample extract for elution, receiving the eluate, and rotary evaporating it to dryness to leave a residue, adding a dissolving solvent to dissolve the residue to obtain the test solution I for the determination of multiple free sterols; The preparation of the test solution II is specifically as follows: weighing the sample, performing saponification reflux, transferring and fixing the volume with the organic solvent, absorbing the supernatant into a separatory funnel, extracting with an extractant, combining the extracts, washing with pure water, dehydrating the organic layer with a dehydrating agent, and rotary evaporating to dryness to leave a residue, adding the dissolving solvent to dissolve the residue to obtain the test solution II for the determination of total sterols.

5. The method for determining the total amount of free sterols and sterol esters in plant sterol esters according to claim 4, characterized in that: 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 at C 18 The solid phase extraction was performed in a small column and eluted with 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.

6. The method for determining the total amount of free sterols and sterol esters in plant sterol esters according to claim 5, characterized in that: 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 volume to methanol in the elution is 2 mL:25 mL; the volume ratio of the sample volume 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 0.25 g:20 mL; the saponification reflux temperature is 95° C., and the reflux time is 90 min; the extraction number of the extractant is 3 times; the pure water washing number is 3 times; and the mass volume ratio of the sample to the dissolving solvent is 0.05 g:50 mL.

7. The method for determining the total amount of free sterols and sterol esters in plant sterol esters according to claim 1, wherein: After the gradient elution, the chromatogram was recorded, and qualitative analysis was performed based on 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 were respectively obtained from the standard curves. The corresponding content of each sterol was calculated, and the total amount of free sterols and the total amount of sterols were calculated. The total amount of phytosterol esters was calculated according to the following formula: Z=(YW)×1.62 Z——total amount of phytosterol esters, in %; Y——total amount of phytosterols, in %; W——total amount of free sterols, in %; 1.62 – Coefficient of conversion of sterols to sterol esters.

8. The method for determining the total amount of free sterols and sterol esters in plant sterol esters according to claim 7, wherein: The standard curve is obtained by gradient elution of the standard curve solution under the same conditions, and the preparation of the standard curve solution includes: A. Accurately weigh 10 mg of rapeseed sterol reference substance, dissolve it in isopropanol and make the volume 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 the volume 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 the volume 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 the volume to 10 mL to obtain a 2 mg / mL β-sitosterol stock solution; B. Accurately pipette 1.0 mL each of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix them, and dilute to 5 mL with the 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, which serve as standard curve solution V; C. Accurately pipette 1.0 mL each of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix them, and dilute 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 each of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix them, and dilute 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, which serve as standard curve III; E. Accurately pipette 1.0 mL each of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix them, and dilute 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 each of the rapeseed sterol stock solution, the stigmasterol stock solution, the campesterol stock solution, and the β-sitosterol stock solution, mix them, and dilute to 100 mL with the diluted 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.

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