A method for simultaneously determining the contents of multiple free sterols and moracolin K in phytosterol ester red yeast rice soft capsules

Abstract

The present invention belongs to the field of quality standards and detection technology of health food, and specifically relates to a detection method for simultaneously determining the contents of multiple free sterols and moracolin K in a plant sterol ester red yeast rice soft capsule. The present invention utilizes ultrasound to extract effective ingredients, and utilizes reverse phase chromatography as a means of separating and detecting target components and eliminating interference from auxiliary materials. Using methanol-water-phosphoric acid mixed solvent as mobile phase A, methanol-isopropanol mixed solvent as mobile phase B, and methanol as mobile phase C, gradient elution is performed in a reverse phase analytical column, and the contents of multiple free sterols and moracolin K are simultaneously determined. The detection method provided by the present invention solves the problems of low separation of multiple sterols, poor accuracy and interference from auxiliary materials in the prior art, and the peak shape of each component is good, and the separation degree is greater than 1.2. Provide data support and reference for the formulation of product quality standards and the screening of preparation processes.

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 simultaneously determining the contents of multiple free sterols and moracolin K in plant sterol ester red yeast rice soft capsules. Background Art

[0002] Plant sterol ester red yeast rice soft capsules use plant sterol esters and functional red yeast rice powder as raw materials, and vegetable oils as auxiliary materials. They are sealed and wrapped into soft capsules using soft capsule materials. They are a health food that helps control blood lipid levels.

[0003] 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 have the function of assisting in inhibiting the absorption of cholesterol in the intestine and effectively reducing total cholesterol and low-density lipoprotein cholesterol in the blood. In 2010, they were approved to be included in the new resource food catalog as a raw material for health food. Phytosterol esters mainly contain stigmasterol, campesterol, β-sitosterol and a small amount of rapeseed sterol. During the production process, because the various free phytosterols are not completely esterified to form phytosterol esters, they coexist in the product with phytosterol esters, accounting for about 2% to 6%. 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.

[0004] Functional red yeast rice powder is a product made from rice and fermented by Monascus purpurogenum to help prevent cardiovascular diseases. Its main active ingredient is molaclin K, which is the main "lipid-regulating factor" of red yeast rice and has become a hot spot in scientific research and the focus of the development of the red yeast rice industry. The testing basis for functional red yeast rice powder is the light industry standard QB / T 2847-2007, and its efficacy index is molaclin K (calculated as lovastatin).

[0005] During the research, development and production of plant sterol ester soft capsules, various sterols and molaclin K can be tested as product quality control indicators. The various sterols need to deduct the free sterols contained in the raw materials themselves and further converted into the amount of plant sterol esters to display the accurate content of sterol esters in the preparation, making the product quality controllable.

[0006] At present, there is no authoritative detection method for the simultaneous determination of multiple sterols and molaclin K in terminal health foods. The literature is blank for the determination or simultaneous determination of molaclin K and multiple sterol contents in products. Soft capsule products mostly use vegetable oil as an excipient, which plays the role of dispersing raw materials, diluting concentration, stabilizing dosage forms, etc. Therefore, the substances brought into the extract interfere with the detection of the target component content. The manufacturers of plant sterol ester raw materials use GC method to detect free sterols. The gas chromatograph needs to be equipped with a cold column head to complete the detection of multiple free sterol indicators, which has high requirements for the detection equipment. The authoritative detection method for lovastatin, QB / T 2847-2007, is only applicable to the determination of molaclin K content in functional red yeast rice powder raw materials. Most of the products with similar prescriptions currently on the market do not use free sterols or total sterols as quality control indicators, or only use the normalization method to roughly quantify the sterol components in the product. There is a lack of effective and accurate quality detection methods for the main raw materials added to the prescription. Summary of the invention

[0007] The purpose of the present invention is to solve the problems of low separation degree of multiple sterols, poor accuracy and interference of auxiliary materials in the prior art, and the lack of method standards for quality detection of multiple functional ingredients in compound preparations of health foods. The present invention discloses a detection method for simultaneously determining the contents of multiple free sterols and moracilliner K in phytosterol ester red yeast rice soft capsules, so as to provide an effective detection basis for controlling the quality of the main functional ingredients (phytosterol esters) of the product. Ultrasonic extraction technology is used to extract the target component. Reverse phase liquid chromatography is used, with methanol-water-phosphoric acid mixed solvent as mobile phase A, methanol-isopropanol mixed solvent as mobile phase B, and methanol as mobile phase C. The three phases are gradient eluted in a suitable ratio through a reverse phase C18 analytical column, and the contents of four free sterols, namely, molaclin K and rapeseed sterol, stigmasterol, campesterol and β-sitosterol, are determined simultaneously. After the above five components are eluted out of the chromatographic column, 100% of mobile phase B (methanol-isopropanol mixed solvent) is used to elute the low polar substances remaining in the chromatographic column out of the chromatographic column at 35.1 to 57 minutes, thereby eliminating the interference of the low polar components and achieving the purpose of accurate quantitative determination of the target component. 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 1.2, thereby solving the problem that multiple sterol components in the prior art cannot be effectively separated, and the contents of multiple components can be accurately detected at the same time, saving time and having good economic benefits, providing reference data for the formulation of quality standards and quality inspection of corresponding terminal health foods, and providing data support for determining process parameters for new product development.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A detection method for simultaneously determining the contents of multiple free sterols and moracolin K in plant sterol ester red yeast rice soft capsules, 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 reverse phase analytical column for gradient elution to simultaneously determine the contents of multiple free sterols and moracolin K.

[0010] Preferably, the gradient elution is performed using a methanol-water-phosphoric acid mixed solvent with a volume ratio of 285:75:0.2 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.

[0011] Preferably, the specific operation of the gradient elution is: at 0 to 12 minutes, the initial volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is 90:10:0; at 12.1 to 14 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 0:10:90; at 14.1 to 35 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 0:10:90; at 35.1 to 57 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 0:100:0; at 57.1 to 67 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 90:10:0.

[0012] 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 30 minutes, and the impurities are eluted after 35 minutes, there is no need to set a gradual change in the last two periods, and the impurities can be eluted directly by using a suitable mobile phase.

[0013] Preferably, the detection wavelength of the gradient elution is 238 nm at 0 to 15 min, and the target component to be measured is morakorin K; after 15 min, it is 210 nm, and the target component to be measured is free sterol; the flow rate of the gradient elution is 1.0 mL / min, the injection volume is 20 μL, and the column temperature is 30 to 34°C.

[0014] Preferably, the reverse phase analytical column is Hypersil ODS2 C 18 Analytical column.

[0015] Preferably, the sample solution is prepared before the gradient elution, specifically by weighing the sample, placing it in a volumetric flask, adding an organic solvent, extracting by shaking, bringing it to room temperature, making up to volume with the organic solvent, shaking it well, letting it stand, filtering and taking the supernatant to obtain the sample solution.

[0016] Preferably, the organic solvent includes one or more of water, methanol, ethanol, and isopropanol; the mass volume ratio of the sample to the total amount of the organic solvent is 2g:100mL; and the oscillation extraction includes ultrasonic extraction.

[0017] Preferably, the organic solvent is 95% ethanol aqueous solution; the output power of the ultrasonic extraction is 500w, and the time is 60min.

[0018] Preferably, after the gradient elution, the chromatogram is recorded, and the qualitative analysis is performed according to the retention time of the reference substances of moracolin K, brassicasterol, stigmasterol, campesterol, and β-sitosterol, and the concentrations of moracolin K, brassicasterol, stigmasterol, campesterol, and β-sitosterol in the test solution are obtained from the standard curve, and the total amount of moracolin K and free sterols is calculated.

[0019] 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:

[0020] A. Accurately weigh 20 mg of lovastatin reference substance, dissolve it in methanol and make up to 10 mL to obtain a lovastatin standard stock solution with a concentration of 2 mg / mL; accurately weigh 10 mg of rapeseed sterol reference substance, dissolve it in isopropanol and make up to 10 mL to obtain a rapeseed sterol stock solution with a concentration of 1 mg / mL; accurately weigh 20 mg of stigmasterol reference substance, dissolve it in isopropanol and make up to 20 mL to obtain a stigmasterol stock solution with a concentration of 1 mg / mL; accurately weigh 10 mg of campesterol reference substance, dissolve it in isopropanol and make up to 10 mL to obtain a campesterol solution with a concentration of 1 mg / mL; accurately weigh 20 mg of β-sitosterol reference substance, dissolve it in isopropanol and make up to 10 mL to obtain a β-sitosterol stock solution with a concentration of 2 mg / mL;

[0021] B. Accurately pipette 1.0 mL of the lovastatin stock solution, 1.0 mL of the rapeseed sterol stock solution, 0.6 mL of the stigmasterol stock solution, 1.2 mL of the campesterol stock solution, and 1 mL of the β-sitosterol stock solution, mix them, and dilute to 5 mL with 95% ethanol to obtain a control solution with a lovastatin concentration of 0.4 mg / mL, a rapeseed sterol concentration of 0.2 mg / mL, a stigmasterol concentration of 0.12 mg / mL, a campesterol concentration of 0.24 mg / mL, and a β-sitosterol concentration of 0.4 mg / mL, as the standard curve solution VI;

[0022] C. Accurately pipette 0.5 mL of the standard curve solution VI, add 95% ethanol to dilute to 1 mL, and prepare a control solution with a lovastatin concentration of 0.2 mg / mL, a rapeseed sterol concentration of 0.1 mg / mL, a stigmasterol concentration of 0.06 mg / mL, a campesterol concentration of 0.12 mg / mL, and a β-sitosterol concentration of 0.2 mg / mL, as the standard curve solution V;

[0023] D. Accurately pipette 0.4 mL of the standard curve solution VI, add 95% ethanol to dilute to 2 mL, and prepare a control solution with a lovastatin concentration of 0.08 mg / mL, a rapeseed sterol concentration of 0.04 mg / mL, a stigmasterol concentration of 0.024 mg / mL, a campesterol concentration of 0.048 mg / mL, and a β-sitosterol concentration of 0.08 mg / mL, as the standard curve solution IV;

[0024] E. Accurately pipette 0.1 mL of the standard curve solution VI, add 95% ethanol to dilute to 1 mL, and prepare a control solution with a lovastatin concentration of 0.04 mg / mL, a rapeseed sterol concentration of 0.02 mg / mL, a stigmasterol concentration of 0.012 mg / mL, a campesterol concentration of 0.024 mg / mL, and a β-sitosterol concentration of 0.04 mg / mL, as the standard curve solution III;

[0025] F. Accurately pipette 0.1 mL of the standard curve solution VI, add 95% ethanol to dilute to 2 mL, and prepare a control solution with a lovastatin concentration of 0.02 mg / mL, a rapeseed sterol concentration of 0.01 mg / mL, a stigmasterol concentration of 0.006 mg / mL, a campesterol concentration of 0.012 mg / mL, and a β-sitosterol concentration of 0.02 mg / mL, as the standard curve solution II;

[0026] G. Accurately pipette 0.1 mL of the standard curve solution VI, add 95% ethanol to dilute to 5 mL, and prepare control solutions with lovastatin concentration of 0.008 mg / mL, rapeseed sterol concentration of 0.004 mg / mL, stigmasterol concentration of 0.0024 mg / mL, campesterol concentration of 0.0048 mg / mL, and β-sitosterol concentration of 0.008 mg / mL as standard curve solution I.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) For the first time, the contents of multiple free sterols and molaclin K were simultaneously determined to improve detection efficiency.

[0029] (2) 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. The three phases were gradient eluted in an appropriate ratio. Phosphoric acid was used as an ion inhibitor to affect the dissociation properties of the substance being tested. In combination with gradient elution, good adsorption and desorption effects were exerted, thereby improving the separation ability of reversed-phase chromatography, so that molaclin K and various sterols were well separated, and the detection results were accurate and reliable.

[0030] (3) After the five target components of molaclin K, brassicasterol, stigmasterol, campesterol, and β-sitosterol in the test solution are eluted out of the chromatographic column, the low-polar substances remaining in the chromatographic column are eluted out of the chromatographic column using 100% mobile phase B (methanol-isopropanol mixed solvent) to eliminate the interference of the low-polar components, thereby achieving the purpose of accurate quantitative determination of the target components and preventing the components introduced by the excipients from interfering with the determination of subsequent injected samples.

[0031] (4) It simplifies the cumbersome sample pretreatment process and enables the simultaneous detection of multiple components using one mobile phase system, saving the time cost of using two mobile phases to detect multiple components separately, reducing the amount of solvent used, reducing environmental pressure, and achieving good economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a chromatogram of the specificity test of the present invention. DETAILED DESCRIPTION

[0033] 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.

[0034] Example 1

[0035] A method for simultaneously determining the contents of multiple free sterols and moracolin K in plant sterol ester red yeast rice soft capsules comprises the following steps:

[0036] (I) Preparation of test solution: Weigh 2 g of the sample, place it in a 100 mL volumetric flask, add 60 mL of 95% ethanol, perform ultrasonic extraction (500w, 60 min), shake occasionally, take out and bring to room temperature, make up to volume with 95% ethanol, shake well, let stand for 10 min, take the supernatant and filter it to obtain the sample test solution.

[0037] (II) HPLC conditions

[0038] Mobile phase: using a methanol-water-phosphoric acid mixed solvent with a volume ratio of 285:75:0.2 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.

[0039] The gradient elution program is as follows: at 0 to 12 minutes, the initial volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is 90:10:0; at 12.1 to 14 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 0:10:90; at 14.1 to 35 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 0:10:90; at 35.1 to 57 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 0:100:0; at 57.1 to 67 minutes, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 90:10:0;

[0040] Chromatographic column: Hypersil ODS2 C18 analytical column (Elite, specification 4.6×250mm, 5μm)

[0041] Detection wavelength: 0-15min, wavelength is 238nm, the target component measured is morakorin K; after 15min, it is 210nm, the target component measured is free sterol;

[0042] Flow rate: 1.0 mL / min;

[0043] Injection volume: 20 μL;

[0044] Column temperature: 30-34°C;

[0045] (III) Preparation of standard solution

[0046] A. Preparation of reference substance stock solution:

[0047] A. Accurately weigh 20 mg of lovastatin reference substance, dissolve it in methanol and make up to 10 mL to obtain a lovastatin standard stock solution with a concentration of 2 mg / mL; accurately weigh 10 mg of rapeseed sterol reference substance, dissolve it in isopropanol and make up to 10 mL to obtain a rapeseed sterol stock solution with a concentration of 1 mg / mL; accurately weigh 20 mg of stigmasterol reference substance, dissolve it in isopropanol and make up to 20 mL to obtain a stigmasterol stock solution with a concentration of 1 mg / mL; accurately weigh 10 mg of campesterol reference substance, dissolve it in isopropanol and make up to 10 mL to obtain a campesterol solution with a concentration of 1 mg / mL; accurately weigh 20 mg of β-sitosterol reference substance, dissolve it in isopropanol and make up to 10 mL to obtain a β-sitosterol stock solution with a concentration of 2 mg / mL;

[0048] B. Accurately pipette 1.0 mL of lovastatin stock solution, 1.0 mL of rapeseed sterol stock solution, 0.6 mL of stigmasterol stock solution, 1.2 mL of campesterol stock solution, and 1 mL of β-sitosterol stock solution, add 95% ethanol to dilute to 5 mL, and prepare control solutions with lovastatin concentration of 0.4 mg / mL, rapeseed sterol concentration of 0.2 mg / mL, stigmasterol concentration of 0.12 mg / mL, campesterol concentration of 0.24 mg / mL, and β-sitosterol concentration of 0.4 mg / mL as standard curve VI;

[0049] C. Accurately pipette 0.5 mL of standard curve VI, add 95% ethanol to dilute to 1 mL, and prepare control solutions with lovastatin concentration of 0.2 mg / mL, rapeseed sterol concentration of 0.1 mg / mL, stigmasterol concentration of 0.06 mg / mL, campesterol concentration of 0.12 mg / mL, and β-sitosterol concentration of 0.2 mg / mL as standard curve V;

[0050] D. Accurately pipette 0.4 mL of standard curve VI, add 95% ethanol to dilute to 2 mL, and prepare control solutions with lovastatin concentration of 0.08 mg / mL, rapeseed sterol concentration of 0.04 mg / mL, stigmasterol concentration of 0.024 mg / mL, campesterol concentration of 0.048 mg / mL, and β-sitosterol concentration of 0.08 mg / mL as standard curve IV;

[0051] E. Accurately pipette 0.1 mL of standard curve VI, add 95% ethanol to dilute to 1 mL, and prepare control solutions with lovastatin concentration of 0.04 mg / mL, rapeseed sterol concentration of 0.02 mg / mL, stigmasterol concentration of 0.012 mg / mL, campesterol concentration of 0.024 mg / mL, and β-sitosterol concentration of 0.04 mg / mL as standard curve III;

[0052] F. Accurately pipette 0.1 mL of standard curve VI, add 95% ethanol to dilute to 2 mL, and prepare control solutions with lovastatin concentration of 0.02 mg / mL, rapeseed sterol concentration of 0.01 mg / mL, stigmasterol concentration of 0.006 mg / mL, campesterol concentration of 0.012 mg / mL, and β-sitosterol concentration of 0.02 mg / mL as standard curve II;

[0053] G. Accurately pipette 0.1 mL of standard curve VI, add 95% ethanol to dilute to 5 mL, and prepare control solutions with lovastatin concentration of 0.008 mg / mL, rapeseed sterol concentration of 0.004 mg / mL, stigmasterol concentration of 0.0024 mg / mL, campesterol concentration of 0.0048 mg / mL, and β-sitosterol concentration of 0.008 mg / mL as standard curve I.

[0054] (IV) Preparation of excipient sample solution: Take the excipient sample and prepare it according to the above scheme (I).

[0055] (V) Preparation of sample spike solution: Take the test solution of the above scheme (I) and standard curve III and mix them in a volume ratio of 1:1.

[0056] (VI) Reference substance mixture: Take the above-mentioned standard curve solution II as the reference substance mixture.

[0057] (VII) Take 20 μL of blank solvent (95% ethanol), standard curve solution I to standard curve solution VI, and test solution respectively, inject into the chromatograph according to the chromatographic conditions in the technical plan to record the chromatogram, and perform qualitative analysis based on the retention time of lovastatin (lactone), rapeseed sterol, stigmasterol, campesterol, and β-sitosterol reference substances. The concentrations of moracolin K, rapeseed sterol, stigmasterol, campesterol, and β-sitosterol in the test solution are obtained from the standard curves, and the corresponding content of each sterol component is calculated, and then the total amount of free sterols in the sample is calculated, such as Figure 1 shown.

[0058] Example 2

[0059] Selection of chromatographic conditions

[0060] In the existing literature, there is no relevant research on the simultaneous determination of Moracolin K and multiple sterols. Moracolin K refers to lovastatin (lactone) QB / T 2847-2007, but it only targets raw materials. At present, there is no authoritative detection method for the detection of plant sterol ester components in health foods. In the published literature, liquid chromatography, gas chromatography, and gas chromatography-mass spectrometry are mainly involved in the detection of multiple 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.

[0061] After a lot of trial and error, the inventor used methanol-water-phosphoric acid mixed solvent as mobile phase A, methanol-isopropanol mixed solvent as mobile phase B, and methanol as mobile phase C, and gradient eluted through a reversed phase C18 analytical column, and simultaneously determined the contents of four free sterols, namely, molaclin K and rapeseed sterol, stigmasterol, campesterol, and β-sitosterol. After the above five components were eluted out of the chromatographic column, 100% of the mobile phase B (methanol-isopropanol mixed solvent) was used to elute the low-polar substances remaining in the chromatographic column out of the chromatographic column at 35.1-57 minutes, eliminating the interference of low-polar components and achieving the purpose of accurate quantitative determination of the target component. The chromatographic conditions developed are shown in (ii) of the technical scheme of Example 1. The chromatographic condition selection exploration process is shown in Table 1.

[0062] Table 1 Chromatographic conditions investigation

[0063]

[0064] As can be seen from Table 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 (ii) of the technical solution 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 (ii) of the technical solution of Example 1.

[0065] Example 3

[0066] Determination of extraction solvent for test solution

[0067] Take the plant sterol ester red yeast rice soft capsule, weigh the sample, and use 85% ethanol, 95% ethanol, and methanol-isopropanol (4:1) as extraction solvents, respectively. Other conditions are the same as those in the technical scheme (i) of Example 1. Record the peak areas of lovastatin (lactone), brassicasterol, stigmasterol, campesterol, and β-sitosterol. The results are shown in Table 2.

[0068] Table 2 Pretreatment ultrasonic extraction solvent investigation results (unit: peak area)

[0069]

[0070] As can be seen from Table 2, the extraction effects of 95% ethanol and methanol-isopropanol (4:1) are comparable, and it is better to choose 95% ethanol, which is cheaper and less toxic.

[0071] Example 4

[0072] Column selection

[0073] There is almost no separation data of each sterol in the existing literature. The inventors of the present application have conducted a lot of exploratory experiments and took a mixed standard solution containing lovastatin (lactone), brassicasterol, stigmasterol, campesterol, and β-sitosterol to investigate most of the chromatographic columns available on the market. The experimental results are shown in Table 3.

[0074] Table 3 Different chromatographic column investigation tests

[0075]

[0076]

[0077] It can be seen from Table 3 that only the chromatographic column described in the technical solution of the present invention can achieve the separation of polysterols under the elution gradient of the present invention.

[0078] Example 5

[0079] Specificity and system suitability experiments

[0080] Accurately take 20 μL of blank solvent (95% ethanol), auxiliary material sample solution, test solution, reference substance mixed solution, and sample spike solution, and perform the measurement according to the technical scheme described in Example 1, and record the chromatogram. Figure 1 shown.

[0081] The determination results show that the blank solvent (95% ethanol) does not interfere with the main peak determination of the five components. Stigmasterol and β-sitosterol are detected in the auxiliary sample solution, which is related to the auxiliary material containing soybean oil, because soybean oil contains a small amount of stigmasterol. The experimental results are consistent with the actual situation; there is no interference near the main peak of the reference mixed standard solution, the test solution and the sample spiked solution, and the retention time of the main peak is consistent, indicating that the specificity of the method meets the requirements; according to the chromatogram and peak area of ​​the reference mixed solution obtained by continuous injection of 6 times, the RSD and separation degree are calculated, and the separation degree of each component peak is greater than 1.2. The experimental results are shown in Table 4.

[0082] Table 4 System suitability experimental data

[0083]

[0084]

[0085] Example 6

[0086] Limit of Detection and Limit of Quantitation

[0087] Lovastatin (lactone), rapeseed sterol, stigmasterol, campesterol, and β-sitosterol reference substances were prepared into solutions with a signal-to-noise ratio of about 10:1 as quantitative limit reference solutions; a solution with a signal-to-noise ratio of 3:1 was prepared as a detection limit reference solution, and the samples were injected and measured according to the chromatographic conditions in the technical scheme of Example 1, and the chromatogram was recorded. The quantitative limit of lovastatin was measured to be 0.073 μg, and the detection limit was 0.025 μg; the quantitative limit of rapeseed sterol was 0.38 μg, and the detection limit was 0.013 μg; the quantitative limit of stigmasterol was 0.021 μg, and the detection limit was 0.0069 μg; the quantitative limit of campesterol was 0.11 μg, and the detection limit was 0.037 μg; the quantitative limit of β-sitosterol was 0.19 μg, and the detection limit was 0.062 μg.

[0088] Example 7

[0089] Linear relationship test

[0090] According to the chromatographic conditions in the technical scheme of Example 1, 20 μL of each of the standard solutions I to VI were accurately drawn and injected into the liquid chromatograph, and 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 lovastatin is y=1161.5518x-0.0710, and the correlation coefficient r 2=0.9998; the standard linear equation of rapeseed sterol is y=108.0571x+0.0656, and the correlation coefficient is r 2 =0.9998; the standard linear equation of stigmasterol is y=114.2753x+0.1396, and the correlation coefficient is r 2 =0.9998; the standard linear equation of β-sitosterol is y=78.1923x+0.1593, and the correlation coefficient is r 2 =0.9998, indicating that the method presents a good linear relationship.

[0091] Example 8

[0092] Precision test

[0093] Take 6 samples of the same batch of plant sterol ester red yeast rice soft capsules, prepare the test solution and the standard curve solution according to the conditions in the technical scheme of Example 1, and measure them according to the chromatographic conditions, record the chromatogram, calculate the concentration of moracolin K, rapeseed sterol, stigmasterol, campesterol and β-sitosterol in each sample according to the measured standard curve, and then calculate the content, average content and relative standard deviation (RSD) of each component. The results are shown in Table 5, indicating that the precision meets the requirements.

[0094] Table 5 Precision test results (unit: mg / g)

[0095]

[0096] Example 9

[0097] Accuracy test

[0098] Reference substance spiked solution: Take the lovastatin stock solution, rapeseed sterol stock solution, campesterol stock solution, stigmasterol stock solution, and β-sitosterol stock solution in the technical solution of Example 1, and dilute them with 95% ethanol to obtain a spiked solution with a lovastatin concentration of 0.06634 mg / mL, a rapeseed sterol concentration of 0.05508 mg / mL, a stigmasterol concentration of 0.04543 mg / mL, a campesterol concentration of 0.04760 mg / mL, and a β-sitosterol concentration of 0.03746 mg / mL.

[0099] Take 6 samples of the same batch of plant sterol ester red yeast soft capsules, accurately weigh 2.4g of the sample, put it in a 100mL volumetric flask, add 60mL of 95% ethanol, ultrasonically extract (500w, 60min), shake from time to time, take it out and put it at room temperature, dilute it with 95% ethanol, shake it well, let it stand for 10min, accurately draw 2.5.0mL of the supernatant, put it in a 5mL volumetric flask, dilute it to the scale with the reference substance spike solution, shake it well, and get 6 parts of the accuracy test solution, respectively measure it according to the chromatographic conditions, record the chromatogram, and calculate the actual measurement. The accuracy test results are shown in Table 6.

[0100] Table 6 Accuracy experimental results

[0101]

[0102] From the test results, it can be seen that the average recoveries of lovastatin (lactone), brassicasterol, stigmasterol, campesterol and β-sitosterol are 100.3%, 104.7%, 97.0%, 96.9% and 93.0% (n=6), respectively, and the RSDs are 1.3%, 0.7%, 5.0%, 4.1% and 4.0%, respectively, and the RSDs are all less than 5%, indicating that the determination method is accurate and reliable.

[0103] Example 10

[0104] Investigate the influence of different analytical column temperatures on separation and determination results in chromatographic conditions

[0105] Take 2 portions of the same batch of samples respectively, and prepare the test solution and the standard curve solution according to the "Technical Scheme of Example 1". Inject the standard curve solution and the test solution at column temperatures of 30°C, 32°C, 34°C, and 36°C, respectively, and record their separation degrees. According to the experimental results, when the column temperature is 30°C-34°C, the separation degree of molaclin K and the front and back peaks is not less than 1.2, but at 36°C, the separation degree is only 1.0; therefore, it is considered that the column temperature is 30°C-34°C more reasonable.

[0106] Take the above test sample solution and standard curve solution, and determine the contents of lovastatin (lactone), brassicasterol, stigmasterol, campesterol and β-sitosterol in the samples at column temperatures of 30°C, 32°C and 34°C, respectively, and calculate the separation degree. The determination results are shown in Tables 7 and 8.

[0107] Table 7 Separation results of different analytical column temperatures

[0108] Column temperature Morakolin K and the separation between the front and rear peaks Brassicasterol and stigmasterol Stigmasterol and Campesterol Campesterol and β-sitosterol 30℃ 1.37 / 1.23 3.16 1.52 1.19 32℃ 1.40 / 1.20 3.12 1.46 1.25 34℃ 1.35 / 1.20 3.13 1.39 1.29 36℃ 1.23 / 1.01 3.10 1.38 1.33

[0109] Table 8 Content determination results at different analytical column temperatures

[0110]

[0111] The contents of lovastatin (lactone), brassicasterol, stigmasterol, campesterol and β-sitosterol in the same batch of samples were determined at column temperatures of 30°C, 32°C and 34°C, respectively. The differences were not obvious and the RSD was less than 10%.

[0112] Embodiment 11

[0113] Study on the influence of different flow rates of mobile phase on separation and determination results in chromatographic conditions

[0114] Two portions of the same batch of samples were taken respectively, and the test solution and the standard curve solution were prepared according to the "Technical Scheme of Example 1". When the flow rates of the mobile phase were 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min, respectively, the contents of lovastatin (lactone), brassicasterol, stigmasterol, campesterol, and β-sitosterol in the samples were determined, and the separation was statistically calculated. The determination results are shown in Tables 9 and 10.

[0115] Table 9 Content determination results of different mobile phase flow rates

[0116]

[0117] Table 10 Separation results of different mobile phase flow rates

[0118]

[0119] From the determination results, it can be seen that when the flow rate is 0.9mL / min or 1.1mL / min, the separation degree between lovastatin (lactone) and the front and rear peaks is low, and the separation degree between the sterols decreases as the flow rate increases. Considering comprehensively, the separation effect is good when the flow rate is 1.0mL / min. When the flow rates are 0.9mL / min, 1.0mL / min, and 1.1mL / min, respectively, the contents of lovastatin (lactone), rapeseed sterol, stigmasterol, campesterol, and β-sitosterol in the same batch of samples are determined, and the differences are not obvious, and the RSD is not greater than 10%.

[0120] Example 12

[0121] Investigate the stability of the test solution within 24 hours

[0122] Take the same batch of samples and prepare 1 test solution according to the "Technical Scheme of Example 1". Repeat the injection at 0h, 2h, 4h, 12h and 24h according to the chromatographic conditions. The peak areas of lovastatin (lactone), rapeseed sterol, stigmasterol, campesterol and β-sitosterol of 5 injections are counted, and the RSD is counted. The measurement results are shown in Table 8.

[0123] From the determination results, it can be seen that as the column temperature increases, the separation degree of lovastatin (lactone) and rapeseed sterol increases, but the separation degree of stigmasterol and campesterol decreases. Taking all factors into consideration, the column temperature is optimal at 32°C, and the separation effect is good. The contents of lovastatin (lactone), rapeseed sterol, stigmasterol, campesterol, and β-sitosterol in the same batch of samples were determined at column temperatures of 30°C, 32°C, and 34°C, respectively. The differences were not obvious, and the RSD was no more than 8%, as shown in Table 11.

[0124] Table 11 Stability test data of the test solution within 24 hours

[0125]

[0126] Example 13

[0127] Assays in multiple batches of samples

[0128] Three batches of plant sterol ester red yeast rice soft capsule samples from Guangzhou Baiyunshan Hanfang Modern Pharmaceutical Co., Ltd. were taken and operated according to the conditions of the technical scheme in Example 1. The contents of lovastatin (lactone), rapeseed sterol, stigmasterol, campesterol and β-sitosterol in the samples were respectively determined. The determination results are shown in Table 12.

[0129] Table 12 Determination results of lovastatin (lactone) and free sterol content in phytosterol ester red yeast soft capsules

[0130]

[0131] From the determination results, it can be seen that the content of lovastatin (lactone) and free sterol in phytosterol ester red yeast rice soft capsules can be quickly detected by the method of the present invention, and the samples of 3 batches of phytosterol ester red yeast rice soft capsules all meet the product internal control standards.

[0132] 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

1. A method for simultaneously determining the contents of multiple free sterols and moracolin K in phytosterol ester red yeast rice soft capsules, wherein the free sterols include rapeseed sterol, stigmasterol, campesterol, and β-sitosterol, characterized in that: Using methanol-water-phosphoric acid mixed solvent as mobile phase A, methanol-isopropanol mixed solvent as mobile phase B, and methanol as mobile phase C, a reversed phase analytical column was used for gradient elution to simultaneously determine the contents of various free sterols and moracolin K; The gradient elution was performed using a methanol-water-phosphoric acid mixed solvent with a volume ratio of 285:75:0.2 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 was C 18 Analytical columns; The specific operation of the gradient elution is as follows: at 0-12 min, the initial volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is 90:10:0; at 12.1-14 min, 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 0:10:90; at 14.1-35 min; the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 0:10:90; at 35.1-57 min, the volume ratio of the mobile phase A, the mobile phase B and the mobile phase C is maintained at 0:100:0; at 57.1-67 min, 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 238 nm at 0-15 min, and the target component to be measured is morakorin K; after 15 min, it is 210 nm, and the target component to be measured is free sterol; the flow rate of the gradient elution is 1.0 mL / min, the injection volume is 20 μL, and the column temperature is 30-34° C.; The reverse phase analytical column is Hypersil ODS2 C 18 Analytical columns; The sample solution is prepared before the gradient elution, specifically: weigh the sample, place it in a volumetric flask, add an organic solvent, shake and extract, let it cool to room temperature, make up to volume with the organic solvent, shake well, let it stand, filter and take the supernatant to obtain the sample solution; the organic solvent is 95% ethanol aqueous solution or a methanol-isopropanol solution with a volume ratio of 4:

1.

2. The method for simultaneously determining the contents of multiple free sterols and moracolin K in the phytosterol ester red yeast rice soft capsules according to claim 1, characterized in that: The mass volume ratio of the sample to the total amount of the organic solvent is 2g:100mL; the oscillation extraction includes ultrasonic extraction.

3. The method for simultaneously determining the contents of multiple free sterols and moracolin K in the phytosterol ester red yeast rice soft capsules as claimed in claim 2, characterized in that: The output power of the ultrasonic extraction is 500w and the time is 60min.

4. The method for simultaneously determining the contents of multiple free sterols and moracolin K in the phytosterol ester red yeast rice soft capsules according to claim 1, characterized in that: After the gradient elution, the chromatogram was recorded, and the qualitative analysis was performed according to the retention time of the reference substances of moracolin K, brassicasterol, stigmasterol, campesterol, and β-sitosterol. The concentrations of moracolin K, brassicasterol, stigmasterol, campesterol, and β-sitosterol in the test solution were obtained from the standard curve, and the total amount of moracolin K and free sterols was calculated.

5. The method for simultaneously determining the contents of multiple free sterols and moracolin K in phytosterol ester red yeast rice soft capsules as claimed in claim 4, characterized in that: 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 20 mg of lovastatin reference substance, dissolve it in methanol and make up to 10 mL to obtain a lovastatin standard stock solution with a concentration of 2 mg / mL; accurately weigh 10 mg of rapeseed sterol reference substance, dissolve it in isopropanol and make up to 10 mL to obtain a rapeseed sterol stock solution with a concentration of 1 mg / mL; accurately weigh 20 mg of stigmasterol reference substance, dissolve it in isopropanol and make up to 20 mL to obtain a stigmasterol stock solution with a concentration of 1 mg / mL; accurately weigh 10 mg of campesterol reference substance, dissolve it in isopropanol and make up to 10 mL to obtain a campesterol stock solution with a concentration of 1 mg / mL; accurately weigh 20 mg of β-sitosterol reference substance, dissolve it in isopropanol and make up to 10 mL to obtain a β-sitosterol stock solution with a concentration of 2 mg / mL; B. Accurately pipette 1.0 mL of the lovastatin standard stock solution, 1.0 mL of the rapeseed sterol stock solution, 0.6 mL of the stigmasterol stock solution, 1.2 mL of the campesterol stock solution, and 1 mL of the β-sitosterol stock solution, mix them, and dilute to 5 mL with 95% ethanol to obtain a control solution with a lovastatin concentration of 0.4 mg / mL, a rapeseed sterol concentration of 0.2 mg / mL, a stigmasterol concentration of 0.12 mg / mL, a campesterol concentration of 0.24 mg / mL, and a β-sitosterol concentration of 0.4 mg / mL, as the standard curve solution VI; C. Accurately pipette 0.5 mL of the standard curve solution VI, add 95% ethanol to dilute to 1 mL, and prepare a control solution with a lovastatin concentration of 0.2 mg / mL, a rapeseed sterol concentration of 0.1 mg / mL, a stigmasterol concentration of 0.06 mg / mL, a campesterol concentration of 0.12 mg / mL, and a β-sitosterol concentration of 0.2 mg / mL, as the standard curve solution V; D. Accurately pipette 0.4 mL of the standard curve solution VI, add 95% ethanol to dilute to 2 mL, and prepare a control solution with a lovastatin concentration of 0.08 mg / mL, a rapeseed sterol concentration of 0.04 mg / mL, a stigmasterol concentration of 0.024 mg / mL, a campesterol concentration of 0.048 mg / mL, and a β-sitosterol concentration of 0.08 mg / mL, as the standard curve solution IV; E. Accurately pipette 0.1 mL of the standard curve solution VI, add 95% ethanol to dilute to 1 mL, and prepare a control solution with a lovastatin concentration of 0.04 mg / mL, a rapeseed sterol concentration of 0.02 mg / mL, a stigmasterol concentration of 0.012 mg / mL, a campesterol concentration of 0.024 mg / mL, and a β-sitosterol concentration of 0.04 mg / mL, as the standard curve solution III; F. Accurately pipette 0.1 mL of the standard curve solution VI, add 95% ethanol to dilute to 2 mL, and prepare a control solution with a lovastatin concentration of 0.02 mg / mL, a rapeseed sterol concentration of 0.01 mg / mL, a stigmasterol concentration of 0.006 mg / mL, a campesterol concentration of 0.012 mg / mL, and a β-sitosterol concentration of 0.02 mg / mL, as the standard curve solution II; G. Accurately pipette 0.1 mL of the standard curve solution VI, add 95% ethanol to dilute to 5 mL, and prepare control solutions with lovastatin concentration of 0.008 mg / mL, rapeseed sterol concentration of 0.004 mg / mL, stigmasterol concentration of 0.0024 mg / mL, campesterol concentration of 0.0048 mg / mL, and β-sitosterol concentration of 0.008 mg / mL as standard curve solution I.

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