Method for determining statins component content in red yeast rice product based on quantitative analysis of multi-components by single marker and application of method

Abstract

The invention discloses a method for determining the content of statin components in a red yeast rice product based on quantitative analysis of multi-components by single marker and application of the method. The method comprises the following steps: (1) preparing a lovastatin reference substance into a reference substance solution; (2) obtaining an alcohol extract of a monascus product to be detected as a test solution; (3) respectively carrying out high performance liquid chromatography detection on the test solution and the reference solution, and calculating the content of lovastatin in the test solution through an external standard method; (4) respectively calculating the content of lovastatin acid and the content of dehydroxylated lovastatin in the test sample according to a formula: Ci = Ai * CS / (fi / s * AS) and the determined correction factor fi / s by taking the content of lovastatin in the test sample as a reference; the fi / s value of the lovastatin acid is 0.6229, and the fi / s value of the hydroxyl-removed lovastatin is 1.1479. The method is rapid, simple and convenient, low in cost and very accurate in result.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality inspection, and specifically relates to a method for determining the content of statins in red yeast rice products based on a one-measurement-multiple-evaluation method and its application. Background Art

[0002] Red yeast rice (monacolin K, MK) is a fermented form of the Aspergillus family fungus Monacolin K, inoculated onto soaked, steamed rice. First described in the "Essentials of Food and Drink," it is a traditional Chinese medicine used for both food and medicine, boasting benefits such as promoting blood circulation, removing blood stasis, and strengthening the spleen and digestion. In the 1970s, Japanese scholar Akira Endo first isolated the active substance lovastatin (monacolin K, MK) from Monacolin K. Modern research indicates that MK exerts its lipid-lowering effects by inhibiting the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key enzyme in cholesterol synthesis. Currently, red yeast rice is a common raw material for lipid-lowering drugs (such as Xuezhikang and Zhibituo) and health supplements (with 156 approved numbers). It significantly lowers blood cholesterol and protects vascular endothelial cells, and holds broad application prospects in the prevention and treatment of arteriosclerosis and coronary heart disease.

[0003] However, studies have shown that the MK content in commercially available red yeast rice products fluctuates widely. The MK content in a dietary supplement containing 600 mg of red yeast rice can vary by as much as 40 times (0.03 mg to 2.18 mg), making it difficult to guarantee its efficacy, safety, and quality stability.

[0004] The current red yeast rice quality evaluation methods have the following problems: (1) The determination methods and evaluation index limits in different quality standards are different, and there is no unified quality standard, which leads to large deviations in the MK content in red yeast rice and its products on the market when tested using different methods; (2) The quality evaluation index is single, only MK or the total amount of MK and MKA (monacolin K acid, MKA) is used as the indicator component, and the content of other statins is not controlled; for example, the 2020 edition of the Chinese Pharmacopoeia only includes the red yeast rice raw material standards for Xuezhikang tablets and capsules, and stipulates the content of MK in the red yeast rice raw material; the Sichuan Provincial Processing Specifications only stipulate the content of MK, while the Zhejiang and Jiangsu Provincial Processing Specifications stipulate the total amount of MK and MKA and the MK / MKA ratio. Therefore, how to scientifically and rationally evaluate the quality of fermented Chinese medicine red yeast rice is a current research difficulty.

[0005] Modern research indicates that red yeast rice contains a range of statins. The quality differences between commercially available red yeast rice products from different manufacturers are primarily due to the statins they contain. Establishing a multi-index content determination method for red yeast rice, using these components as quality control indicators, has important practical application for scientifically evaluating red yeast rice quality. However, multi-index content determination methods require a large number of expensive reference materials, significantly increasing testing costs for pharmaceutical manufacturers and testing agencies.

[0006] In order to solve this problem, the literature (Zhu Yingli et al., Simultaneous determination of lovastatin and lovastatin acid in red yeast rice and Poria cocos tablets by a single-marker method, Food Industry Science and Technology, 2023, 44(14): 290-296.) and the literature (Wang Ying et al., Determination of lovastatin and lovastatin acid in red yeast rice products by a single-marker method, West China Journal of Pharmaceutical Sciences, 2020, 36(6): 664-667.) both disclosed the use of a single-marker method (quantitative determination analysis multi-component by a single-marker, QAMS), using lovastatin as a reference to establish a correction factor relationship between it and lovastatin acid, and then achieve the simultaneous quantitative determination of lovastatin and lovastatin acid using one standard.

[0007] However, the above literature method only selected lovastatin and lovastatin acid as the detection objects, but the content of dehydroxylovastatin in different red yeast rice products varies greatly, which will affect the accuracy of the detection results of the total amount of statins in red yeast rice products. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for determining the content of statins in red yeast rice products based on a one-measurement-multiple-evaluation method and its application. This method can quickly, simply, cost-effectively and accurately determine the content of three statins in red yeast rice products, providing a reliable way to evaluate the quality of red yeast rice products.

[0009] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0010] A method for determining the content of statins in red yeast rice products based on a one-test-multiple-evaluation method, the method comprising the following steps:

[0011] (1) preparing a lovastatin reference substance into a reference substance solution;

[0012] (2) Obtaining an alcohol extract of the red yeast rice product to be tested as the test solution;

[0013] (3) The test solution and the reference solution were tested by high performance liquid chromatography, and the content of lovastatin in the test sample was calculated by the external standard method;

[0014] (4) Taking the content of lovastatin in the test sample as a reference, according to the formula: C i =A i ×C S / (f i / s ×A S ) Calculate the contents of lovastatin acid and deshydroxylovastatin in the test sample respectively;

[0015] In the formula, C i A is the content of lovastatin acid or deshydroxylovastatin in the test sample; i A is the peak area of lovastatin acid or deshydroxylovastatin in the test sample; S is the peak area of lovastatin in the sample; C S is the concentration of lovastatin in the test sample measured by the external standard method; f i / s is the correction factor;

[0016] Among them, the f of lovastatin acid i / s The value is 0.6229, and the f of deshydroxylovastatin i / s The value is 1.1479.

[0017] The present invention uses lovastatin as a reference substance and adopts a multi-point calibration method to obtain the relative correction factor f of lovastatin acid and deshydroxylovastatin. i / s It has been verified that the relative correction factor has high accuracy and will not deviate significantly under different chromatographic conditions (including instruments, chromatographic columns, flow rates, column temperatures, and detection wavelengths). In addition, the relative retention times can be used to locate the chromatographic peaks of lovastatin acid and deshydroxylovastatin. In this way, a method for determining the content of statins in red yeast rice products can be established based on the one-measurement-multiple-evaluation method. During the detection, it is only necessary to use the external standard method to determine the content of lovastatin, and the relative correction factor can be used to calculate the content of lovastatin acid and deshydroxylovastatin. This method is fast, simple, low-cost, and the results are very accurate.

[0018] Preferably, in step (2) of the above method for determining the content of statins in red yeast rice products, ethanol or methanol is used as the extraction reagent.

[0019] Preferably, in step (2) of the above method for determining the content of statins in red yeast rice products, 50% ethanol or 50% methanol is used as the extraction reagent; more preferably 50% ethanol.

[0020] Preferably, in step (2) of the above method for determining the content of statins in red yeast rice, the volume mass ratio of the extraction reagent to the red yeast rice slices to be tested is (25-50) mL / g; further preferably, the volume mass ratio of the extraction reagent to the red yeast rice product to be tested is 25 mL / g.

[0021] Preferably, in step (2) of the above method for determining the content of statins in red yeast rice, ultrasonic extraction or reflux extraction is used to obtain the alcohol extract of the red yeast rice slices to be tested.

[0022] More preferably, ultrasonic extraction for 30-60 min is used to obtain the alcohol extract of the red yeast rice product to be tested; during ultrasonic extraction, the optimal parameters are: ultrasonic extraction for 30 min at a power of 250 W and a frequency of 40 kHz.

[0023] Preferably, in step (3) of the above method for determining the content of statins in red yeast rice, the HPLC detection conditions are:

[0024] Chromatographic column: Zafex-Supfex-JX-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm);

[0025] Mobile phase: A is 0.1% phosphoric acid solution, B is acetonitrile, and C is methanol;

[0026] Gradient elution: 0-25 min, 40% A→40% A, 53% B→53% B, 7%→7% C;

[0027] 25~35min, 40%A→10%A, 53%B→90%B, 7%→0%C;

[0028] 35~45min, 10%A→10%A, 90%B→90%B, 0%→0%C;

[0029] Flow rate: 1 mL min -1 ;

[0030] Detection wavelength: 238nm;

[0031] Column temperature: 20°C;

[0032] Injection volume: 10 μL.

[0033] The present invention also provides an application of the above method in the quality evaluation of red yeast rice products, which includes: evaluating the quality of the red yeast rice product to be tested based on the contents of lovastatin, lovastatin acid and deshydroxylovastatin measured by the above method.

[0034] Compared with the prior art, the technical effects of the present invention are embodied in:

[0035] The present invention uses lovastatin as a reference substance and adopts a multi-point calibration method to obtain the relative correction factor f of lovastatin acid and deshydroxylovastatin. i / sThe relative correction factor has been verified to be highly accurate, with no significant deviations under different chromatographic conditions (including instrumentation, chromatographic column, flow rate, column temperature, and detection wavelength). Furthermore, the relative retention times can be used to locate the chromatographic peaks of lovastatin acid and deshydroxylovastatin. This method can be used to determine the contents of three statins in red yeast rice products based on the single-measurement, multiple-evaluation method. During testing, lovastatin only needs to be determined using the external standard method, and the relative correction factor can be used to calculate the contents of lovastatin acid and deshydroxylovastatin. This method enables rapid, simple, low-cost, and accurate determination of statins in red yeast rice products, providing a reliable approach for evaluating their quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The UV absorption graphs of lovastatin acid, lovastatin and deshydroxylovastatin;

[0037] Figure 2 HPLC chromatograms of lovastatin acid, lovastatin and deshydroxylovastatin mixed reference solution and red yeast rice test solution;

[0038] Among them, 1 is the chromatographic peak of lovastatin acid, 2 is the chromatographic peak of lovastatin, and 3 is the chromatographic peak of deshydroxylovastatin;

[0039] Figure 3 The standard curves are for lovastatin acid, lovastatin and deshydroxylovastatin. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0041] In the embodiments of the present invention, the following instruments, reagents and samples were used:

[0042] Instruments: Agilent liquid chromatograph; Thermo Fisher U3000 RSLC liquid chromatograph; Minispin 12,000 rpm centrifuge (Eppendorf, Germany); Milli-Q Advantage A ultrapure water machine (Millipore, USA); XPE-205 analytical balance (Mettler Toledo, accuracy 1 / 100,000); Elmasonic P ultrasonic cleaner (Elma, Germany).

[0043] Reagents: acetonitrile (MERCK, chromatographic grade); methanol (Sinopharm Chemical Reagent Co., Ltd., analytical grade); water (Mili-Q homemade water). Lovastatin (batch number 100600-202006, purity 99.6%) was purchased from the China Food and Drug Administration, lovastatin acid sodium salt (batch number: J28HS188558, purity 90.0%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and deshydroxylovastatin (batch number: WP24022008, 98.0%) was purchased from Sichuan Weikeqi Biotechnology Co., Ltd. All were used for content determination.

[0044] Wherein, the structural formula of lovastatin acid sodium salt is: The structural formula of lovastatin is: The structural formula of deshydroxylovastatin is:

[0045] Samples: 23 red yeast rice samples. The sources and batch numbers of the samples are shown in Table 1.

[0046] Table 1 Red yeast rice slice sample information

[0047]

[0048]

[0049] Example 1 Establishment of a method for determining the content of statins in red yeast rice products based on a one-test-multiple-evaluation method

[0050] 1. Determination of chromatographic conditions

[0051] 1.1 Preparation of reference solution

[0052] Take appropriate amounts of lovastatin acid sodium salt, lovastatin, and deshydroxylovastatin, accurately weigh them, and use 50% ethanol to prepare the following mixed reference solutions: lovastatin acid reference solution 37.50 μg / mL, lovastatin reference solution 41.59 μg / mL, and deshydroxylovastatin reference solution 18.27 μg / mL.

[0053] 1.2 Determine the chromatographic conditions

[0054] Take an appropriate amount of reference solution and inject it into Thermo Fisher U3000 RSLC liquid chromatograph, and obtain the UV absorption graph by the diode array detector, such as Figure 1 shown.

[0055] Depend on Figure 1 It can be determined that lovastatin acid, lovastatin and deshydroxylovastatin all have maximum absorption at 238 nm.

[0056] Therefore, the following HPLC analysis will be performed under the following basic conditions (individual parameters will be optimized below):

[0057] Chromatographic column: Zafex-Supfex-JX-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm);

[0058] Mobile phase: A is 0.1% phosphoric acid solution, B is acetonitrile, and C is methanol;

[0059] Gradient elution: 0-25 min, 40% A→40% A, 53% B→53% B, 7%→7% C;

[0060] 25~35min, 40%A→10%A, 53%B→90%B, 7%→0%C;

[0061] 35~45min, 10%A→10%A, 90%B→90%B, 0%→0%C;

[0062] Flow rate: 1 mL min -1 ;

[0063] Detection wavelength: 238nm;

[0064] Column temperature: 20°C;

[0065] Injection volume: 10 μL.

[0066] 2. Investigation of extraction conditions of test solution

[0067] 2.1 Investigation of extraction methods

[0068] Take 170.4 g of red yeast rice sample S, add 25 mL of 50% ethanol, and perform ultrasonic extraction (power 250 W, frequency 40 kHz, 30 min) and reflux extraction (30 min) respectively to obtain the test solution; then the above-mentioned chromatographic conditions are used for analysis, and the analysis results are shown in Table 2.

[0069] Table 2 Comparison results of extraction methods (mg / g)

[0070]

[0071] The results showed that there was no significant difference in the contents of the three components in the test solution during ultrasonic extraction and reflux extraction; however, because ultrasonic extraction is simpler, ultrasonic extraction was chosen below.

[0072] 2.2 Extraction solvent investigation

[0073] Take 170.4 g of red yeast rice sample S, add 25 mL of ethanol, 50% ethanol, methanol, 20% methanol, and 50% methanol respectively, and perform ultrasonic extraction (power 250 W, frequency 40 kHz, 30 min) to obtain the test solution; then use the above-mentioned chromatographic conditions for analysis, and the analysis results are shown in Table 3.

[0074] Table 3 Comparison results of extraction solvents (mg / g)

[0075]

[0076] The results showed that 50% ethanol had the best extraction efficiency for lovastatin acid and lovastatin, while methanol had the best extraction efficiency for deshydroxylovastatin. After comprehensive consideration, 50% ethanol was selected as the extraction solvent, as all three components had good extraction efficiency.

[0077] 2.3 Extraction time investigation

[0078] Take 170.4 g of red yeast rice sample S, add 25 mL of 50% ethanol, and perform ultrasonic extraction. The extraction time is 30 min, 60 min, and 90 min respectively to obtain the test solution; then the above-mentioned chromatographic conditions are used for analysis, and the analysis results are shown in Table 4.

[0079] Table 4 Comparison of extraction time (mg / g)

[0080]

[0081] The results showed that there was no significant difference in the content determination results of the three components when the extraction time was 30 minutes, 60 minutes, and 90 minutes, so the extraction time selected below was 30 minutes.

[0082] 2.4 Extraction volume investigation

[0083] Take 170.4 g of red yeast rice sample S, add 25 mL, 50 mL, and 100 mL of 50% ethanol respectively, and perform ultrasonic extraction (power 250 W, frequency 40 kHz, 30 min) to obtain the test solution; then use the above-mentioned chromatographic conditions for analysis, and the analysis results are shown in Table 5.

[0084] Table 5 Comparison results of different extraction volumes (mg / g)

[0085]

[0086] The results showed that there was little difference in extraction efficiency between 25 mL and 50 mL. Considering that the peak areas of the three components were moderate, the extraction volume selected below was 25 mL.

[0087] Based on the above experimental results, the extraction method of the test solution was finally determined as follows: take about 0.4 g of red yeast rice sample powder (passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 50% ethanol, seal it, shake it well, weigh it, and perform ultrasonic extraction (power 250 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50% ethanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0088] 3. Preparation of mixed reference solution

[0089] Take appropriate amounts of lovastatin acid, lovastatin, and deshydroxylovastatin, accurately weigh them, and add 50% ethanol to prepare a mixed reference solution containing 37.50 μg of lovastatin acid, 41.59 μg of lovastatin, and 18.27 μg of deshydroxylovastatin per 1 mL.

[0090] 4. Chromatographic analysis

[0091] Take the test solution and the mixed reference solution respectively and analyze them under the above chromatographic conditions. The results are as follows: Figure 2 shown.

[0092] It can be seen that the extraction method and chromatographic conditions determined in this example are good.

[0093] 5. Methodological Investigation

[0094] 5.1 Investigation of linear relationships

[0095] Accurately pipette the mixed reference solution into 1, 2, 4, 8, 10, 15, 20, 25, and 30 μL samples, and analyze using the above chromatographic conditions. Regress the chromatographic peak area (Y) against the injection volume (X) to calculate the regression equations for the three components. The linear plot is shown in Figure 3 , the results are shown in Table 6.

[0096] Table 63 Linear determination results of components

[0097]

[0098] The results showed that the injection amount of lovastatin acid, lovastatin and deshydroxylovastatin showed good linear relationship in the range of 37.500ng~1.275ng, 41.589ng~1247.669ng and 18.267ng~548.016ng, respectively.

[0099] 5.2 Precision test

[0100] The same mixed reference solution was accurately pipetted and injected six times, and the peak areas of the three components were measured. The results are shown in Table 7.

[0101] Table 7 Precision test

[0102]

[0103] The results showed that when the same mixed reference solution was analyzed multiple times, the RSDs of the peak areas of lovastatin acid, lovastatin and deshydroxylovastatin were 0.17%, 0.22% and 0.10%, respectively, indicating good instrument precision.

[0104] 5.3 Stability test

[0105] Accurately pipette the same sample solution and inject it once at regular intervals within 64 hours. The injection volume is 10 μl. The analysis results are shown in Table 8.

[0106] Table 83 Component Stability Test Results

[0107]

[0108] The results showed that when tested at different times, the RSDs of the peak areas of the three components were all less than 3%, indicating that the test solution was basically stable within 64 hours.

[0109] 5.4 Repeatability test

[0110] Six portions of the same batch of test sample S17 (batch number: 231010) were taken and the test sample solutions were prepared according to the above-mentioned preparation method. Then, chromatographic analysis was performed to determine the contents of the three statin components. The determination results are shown in Table 9.

[0111] Table 9 Red yeast rice repeatability test results (mg / g)

[0112]

[0113] The results showed that when the same test sample was analyzed in parallel, the average contents of lovastatin acid, lovastatin and deshydroxylovastatin in the six test sample solutions were 2.315 mg / g, 2.843 mg / g and 0.944 mg / g, respectively, and the RSDs were 2.0%, 2.7% and 2.8% (all less than 3%), respectively, indicating that the method had good repeatability.

[0114] 5.5 Recovery test

[0115] Accurately weigh 6 portions of the test sample S17 (batch number: 231010), add appropriate amounts of lovastatin acid, lovastatin and deshydroxylovastatin, respectively, prepare the test sample solution according to the above method, and determine the content of the three statins contained therein. The results are shown in Table 10.

[0116] Table 10 Red yeast rice recovery test results (n=6)

[0117]

[0118]

[0119] 6. QAMS method research

[0120] 6.1 Calculation of relative correction factor

[0121] 0.5, 1, 5, 10, 15, 20, 30, and 40 μL of the mixed reference solution were accurately aspirated and injected into the ThermoU3000 RSLC liquid chromatograph. The standard curve was drawn with the chromatographic peak area (Y) as the ordinate and the injection volume (X, ng) as the abscissa, and the regression equation and linear range were calculated. Lovastatin was used as the reference substance, and the relative correction factors of lovastatin acid and deshydroxylovastatin at various concentrations were calculated by the multi-point calibration method. The results are shown in Table 11.

[0122] Table 11 Correction factor calculation results

[0123]

[0124] 6.2 Effects of Different Instruments and Columns on Relative Correction Factors

[0125] Using lovastatin as internal reference, the Zafex-Supfex-JX-C 18 (4.6×250mm, 5μm), Cosmosil5C 18 -MS-II (4.6×250mm, 5μm), phenotypex luna C 18 The effects of three brands of chromatographic columns (4.6×250mm, 5μm) on the correction factor are shown in Table 12.

[0126] Table 12 Effects of different instruments and chromatographic columns

[0127]

[0128] The results showed that the RSDs of the relative correction factors of lovastatin acid and deshydroxylovastatin on different instruments and columns were all less than 3.0%, indicating good reproducibility. The instruments and chromatographic columns had little effect on the relative correction factors of lovastatin acid and deshydroxylovastatin.

[0129] 6.3 Effect of different flow rates on relative correction factors

[0130] The mixed reference solution was taken and the chromatograph was carried out on a Thermo Fisher U3000 liquid chromatograph in a Zafex-Supfex-JX-C 18The chromatographic column was tested at different flow rates (0.9, 1.0, 1.1 mL·min -1 ) for each component f i / s The results are shown in Table 13.

[0131] Table 13 Comparison of correction factors for different flow rates

[0132]

[0133] The results showed that lovastatin acid and deshydroxylovastatin f i / s The RSDs were all less than 3.0%, indicating that flow rate had no significant effect on their relative correction factors.

[0134] 6.4 Effect of Different Column Temperatures on Relative Correction Factors

[0135] The mixed reference solution was taken and the chromatograph was carried out on a Thermo Fisher U3000 liquid chromatograph in a Zafex-Supfex-JX-C 18 The effects of different column temperatures (20℃, 25℃, 30℃, 35℃) on the f-value of lovastatin acid and deshydroxylovastatin were investigated on the chromatographic column. i / s The results are shown in Table 14.

[0136] Table 14 Comparison of relative correction factors at different column temperatures

[0137]

[0138] The results showed that at different column temperatures, the f i / s The RSDs were all less than 2.0%, indicating that different column temperatures had no significant effect on their relative correction factors.

[0139] 6.5 Effect of different detection wavelengths on relative correction factors

[0140] The mixed reference solution was taken and the chromatograph was carried out on a Thermo Fisher U3000 liquid chromatograph in a Zafex-Supfex-JX-C 18 The effects of different detection wavelengths (236 nm, 237 nm, 238 nm, 239 nm, and 240 nm) on the performance of lovastatin acid and deshydroxylovastatin were investigated on the chromatographic column. i / s The results are shown in Table 15.

[0141] Table 15 Effect of different detection wavelengths on relative correction factors

[0142]

[0143]

[0144] The results showed that at different detection wavelengths, lovastatin acid and deshydroxylovastatin fi / s The RSDs of the two groups were all less than 1.0%, indicating that different detection wavelengths had a significant effect on their f i / s No significant effect.

[0145] 6.6 Chromatographic Peak Positioning

[0146] The mixed reference solution was injected and measured according to the chromatographic conditions described above. The relative retention times of lovastatin acid and deshydroxylovastatin were calculated using lovastatin as a reference. The results are shown in Table 16.

[0147] Table 16 Relative retention times of lovastatin acid and deshydroxylovastatin

[0148]

[0149] The results showed that the RSDs of the relative retention times of lovastatin acid and deshydroxylovastatin were all less than 2.0% under different instruments and chromatographic columns, indicating that the relative retention time method could be used to locate the chromatographic peaks of lovastatin acid and deshydroxylovastatin.

[0150] 7. Method for determining the content of statins in red yeast rice products based on the one-test-multiple-evaluation method

[0151] 7.1 Establishment of the determination method

[0152] Based on the above experimental data, this embodiment proposes a method for determining the content of statins in red yeast rice products based on a one-measurement, multiple-evaluation method, which includes the following steps:

[0153] (1) preparing a lovastatin reference substance into a reference substance solution;

[0154] Specifically, the reference solution can be prepared by first dissolving the lovastatin reference substance in 50% ethanol to prepare a stock solution, and then diluting it in a gradient manner with 50% ethanol to obtain a series of reference solutions;

[0155] Alternatively, the lovastatin reference substance may be dissolved in 50% ethanol and directly prepared, and the injection volume may be changed gradually during HPLC analysis;

[0156] (2) Obtaining an alcohol extract of the red yeast rice product to be tested as the test solution;

[0157] Specifically, take about 0.4 g of red yeast rice sample powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 50% ethanol, seal it tightly, shake it well, weigh it, and perform ultrasonic extraction (power 250 W, frequency 40 kHz) for 30 min. Let it cool, weigh it again, make up the lost weight with 50% ethanol, shake it well, filter it, and take the filtrate to obtain the test solution;

[0158] (3) The test solution and the reference solution were tested by high performance liquid chromatography, and the content of lovastatin in the test sample was calculated by the external standard method;

[0159] The chromatographic conditions used for high performance liquid chromatography (HPLC) are as follows:

[0160] Chromatographic column: Zafex-Supfex-JX-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm);

[0161] Mobile phase: A is 0.1% phosphoric acid solution, B is acetonitrile, and C is methanol;

[0162] Gradient elution: 0-25 min, 40% A→40% A, 53% B→53% B, 7%→7% C;

[0163] 25~35min, 40%A→10%A, 53%B→90%B, 7%→0%C;

[0164] 35~45min, 10%A→10%A, 90%B→90%B, 0%→0%C;

[0165] Flow rate: 1 mL min -1 ;

[0166] Detection wavelength: 238nm;

[0167] Column temperature: 20°C;

[0168] Injection volume: 10 μL;

[0169] The reference solution was subjected to high performance liquid chromatography (HPLC), and the injection volume (X) was regressed with the chromatographic peak area (Y) to obtain a standard curve. The test solution was subjected to high performance liquid chromatography (HPLC), and the chromatographic peak area of lovastatin was obtained according to the retention time of lovastatin. The content of lovastatin was calculated according to the HPLC curve.

[0170] (4) Taking the content of lovastatin in the test sample as a reference, according to the formula: C i =A i ×C S / (f i / s ×A S ) Calculate the contents of lovastatin acid and deshydroxylovastatin in the test sample respectively;

[0171] In the formula, C i A is the content of lovastatin acid or deshydroxylovastatin in the test sample; i A is the peak area of lovastatin acid or deshydroxylovastatin in the test sample; S is the peak area of lovastatin in the test sample; C Sis the concentration of lovastatin in the test sample measured by the external standard method; f i / s is the correction factor;

[0172] Among them, the f of lovastatin acid i / s The value is 0.6229, and the f of deshydroxylovastatin i / s The value is 1.1479.

[0173] 7.1 Accuracy Analysis

[0174] The above-mentioned “method for determining the content of statins in red yeast rice products based on a one-test-multiple-evaluation method” was used to determine the contents of lovastatin acid, lovastatin and deshydroxylovastatin in 23 batches of red yeast rice from different companies (see Table 1), and the results were compared with those determined by the external standard method. The results are shown in Table 17.

[0175] Table 17 Determination results of one-measurement-multiple-evaluation method and external standard method (%)

[0176]

[0177]

[0178] The t-test was used to compare the content determination values of the external standard method and the single-test, multiple-evaluation method. The results showed that the p-values for lovastatin acid and deshydroxylovastatin were 0.9960 and 0.8762, respectively. The p-values for the contents of the two components were both greater than 0.05, indicating no significant differences. That is, there was no significant difference between the single-test, multiple-evaluation method and the external standard method. This shows that the single-test, multiple-evaluation method established in this example can be applied to the analysis and determination of the contents of the three statins in red yeast rice products.

[0179] Example 2 Quality Evaluation Method for Red Yeast Rice Products

[0180] This embodiment provides a method for evaluating the quality of red yeast rice products, comprising:

[0181] S1: Determine the content of lovastatin using the method of Example 1, and calculate the contents of lovastatin acid and deshydroxylovastatin based on the corresponding relative correction factors;

[0182] S2: comparing the measured contents of lovastatin, lovastatin acid and deshydroxylovastatin with preset values, and evaluating the quality of the red yeast rice product to be tested based on the comparison results.

Claims

1. A method for determining the content of statins in red yeast rice products based on a one-measurement-multiple-evaluation method, characterized in that: The following steps are involved: (1) preparing a lovastatin reference substance into a reference substance solution; (2) Obtaining an alcohol extract of the red yeast rice product to be tested as the test solution; (3) The test solution and the reference solution were tested by high performance liquid chromatography, and the content of lovastatin in the test sample was calculated by the external standard method; (4) Taking the content of lovastatin in the test sample as a reference, according to the formula: C i =A i ×C S / (f i / s ×A S ) Calculate the contents of lovastatin acid and deshydroxylovastatin in the test sample respectively; In the formula, C i A is the content of lovastatin acid or deshydroxylovastatin in the test sample; i A is the peak area of lovastatin acid or deshydroxylovastatin in the test sample; S is the peak area of lovastatin in the test sample; C S is the concentration of lovastatin in the test sample measured by the external standard method; f i / s is the correction factor; Among them, the f of lovastatin acid i / s The value is 0.6229, and the f of deshydroxylovastatin i / s The value is 1.1479.

2. The method for determining the content of statins in red yeast rice products according to claim 1, wherein: In step (2), ethanol or methanol is used as the extraction reagent.

3. The method for determining the content of statins in red yeast rice products according to claim 2, wherein: In step (2), 50% ethanol or 50% methanol is used as the extraction reagent.

4. The method for determining the content of statins in red yeast rice products according to claim 2, wherein: In step (2), the volume mass ratio of the extraction reagent to the red yeast rice product to be tested is (25-50) mL / g.

5. The method for determining the content of statins in red yeast rice products according to claim 4, wherein: In step (2), the volume mass ratio of the extraction reagent to the red yeast rice product to be tested is 25 mL / g.

6. The method for determining the content of statins in red yeast rice products according to claim 1, wherein: In step (2), an ethanol extract of the red yeast rice product to be tested is obtained by ultrasonic extraction or reflux extraction.

7. The method for determining the content of statins in red yeast rice products according to claim 1, wherein: In step (2), an ethanol extract of the red yeast rice product to be tested is obtained by ultrasonic extraction for 30-60 min.

8. The method for determining the content of statins in red yeast rice products according to claim 7, wherein: The parameters of ultrasonic extraction are: ultrasonic extraction at a power of 250W and a frequency of 40 kHz for 30 minutes.

9. The method for determining the content of statins in red yeast rice products according to claim 1, wherein: In step (3), the HPLC detection conditions are: Chromatographic column: Zafex-Supfex-JX-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm); Mobile phase: A is 0.1% phosphoric acid solution, B is acetonitrile, and C is methanol; Gradient elution: 0-25 min, 40% A→40% A, 53% B→53% B, 7%→7% C; 25~35min, 40%A→10%A, 53%B→90%B, 7%→0%C; 35~45min, 10%A→10%A, 90%B→90%B, 0%→0%C; Flow rate: 1 mL min -1 ; Detection wavelength: 238nm; Column temperature: 20°C; Injection volume: 10 μL.

10. Application of the method according to claims 1 to 9 in the quality evaluation of red yeast rice products, characterized in that: include: The quality of the red yeast rice product to be tested was evaluated based on the measured contents of lovastatin, lovastatin acid and deshydroxylovastatin.

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