A method for establishing the fingerprint of red yeast rice by high performance liquid chromatography and its application in the quality detection of Shi Guogong medicinal liquor

Abstract

The application discloses a method for establishing a Hongqu fingerprint spectrum by using a high performance liquid chromatography technology and application of the method in quality detection of Shiguogong medicinal liquor, the Hongqu fingerprint spectrum obtained by using the method has good separation of each chromatographic peak, a smooth baseline, a good peak type, and good precision, repeatability, specificity and stability, seven fingerprint peaks of the Hongqu fingerprint spectrum established by the application are confirmed, and by comparison with reference substances and in combination with ultraviolet absorption spectrum characteristics, characteristic peaks of main chemical components, namely, open-loop lovastatin and lovastatin in the Hongqu are identified, the method can be used for quality detection of the Hongqu, and provides a scientific basis for improving a production process and quality control of the Hongqu, and can also be applied to quality detection of Hongqu components in the Shiguogong medicinal liquor, and has important significance for guaranteeing the quality of the Hongqu in the Shiguogong medicinal liquor.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine identification technology, and more specifically, to a method for establishing a red yeast rice fingerprint spectrum using high performance liquid chromatography and its application in the quality testing of Shi Guogong medicinal wine. Background Technology

[0002] Shi Guogong Medicated Wine is composed of Polygonatum odoratum, vinegar-processed turtle shell, stir-fried Atractylodes macrocephala, Achyranthes bidentata, Taxillus chinensis, silkworm excrement, Ligusticum chuanxiong, Saposhnikovia divaricata, Chaenomeles speciosa, Angelica sinensis, Carthamus tinctorius, Glycyrrhiza uralensis, Notopterygium incisum, Angelica pubescens, Dipsacus asper, deer antler glue, and red yeast rice. It has the effects of dispelling wind and dampness, promoting blood circulation and unblocking meridians, and is mainly used for wind-cold-dampness arthralgia, joint pain, and numbness of the limbs.

[0003] The red yeast rice contained in Shi Guogong medicinal wine is a fermented product with both edible and medicinal effects, made by inoculating rice (with the husk removed) with the Aspergillus violaceus fungus. It possesses excellent lipid-lowering activity and is a raw material for natural statin drugs such as Xuezhikang and Zhibituo, clinically used to treat hyperlipidemia and atherosclerosis. Studies have shown that the main active ingredient in the lipid-lowering effect of red yeast rice is its secondary metabolite, lovastatin.

[0004] While existing technologies disclose methods for determining the lovastatin content in red yeast rice using HPLC (Gao Wenming, Lin Yuesong, Dai Guoliang, Zhao Lingang. Simultaneous determination of three components, including adenosine, in red yeast rice from different sources by HPLC [J]. Pharmaceutical and Clinical Research, 2022, 30(2):140-142), lovastatin exists in both acidic (closed-ring) and lactone (open-ring) forms, both of which are present in red yeast rice. Therefore, existing technologies cannot comprehensively and accurately evaluate the quality and efficacy of red yeast rice. The efficacy and safety of Shi Guogong medicinal wine depend on the quality and proportion of each component, and red yeast rice, as a key component, directly affects the therapeutic effect of Shi Guogong medicinal wine due to its quality and content.

[0005] However, there is currently no method for establishing fingerprint chromatograms of red yeast rice using high-performance liquid chromatography (HPLC). This is mainly because, compared to ultra-high-performance liquid chromatography (UHPLC), HPLC presents challenges in detecting trace components in red yeast rice, increasing the complexity and uncertainty of fingerprint chromatogram establishment. Nevertheless, HPLC also offers advantages such as high separation efficiency and fast analysis speed. Furthermore, the instruments and operating methods for HPLC are relatively simple and widely available. For the Chinese medicinal materials industry, popularizing a simple and efficient fingerprint chromatogram establishment method would help improve the quality control level of Chinese medicinal materials and promote the healthy development of the Chinese medicinal materials industry.

[0006] Because existing technologies cannot fully reflect the characteristics of various components in red yeast rice, such as the acidic and lactone forms of lovastatin, as well as other potential active ingredients, they are even less suitable for batch-to-batch quality control and comparison, and further evaluation of the consistency and stability of red yeast rice quality in Shi Guogong medicinal wine. Therefore, to further detect the quality of red yeast rice in Shi Guogong medicinal wine, control its overall efficacy, reveal the interactions between its components, and improve its quality control, there is an urgent need in this field for a high-performance liquid chromatography (HPLC) method that can establish a fingerprint spectrum of red yeast rice in Shi Guogong medicinal wine and can be applied to its detection. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for establishing a red yeast rice fingerprint spectrum using high performance liquid chromatography and its application in the quality detection of Shi Guogong medicinal wine.

[0008] The first objective of this invention is to provide a method for establishing a fingerprint spectrum of red yeast rice using high performance liquid chromatography.

[0009] The second objective of this invention is to provide a method for establishing a red yeast rice comparative fingerprint spectrum.

[0010] The third objective of this invention is to provide a method for quality testing of red yeast rice.

[0011] The fourth objective of this invention is to provide a method for quality testing of Shi Guogong medicinal wine.

[0012] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0013] A method for establishing a red yeast rice fingerprint using high-performance liquid chromatography includes the following steps:

[0014] Reference solution: Lovastatin solution (open-chain);

[0015] Test solution: methanol extract of red yeast rice;

[0016] HPLC chromatographic conditions: An Agilent ZORBAX SB-Aq column was used. Mobile phase A consisted of 0.08–0.12% (v / v) phosphoric acid aqueous solution, and mobile phase B consisted of acetonitrile. During the gradient elution program, the volume percentage of mobile phase B in the mobile phase system was as follows:

[0017] From 0 to 26 minutes, the mobile phase B increased from 1% to 15%.

[0018] Over 26–40 minutes, mobile phase B increased from 15% to 50%.

[0019] The mobile phase B increased from 50% to 60% over 40–58 minutes.

[0020] 58–60 min, mobile phase B decreased from 60% to 1%;

[0021] The detection wavelength is a variable wavelength detection:

[0022] The detection wavelength was 257 nm, and the time interval was 0–36 min.

[0023] 36–60 min, detection wavelength 238 nm;

[0024] The fingerprint chromatograms of red yeast rice were obtained by performing HPLC detection on the reference solution and the test solution respectively according to the HPLC chromatographic conditions.

[0025] Preferably, the reference solution is an open-ring lovastatin solution obtained by ring-opening a lovastatin solution.

[0026] More preferably, the method for preparing the reference solution is as follows:

[0027] Lovastatin and acetonitrile were mixed to prepare a lovastatin-acetonitrile solution with a concentration of 0.3-0.7 mg / ml. Sodium hydroxide solution with a concentration of 0.15-0.25 mol / L was added to the lovastatin-acetonitrile solution to obtain a mixed solution. The volume ratio of the lovastatin-acetonitrile solution to the sodium hydroxide solution was 1:10-20.

[0028] After ultrasonic conversion of the mixture for 30–60 minutes, add phosphoric acid aqueous solution to adjust the pH to 6.5–7.5 to obtain a solution. Dilute the solution with acetonitrile 1–3 times to obtain a reference solution.

[0029] More preferably, the concentration of lovastatin in the lovastatin-acetonitrile solution is 0.5 mg / ml.

[0030] More preferably, a 0.2 mol / L sodium hydroxide solution is added to the lovastatin-acetonitrile solution to obtain a mixed solution.

[0031] Even more preferably, the ultrasonic conversion mixture is carried out for 30 minutes.

[0032] More preferably, an aqueous solution of phosphoric acid is added to adjust the pH to 7 to obtain a solution.

[0033] Most preferably, the solution is obtained by adding 3 mol / L of phosphoric acid aqueous solution to adjust the pH value to 7.

[0034] More preferably, the solution is diluted by 1 part using acetonitrile.

[0035] The method for preparing the reference solution should also be within the scope of protection of this invention.

[0036] Preferably, the preparation method of the test solution is as follows: take the red yeast rice to be tested, crush it, sieve it, and then sonicate it with a methanol aqueous solution with a volume fraction of 50-75% for 10-20 minutes. After centrifugation, take the supernatant to obtain the test solution.

[0037] More preferably, the supernatant is filtered, and the filtrate is used to obtain the test solution.

[0038] Most preferably, the filtration is performed using a 0.45 μm filter membrane.

[0039] More preferably, the red yeast rice to be tested is pulverized and passed through a 50-mesh sieve.

[0040] More preferably, the mixture is ultrasonically treated with a methanol aqueous solution of 50-75% by volume for 15 minutes.

[0041] Most preferably, the mixture is ultrasonically treated with a 50% (v / v) methanol aqueous solution for 15 minutes.

[0042] More preferably, the ratio of the red yeast rice to be tested to the methanol aqueous solution is 0.1g:10-20ml.

[0043] Most preferably, the ratio of the red yeast rice to be tested to the methanol aqueous solution is 0.1g:10ml.

[0044] More preferably, the ultrasonic processing parameters are a power of 200-300W and a frequency of 30-50kHz.

[0045] Most preferably, the ultrasonic processing parameters are 250W power and 40kHz frequency.

[0046] Preferably, the column temperature of the chromatographic column is 28–40°C.

[0047] More preferably, the column temperature of the chromatographic column is 28–32°C.

[0048] Preferably, the gradient elution process has a flow rate of 0.9–1.2 ml / min.

[0049] More preferably, the gradient elution process has a flow rate of 1 ml / min.

[0050] Preferably, in the HPLC chromatographic conditions, the injection volume for detection is 10 μl.

[0051] Preferably, the mobile phase B is an aqueous solution of formic acid with a volume percentage of 0.10%.

[0052] Preferably, in the red yeast rice fingerprint spectrum obtained using the method, the theoretical plate number, calculated based on the open-ring lovastatin peak obtained from the reference solution, should be no less than 8000.

[0053] The application of any of the methods described in the quality testing of Shi Guogong medicinal wine should also be within the scope of protection of this invention.

[0054] A method for establishing a reference fingerprint spectrum for red yeast rice, the method comprising: performing high performance liquid chromatography (HPLC) on a red yeast rice standard sample according to any of the methods described above to obtain a reference fingerprint spectrum, wherein the reference fingerprint spectrum contains 7 fingerprint peaks: peak 1 to peak 7 in sequence, with peak 4 corresponding to the reference solution as peak S, and the relative retention times of the remaining peaks within ±10% of a specified value, wherein the specified values ​​are: peak 1 0.219; peak 2 0.528; peak 3 0.853; peak S 1.000; peak 5 1.049; peak 6 1.066; peak 7 1.108.

[0055] Using the fingerprint spectrum obtained by the method described in this invention, and by comparing it with a reference standard and combining it with the characteristics of ultraviolet absorption spectroscopy, characteristic peaks of two chemical components were identified among the seven fingerprint peaks: peak 4, a characteristic peak of open-ring lovastatin, and peak 7, a characteristic peak of lovastatin.

[0056] Preferably, according to any of the methods described, high-performance liquid chromatography is used to detect 3 to 6 different batches of red yeast rice standard samples to obtain fingerprint spectra of different batches of red yeast rice standard samples. The "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" is used to automatically match the spectra through the median method and multi-point correction to generate a reference fingerprint spectra.

[0057] More preferably, high-performance liquid chromatography was used to detect four different batches of red yeast rice standard samples.

[0058] Specifically, the “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)” was used for analysis. A batch of red yeast rice standard sample fingerprint chromatograms was randomly selected as reference chromatograms. The time window width was set to 0.1 min. The median method and multi-point correction were used to automatically match the chromatograms and generate a control fingerprint chromatogram.

[0059] A method for quality testing of red yeast rice includes the following steps: establishing a fingerprint spectrum of the red yeast rice to be tested according to any of the methods described above, obtaining a fingerprint spectrum of the sample to be tested, wherein the fingerprint spectrum of the sample to be tested contains 7 fingerprint peaks from the control fingerprint spectrum, and the similarity between the 7 fingerprint peaks from the control fingerprint spectrum and the corresponding fingerprint peaks from the fingerprint spectrum of the sample to be tested is not less than 0.90, and the red yeast rice to be tested is a qualified product.

[0060] Preferably, the method of the chromatographic fingerprint spectrum similarity evaluation system of traditional Chinese medicine is used. The similarity between the 7 fingerprint peaks in the reference fingerprint spectrum and the corresponding fingerprint peaks in the fingerprint spectrum of the sample to be tested is calculated. If the similarity of the 7 fingerprint peaks is not less than 0.90, the sample to be tested is a qualified product.

[0061] The method for establishing a fingerprint spectrum of red yeast rice using high performance liquid chromatography and the method for quality detection of red yeast rice, when applied to the overall evaluation of red yeast rice, should also be within the scope of protection of this invention.

[0062] A method for quality testing of Shi Guogong medicinal wine includes the following steps:

[0063] Reference solution: Lovastatin solution (open-chain);

[0064] Test solution: methanol extract of Shi Guogong medicinal wine;

[0065] HPLC chromatographic conditions: An Agilent ZORBAX SB-Aq column was used. Mobile phase A consisted of 0.08–0.12% (v / v) phosphoric acid aqueous solution, and mobile phase B consisted of acetonitrile. During the gradient elution program, the volume percentage of mobile phase B in the mobile phase system was as follows:

[0066] From 0 to 26 minutes, the mobile phase B increased from 1% to 15%.

[0067] Over 26–40 minutes, mobile phase B increased from 15% to 50%.

[0068] The mobile phase B increased from 50% to 60% over 40–58 minutes.

[0069] 58–60 min, mobile phase B decreased from 60% to 1%;

[0070] The detection wavelength is a variable wavelength detection:

[0071] The detection wavelength was 257 nm, and the time interval was 0–36 min.

[0072] 36–60 min, detection wavelength 238 nm;

[0073] The fingerprint chromatograms of Shi Guogong medicinal wine were obtained by performing HPLC detection on the reference solution and the test solution respectively according to the HPLC chromatographic conditions.

[0074] The fingerprint spectrum of Shi Guogong medicinal wine contains 5 fingerprint peaks. The corresponding peak of the reference solution is designated as the S peak. The specified values ​​for the relative retention times of the 5 fingerprint peaks are 0.528, 0.853, and for the S peak, 1.000, 1.066, and 1.108, respectively. If the relative retention times of the 5 fingerprint peaks are within ±10% of the specified values, then the quality of the red yeast rice component in the Shi Guogong medicinal wine to be tested is qualified.

[0075] Preferably, the reference solution is prepared using the method for preparing the reference solution.

[0076] Preferably, the preparation method of the test solution is as follows: take the Shi Guogong medicinal wine to be tested, evaporate it to dryness to obtain the residue, treat the residue with a methanol aqueous solution with a volume fraction of 50-75% by ultrasonic treatment for 10-20 minutes, centrifuge and take the supernatant to obtain the test solution.

[0077] More preferably, the supernatant is filtered, and the filtrate is used to obtain the test solution.

[0078] Most preferably, the filtration is performed using a 0.45 μm filter membrane.

[0079] More preferably, the mixture is ultrasonically treated with a methanol aqueous solution of 50-75% by volume for 15 minutes.

[0080] Most preferably, the mixture is ultrasonically treated with a 50% (v / v) methanol aqueous solution for 15 minutes.

[0081] More preferably, the ratio of the amount of the Shi Guogong medicinal wine to be tested to the amount of the methanol aqueous solution is 3-6g:5-10ml.

[0082] Most preferably, the ratio of the amount of the Shi Guogong medicinal wine to be tested to the amount of the methanol aqueous solution is 3-6g:5ml.

[0083] More preferably, the ultrasonic processing parameters are a power of 200-300W and a frequency of 30-50kHz.

[0084] Most preferably, the ultrasonic processing parameters are 250W power and 40kHz frequency.

[0085] Preferably, the HPLC chromatographic conditions are the same as those in the method for establishing a red yeast rice fingerprint using high-performance liquid chromatography.

[0086] The method for quality testing of Shi Guogong medicinal wine, when applied to the quality testing of red yeast rice components in Shi Guogong medicinal wine, should also be within the scope of protection of this invention.

[0087] Compared with the prior art, the present invention has the following beneficial effects:

[0088] This invention establishes an HPLC fingerprint spectrum with good separation and comprehensive chromatographic peaks for red yeast rice. A total of 7 fingerprint peaks were identified. Each chromatographic peak was well separated, with a stable baseline and good peak shape. It also has good precision, repeatability, specificity and stability. Through practical application, it has been confirmed that this fingerprint spectrum establishment method is suitable for the quality detection of red yeast rice components in Shi Guogong medicinal wine, which is of great significance for ensuring the quality of red yeast rice in Shi Guogong medicinal wine.

[0089] The fingerprint spectrum establishment method described in this invention can detect the traditional Chinese medicine chemical components of red yeast rice. With the help of the traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, the similarity between the fingerprint spectrum of the red yeast rice sample to be tested and the control fingerprint spectrum can be calculated. This similarity result can be used to evaluate the consistency of chemical components between the red yeast rice sample to be tested and the red yeast rice standard sample.

[0090] The quality testing method for red yeast rice described in this invention can be used to optimize the production process and quality control procedures of red yeast rice. By analyzing the similarity of the fingerprint spectrum of red yeast rice samples under different process conditions, key factors affecting the quality of red yeast rice can be identified, thereby optimizing the production process, improving the quality and stability of red yeast rice, and providing a scientific basis for improving the production process and quality control of red yeast rice. Attached Figure Description

[0091] Figure 1 The chromatogram is obtained under the initial liquid chromatography column elution conditions.

[0092] Figure 2 The chromatogram obtained by adjusting the elution conditions of the liquid chromatography column.

[0093] Figure 3 The chromatogram obtained after readjusting the elution conditions of the liquid chromatography column.

[0094] Figure 4 Chromatograms obtained at different column temperatures.

[0095] Figure 5 Chromatograms obtained at different detection wavelengths: A in Figure 5 is the fingerprint spectrum of the test sample obtained by detection at a wavelength of 237 nm; B in Figure 5 is the fingerprint spectrum of the test sample obtained by detection at a wavelength of 238 nm; C in Figure 5 is the fingerprint spectrum of the test sample obtained by detection at a wavelength of 254 nm; D in Figure 5 is the fingerprint spectrum of the test sample obtained by detection at a wavelength of 257 nm.

[0096] Figure 6 Chromatograms obtained using methanol-water solutions of different volume fractions as extraction solvents.

[0097] Figure 7 HPLC comparative chromatograms were used to confirm the main chemical components of red yeast rice.

[0098] Figure 8 The images show a comparison of the ultraviolet (UV) absorption spectra of the reference standard and the test sample. The UV absorption spectrum of the reference standard is located on the left, and the UV absorption spectrum of the test sample is located on the right. From top to bottom, the images show the UV absorption spectra of open-ring lovastatin and lovastatin, respectively.

[0099] Figure 9 The chromatogram of the open-ring lovastatin reference standard obtained under the initial preparation conditions is shown.

[0100] Figure 10 Chromatograms of open-ring lovastatin reference standards obtained with different reference solvents: Figure 10 Figure A in the figure is a chromatogram obtained using acetonitrile as a reference solvent; Figure 10 Figure B in the figure is a chromatogram obtained using methanol as a reference solvent.

[0101] Figure 11 Chromatograms of open-ring lovastatin reference standards obtained with different pH adjusters: Figure 11 Figure A in the figure is a chromatogram obtained by using a 37% hydrochloric acid solution as a pH adjuster reference. Figure 11 Figure B in the figure is a chromatogram obtained by using a 3 mol / L phosphoric acid aqueous solution as a pH adjuster reference.

[0102] Figure 12 The fingerprint spectrum of different batches of red yeast rice is a common pattern diagram.

[0103] Figure 13 This is a reference fingerprint spectrum for red yeast rice.

[0104] Figure 14 The figures show HPLC comparison chromatograms of blank solvent, reference solution, and test sample solution, wherein the test sample is red yeast rice.

[0105] Figure 15 The chromatogram of a 6g sample of Shiguogong medicinal wine after ultrasonic treatment, obtained using the HPLC chromatographic conditions described in this invention.

[0106] Figure 16 The chromatogram of a 12g sample of Shiguogong medicinal wine after ultrasonic treatment, obtained using the HPLC chromatographic conditions described in this invention.

[0107] Figure 17 The chromatogram of a 6g sample of Shiguogong medicinal wine after ultrasonic treatment is shown in the HPLC chromatographic conditions described in the prior art.

[0108] Figure 18 The chromatogram of a 12g sample of Shiguogong medicinal wine after ultrasonic treatment is obtained using the HPLC chromatographic conditions described in the prior art.

[0109] Figure 19 S1 in the figure is the chromatogram of the red yeast rice sample obtained under the HPLC chromatographic conditions described in this invention; Figure 19 S2 in the figure is the chromatogram of the red yeast rice sample obtained using the HPLC chromatographic conditions described in the prior art. Detailed Implementation

[0110] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0111] The Agilent 1260 high-performance liquid chromatograph was purchased from Agilent Technologies Inc.; the Waterse2695 high-performance liquid chromatograph was purchased from Waters Technologies (Shanghai) Co., Ltd.; the TG16-WS benchtop high-speed centrifuge was purchased from Hunan Xiangli Scientific Instruments Co., Ltd.; the KQ300DE CNC ultrasonic cleaner was purchased from Kunshan Ultrasonic Instruments Co., Ltd.; and the ME204E / 02 electronic balance was purchased from Mettler Toledo Instruments (Shanghai) Co., Ltd.

[0112] The Agilent ZORBAX SB-Aq column (250mm*4.6mm, 5μm) was purchased from Agilent Technologies Inc.; the Ultimate LP-C18 column (250mm*4.6mm, 5μm) was purchased from Yuexu Technology (Shanghai) Co., Ltd.; the Inertsil ODS-3 column (250mm*4.6mm, 5μm) was purchased from Shimadzu Corporation; and the XSelect HSS T3 column (250mm*4.6mm, 5μm) was purchased from Waters Technologies Inc.

[0113] Lovastatin (batch number 100600-202006, purity 99.6%) was purchased from the National Institutes for Food and Drug Control. Water was distilled; acetonitrile and phosphoric acid were chromatographic grade; methanol, sodium hydroxide, and hydrochloric acid were analytical grade.

[0114] The samples used in the following examples:

[0115] The red yeast rice samples with batch number Y188-2010001-Hangzhou were produced in Hangzhou; the samples with batch number 20230504-Hubei were produced in Hubei; the samples with batch number 20230504-Zhejiang were produced in Zhejiang; and the samples with batch number 20230504-Shandong were produced in Shandong.

[0116] Example 1: Effect of different chromatographic conditions on the fingerprint of red yeast rice

[0117] 1. Preparation of the test solution

[0118] After crushing the red yeast rice sample and passing it through a 50-mesh sieve, accurately weigh 0.1 g and place it in a volumetric flask. Add a 50% methanol aqueous solution to 10 ml, sonicate for 15 minutes, shake well, centrifuge at 12000 rpm for 10 minutes, take the supernatant and filter it through a 0.45 μm filter membrane. Take the filtrate to obtain the test solution.

[0119] 2. Effect of different elution conditions on peak shape

[0120] (1) Experimental method:

[0121] Accurately pipette 10 μl of the test solution prepared in "1. Preparation of the test solution" and inject it into the high-performance liquid chromatograph (HPLC) for analysis. Obtain the chromatogram:

[0122] Using a 0.1% (v / v) aqueous solution of phosphoric acid as mobile phase A and acetonitrile as mobile phase B, elution was performed under the conditions shown in Tables 1, 2, or 3, respectively.

[0123] Other chromatographic conditions were as follows: octadecylsilane-bonded silica gel as the packing material; Agilent ZORBAX SB-Aq column (25 cm in length, 4.6 mm in inner diameter, and 5 μm in particle size); flow rate of 1.0 mL per minute; detection wavelength of 254 nm; and column temperature of 40 °C.

[0124] Table 1 Initial HPLC column elution conditions

[0125]

[0126] Table 2 Elution conditions for liquid chromatography column

[0127]

[0128] Table 3 Elution conditions for liquid chromatography column

[0129]

[0130] (2) Experimental Results

[0131] The results are as follows Figures 1-3 As shown:

[0132] The chromatograms obtained under the elution conditions shown in Table 1 are as follows: Figure 1 As shown, by Figure 1 It can be seen that under these elution conditions, there are basically no chromatographic peaks appearing in the 24-40 minute time period and after 58 minutes;

[0133] The chromatograms obtained under the elution conditions shown in Table 2 are as follows: Figure 2 As shown, the baseline is higher in the 20-28 minute segment, but the peak distribution is uniform and the separation is good in other time periods.

[0134] The chromatograms obtained under the elution conditions shown in Table 3 are as follows: Figure 3 As shown, the chromatographic peak signals are relatively uniform, most chromatographic peaks are well separated, and the main chromatographic peaks have been basically collected. Therefore, it is preliminarily determined that the elution conditions in Table 3 will be used for subsequent experiments.

[0135] 3. Effect of different column temperatures on elution results

[0136] (1) Experimental methods

[0137] Using the elution conditions in Table 3, the test solution was analyzed by high-performance liquid chromatography (HPLC) according to the experimental method in "2. Effect of different elution conditions on peak shape" of this embodiment, except that the column temperature of the HPLC column was changed:

[0138] The column temperature for experimental group 1 was 40℃; the column temperature for experimental group 2 was 35℃; and the column temperature for experimental group 3 was 30℃.

[0139] Compare the effects of different column temperatures on elution results.

[0140] (2) Experimental Results

[0141] Chromatographic values ​​obtained at different liquid chromatography column temperatures are as follows: Figure 4 As shown in the results, there was no significant difference in the elution of chromatographic peaks in the chromatograms at different column temperatures. Since the column temperature of 30℃ is closer to room temperature and the experimental conditions are easier to control, the column temperature of 30℃ was chosen for subsequent experiments.

[0142] 4. The effect of different detection wavelengths on chromatograms

[0143] (1) Experimental methods

[0144] The fine powder of the red yeast rice sample after pulverizing and passing through a 50-mesh sieve was scanned at wavelengths of 210–400 nm. According to relevant literature, the main components of red yeast rice are lovastatin and open-ring lovastatin. These two compounds have the highest absorbance at a wavelength of around 238 nm. Existing technologies also use a wavelength of around 256 nm as the detection wavelength. Therefore, wavelengths of 237 nm, 238 nm, 254 nm, and 257 nm were selected for analysis.

[0145] Using the elution conditions in Table 3, the test solution was analyzed by high-performance liquid chromatography according to the experimental method in "2. Effect of different elution conditions on peak shape" of this embodiment, only changing the detection wavelength:

[0146] Experimental group 1 was detected at a wavelength of 237 nm; Experimental group 2 was detected at a wavelength of 238 nm.

[0147] Experimental group 3 was detected at a wavelength of 254 nm; Experimental group 4 was detected at a wavelength of 257 nm.

[0148] Compare the effects of different detection wavelengths on chromatograms.

[0149] (2) Experimental Results

[0150] The chromatograms obtained from experimental groups 1-4 are as follows: Figure 5As shown in the figure, A in Figure 5 represents the chromatogram obtained from experimental group 1; B in Figure 5 represents the chromatogram obtained from experimental group 2; C in Figure 5 represents the chromatogram obtained from experimental group 3; and D in Figure 5 represents the chromatogram obtained from experimental group 4. The results show that before 36 minutes, experimental group 3 had more peaks, while after 36 minutes, experimental group 1 had more peaks. Furthermore, the peak heights of experimental group 4 were higher than those of experimental group 3, and the peak heights of experimental group 2 were higher than those of experimental group 1.

[0151] Therefore, variable wavelength detection was chosen. The specific detection wavelengths are shown in Table 4. Table 4 was used as the detection wavelength for subsequent experiments.

[0152] Table 4 Variable Wavelength Detection Conditions

[0153]

[0154]

[0155] Example 2: A method for detecting red yeast rice using high performance liquid chromatography

[0156] High-performance liquid chromatography (HPLC) was used to detect red yeast rice. The specific detection method is as follows:

[0157] An Agilent ZORBAX SB-Aq column (25 cm long, 4.6 mm inner diameter, 5 μm particle size) was used as the stationary phase with octadecylsilane-bonded silica gel as the stationary phase. Elution was performed using 0.1% (v / v) phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, according to the conditions in Table 3. The flow rate was 1.0 mL / min, the column temperature was 30 °C, and wavelength-varying detection was performed according to the conditions in Table 4.

[0158] Example 3: Effect of different concentrations of extraction solvent on the fingerprint spectrum of red yeast rice

[0159] I. Experimental Methods

[0160] Extract the test sample using methanol-water solutions of different volume fractions: The red yeast rice sample was pulverized and passed through a 50-mesh sieve to obtain the test sample. Four portions of the test sample, each 0.1 g, were accurately weighed and placed in separate volumetric flasks. Methanol-water solutions of different volume fractions were added to each volumetric flask to a final volume of 10 ml.

[0161] Experimental group 1 was treated with a 25% (v / v) methanol aqueous solution;

[0162] Experimental group 2 was treated with a 50% (v / v) methanol aqueous solution.

[0163] Experimental group 3 was treated with a 75% (v / v) methanol aqueous solution.

[0164] The sample was ultrasonically treated for 15 minutes at a power of 250W and a frequency of 40kHz. After shaking, it was centrifuged at 12000 rpm for 10 minutes. The supernatant was filtered through a 0.45μm filter membrane. The filtrate was then used to obtain the test solution.

[0165] Accurately pipette 10 μl of each of the prepared test solutions from different experimental groups and inject them into the liquid chromatograph. Perform high-performance liquid chromatography analysis using the method in Example 2.

[0166] II. Experimental Results

[0167] Chromatographic results obtained using methanol-water solutions of different volume fractions as extraction solvents are as follows: Figure 6 As shown in the results, the chromatograms obtained from experimental groups 1, 2, and 3 exhibited significant differences, with experimental group 2 showing better peak shape and higher peak height. Therefore, a 50% (v / v) methanol-water solution was selected for the extraction of the sample for subsequent experiments.

[0168] Example 4: Preparation method of test solution

[0169] After pulverizing the test sample and passing it through a 50-mesh sieve, accurately weigh 0.1 g and place it in a volumetric flask. Add 50% methanol aqueous solution to 10 ml, sonicate at 250 W and 40 kHz for 15 minutes, shake well, centrifuge at 12000 rpm for 10 minutes, take the supernatant and filter it through a 0.45 μm filter membrane. Take the filtrate to obtain the test solution.

[0170] Example 5: Chromatographic Peak Assignment

[0171] I. Experimental Methods

[0172] Based on literature review, peak localization studies were conducted on the main chemical components of red yeast rice: open-ring lovastatin and lovastatin reference standards. The specific experimental steps are as follows:

[0173] S1. The test solution was prepared according to the preparation method of Example 4, and the test sample was red yeast rice;

[0174] Preparation of the reference solution:

[0175] Preparation of lovastatin reference solution: Accurately weigh an appropriate amount of lovastatin and add acetonitrile to prepare a lovastatin reference solution with a concentration of 0.5 mg / ml;

[0176] Preparation of the open-ring lovastatin reference solution: Accurately measure 1 ml of lovastatin reference solution into a 20 ml volumetric flask, add 10 ml of 0.2 mol / L sodium hydroxide solution, sonicate at 250 W and 40 kHz for 30 minutes, remove, adjust the pH to 7 with 3 mol / L phosphoric acid, dilute to the mark with acetonitrile, shake well, filter through a 0.45 μm filter membrane, and collect the filtrate to obtain the open-ring lovastatin reference solution;

[0177] S2. Accurately pipette 10 μl each of the test solution, the open-ring lovastatin reference solution and the lovastatin reference solution prepared in step S1, and inject them into the liquid chromatograph respectively, and perform detection according to the method of Example 2;

[0178] Under conditions of 210–400 nm, the test solution and the reference solution prepared in step S1 were scanned across the entire wavelength to obtain a comparison of ultraviolet absorption spectra.

[0179] S3. Identify the main fingerprint peaks by comparing the retention times and UV absorption spectra of the peaks eluted from the reference solution and the test solution.

[0180] II. Experimental Results

[0181] The experimental results are shown in Figure 7 and Figure 8 ,in, Figure 7 A total of 7 fingerprint peaks were identified; Figure 8 The left column shows the UV absorption spectra of the reference solutions. Figure 8 The column on the right shows a comparison of the ultraviolet absorption spectra of the test solutions. Figure 8 The images shown from top to bottom are a comparison of the UV absorption spectra of open-ring lovastatin and lovastatin.

[0182] according to Figure 7 and Figure 8 The fingerprint peak identification results are summarized in Table 5.

[0183] Table 5. Fingerprint Peak Identification Results

[0184]

[0185] Depend on Figure 7 It is known that the chromatographic peak of open-ring lovastatin has a moderate retention time and a high response value. Therefore, open-ring lovastatin was selected as the reference peak and labeled as peak S. At the same time, the reference standard open-ring lovastatin was used as the reference substance.

[0186] Example 6: Effect of the preparation method of the reference solution on the peak shape of the reference solution

[0187] 1. The effect of reference preparation method on peak shape

[0188] (1) Experimental methods

[0189] S1. Preparation of the reference solution, the specific steps are as follows:

[0190] Take an appropriate amount of lovastatin and add 2 mol / L sodium hydroxide solution to prepare a lovastatin-sodium hydroxide solution with a concentration of 0.01 mg / ml. Place the lovastatin-sodium hydroxide solution under 50℃ with a power of 250W and a frequency of 40kHz for 1 hour for ultrasonic conversion, and then let it stand at room temperature for 1 hour. Then add hydrochloric acid to adjust the pH value to 7, shake well, and filter through a 0.45μm filter membrane. Take the filtrate to obtain the reference solution.

[0191] S2. Accurately pipette 10 μl of the reference solution prepared in step S1 and inject it into the liquid chromatograph. Perform high performance liquid chromatography analysis using the method of Example 2.

[0192] (2) Experimental Results

[0193] The obtained chromatogram is as follows Figure 9 As shown in the figure, the results indicate that the obtained open-ring lovastatin peak has severe tailing, with a tailing factor of 1.7, and the peak shape is poor, affecting the accuracy and reliability of chromatographic analysis.

[0194] 2. Effect of different solvents on the peak shape of open-ring lovastatin

[0195] (1) Experimental method:

[0196] The specific steps for changing the lovastatin solvent in the preparation of the reference solution are as follows:

[0197] S1. Experimental group 1 used acetonitrile as a solvent: Take an appropriate amount of lovastatin and add acetonitrile to prepare a lovastatin-acetonitrile solution with a concentration of 1 mg / ml. Take 1 ml of the lovastatin-acetonitrile solution into a 20 ml volumetric flask, add 10 ml of 0.2 mol / L sodium hydroxide solution to the 20 ml volumetric flask, and then sonicate at 250 W and 40 kHz for 30 minutes. Then adjust the pH value to 7 with 37% hydrochloric acid solution and add acetonitrile to make up to 20 ml to obtain reference solution 1.

[0198] Experimental group 2 used methanol as a solvent: 10 mg of lovastatin was placed in a 50 ml volumetric flask, and an appropriate amount of pure methanol was added until the lovastatin was completely dissolved. Then, 20 ml of 0.2 mol / L sodium hydroxide solution was added to the 50 ml volumetric flask, and the mixture was ultrasonically converted at a power of 250 W and a frequency of 40 kHz for 30 minutes. Subsequently, the pH value was adjusted to 7 using 3 mol / L phosphoric acid aqueous solution, and pure methanol was added to make up to 20 ml to obtain reference solution 2.

[0199] S2. Accurately pipette 10 μl each of reference solution 1 and reference solution 2 obtained in step S1, inject them into the liquid chromatograph, and perform high performance liquid chromatography analysis using the method of Example 2.

[0200] (2) Experimental Results

[0201] Experimental results are as follows Figure 10 As shown, Figure 10 Figure A in the figure is the chromatogram obtained from experimental group 1; Figure 10 Figure B in the figure shows the chromatogram obtained from experimental group 2. The open-ring lovastatin peaks obtained from both experimental groups 1 and 2 have good peak shapes and tailing factors of 1.3. However, the chromatogram obtained from experimental group 2 has a small peak at a retention time of 51.6 min. Figure 13 (As shown in the elliptical portion of Figure B in the diagram), therefore acetonitrile was chosen as the solvent for subsequent experiments.

[0202] 3. Effects of different pH adjusters on the peak shape of open-ring lovastatin

[0203] (1) Experimental method:

[0204] The pH adjuster in the preparation of the reference solution was changed, and the specific steps are as follows:

[0205] S1. Take an appropriate amount of lovastatin and add acetonitrile to prepare a lovastatin-acetonitrile solution with a concentration of 0.5 mg / ml. Take 1 ml of the lovastatin-acetonitrile solution into a 20 ml volumetric flask, add 10 ml of 0.2 mol / L sodium hydroxide solution to the 20 ml volumetric flask, and then sonicate at 250 W and 40 kHz for 30 minutes. Then use:

[0206] Experimental group 1: 37% hydrochloric acid solution; Experimental group 2: 3 mol / L phosphoric acid aqueous solution;

[0207] Adjust the pH value to 7 respectively, add acetonitrile to make up to 20 ml, and prepare reference solution 3 and reference solution 4;

[0208] S2. Accurately pipette 10 μl each of reference solution 3 and reference solution 4 obtained in step S1, inject them into the liquid chromatograph, and perform high performance liquid chromatography analysis using the method of Example 2.

[0209] (2) Experimental Results

[0210] Experimental results are as follows Figure 11 As shown, Figure 11 Figure A in the figure is the chromatogram obtained from experimental group 1; Figure 11 Figure B in the figure shows the chromatogram obtained from experimental group 2. The open-ring lovastatin peaks obtained from both experimental groups 1 and 2 showed good peak shapes, with a tailing factor of 1.3; however, experimental group 1 had a small peak at retention time of 52 min (…). Figure 11 (As shown in the elliptical part), and 3 mol / L phosphoric acid aqueous solution is more likely to ensure the safety of the experiment than hydrochloric acid. Therefore, 3 mol / L phosphoric acid aqueous solution was chosen as the pH adjuster in the preparation of the reference solution.

[0211] Example 7: Preparation method of reference solution

[0212] The preparation method of the open-ring lovastatin reference solution suitable for HPLC detection is as follows:

[0213] Take an appropriate amount of lovastatin and add acetonitrile to prepare a lovastatin-acetonitrile solution with a concentration of 0.5 mg / ml. Take 1 ml of the lovastatin-acetonitrile solution into a 20 ml volumetric flask, add 10 ml of 0.2 mol / L sodium hydroxide solution to the 20 ml volumetric flask, and then sonicate at 250 W and 40 kHz for 30 minutes. Then adjust the pH value to 7 with 3 mol / L phosphoric acid aqueous solution, add acetonitrile to make up to 20 ml, and obtain the reference solution.

[0214] Example 8: Method for Establishing Red Yeast Rice Fingerprint Spectrum

[0215] (1) Preparation of the test solution

[0216] Accurately weigh 0.1g of the fine powder to be tested after passing it through a 50-mesh sieve. Add 50% methanol aqueous solution to 10ml, sonicate at 250W power and 40kHz frequency for 15 minutes, shake well, centrifuge at 12000 rpm for 10 minutes, take the supernatant and filter it through a 0.45μm filter membrane. Take the filtrate to obtain the test solution.

[0217] (2) Preparation of reference solution

[0218] Take an appropriate amount of lovastatin and add acetonitrile to prepare a lovastatin-acetonitrile solution with a concentration of 0.5 mg / ml. Take 1 ml of the lovastatin-acetonitrile solution into a 20 ml volumetric flask, add 10 ml of 0.2 mol / L sodium hydroxide solution to the 20 ml volumetric flask, and then sonicate at 250 W and 40 kHz for 30 minutes. Then adjust the pH value to 7 with 3 mol / L phosphoric acid aqueous solution, add acetonitrile to make up to 20 ml, and obtain the reference solution.

[0219] (3) Determination method

[0220] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatograms.

[0221] (4) Chromatographic conditions

[0222] An Agilent ZORBAX SB-Aq column (25 cm length, 4.6 mm inner diameter, 5 μm particle size) was used as the chromatographic column; 0.1% (v / v) aqueous phosphoric acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B, eluted according to the conditions in Table 3; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and wavelength-variable detection was performed according to the conditions in Table 4. The theoretical plate number, calculated based on the decanlovastatin peak, should be no less than 8000.

[0223] Example 9: Establishment of Red Yeast Rice Comparative Fingerprint

[0224] (1) Calibration of common peaks

[0225] Using the method of Example 8, four batches of red yeast rice standard samples with batch numbers Y188-2010001-Hangzhou, 20230504-Hubei, 20230504-Zhejiang, and 20230504-Shandong were measured, and the obtained fingerprint spectra were analyzed. The chromatographic peaks with good stability and suitable response values ​​in the fingerprint spectra of the four batches of red yeast rice standard samples were selected as common peaks. A total of 7 common peaks were identified. The 7 common peaks are the fingerprint peaks in Example 5. The source of the medicinal ingredients and the peak numbers are shown in Table 5.

[0226] The fingerprint spectra of 4 batches of samples are shown below. Figure 12 , Figure 12 In the table, S1 is the fingerprint chromatogram of sample Y188-2010001-Hangzhou; S2 is the fingerprint chromatogram of sample 20230504-Hubei; S3 is the fingerprint chromatogram of sample 20230504-Shandong; S4 is the fingerprint chromatogram of sample 20230504-Zhejiang; R is the reference fingerprint chromatogram automatically matched by the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" based on S1 to S4.

[0227] The results showed that the fingerprint spectra of the four batches of samples contained seven common peaks, which were the fingerprint peaks in Example 5. The peak corresponding to the open-ring lovastatin reference was designated as the S peak. The relative retention times of each common peak and the S peak were calculated. The average relative retention times of each common peak were as follows: Peak 1 0.219; Peak 2 0.528; Peak 3 0.853; S peak 1.000; Peak 5 1.049; Peak 6 1.066; Peak 7 1.108.

[0228] (2) Establishment of comparative fingerprint patterns

[0229] The analysis was performed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". Using S1 from "(1) Calibration of Common Peaks" in this embodiment as the reference chromatogram, with a time window width of 0.1 min, the median method and multi-point correction were used for automatic chromatogram matching to generate a control fingerprint chromatogram. The control fingerprint chromatogram is shown below. Figure 13, Figure 13 The seven common peaks are the fingerprint peaks in Example 5, therefore according to Figure 13 As shown in Table 5, Figure 13 In the chromatogram, peak 4 (S peak) is the chromatogram peak of open-ring lovastatin; peak 7 is the chromatogram peak of lovastatin.

[0230] (3) Similarity calculation

[0231] The similarity between the four batches of red yeast rice samples and the control fingerprint spectrum obtained from “(2) Establishment of control fingerprint spectrum” was calculated using the “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 version)” with seven common peaks. The results are shown in Table 6.

[0232] Table 6. Similarity Results of Fingerprint Spectra of Four Batches of Red Yeast Rice Samples

[0233]

[0234] According to the results in Table 6, the similarity between the fingerprint spectra of the four batches of red yeast rice samples and the control fingerprint spectra was greater than 0.90, indicating that the similarity between samples from different batches was good.

[0235] Therefore, the fingerprint chromatogram of the test sample should show a chromatographic peak with the same retention time as the reference peak, and should show 7 common peaks (i.e. fingerprint peaks). According to the method of "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)", the similarity is calculated based on the common peaks. If the similarity is not less than 0.90, the test sample can be judged as a qualified sample.

[0236] Example 10: Method and Methodological Investigation for Establishing Red Yeast Rice Fingerprint Spectra

[0237] 1. Exclusivity and wholeness

[0238] (1) Experimental methods

[0239] The fingerprint chromatogram determination method described in Example 8 was used, except that acetonitrile was used as mobile phase A and 0.1% (v / v) phosphoric acid aqueous solution was used as mobile phase B, and elution was performed according to the liquid chromatography elution conditions shown in Table 7.

[0240] A 50% (v / v) methanol aqueous solution was selected as the blank solvent to establish chromatograms of the test sample, reference solution, and blank solvent, wherein the test sample was red yeast rice.

[0241] Table 7 Elution conditions for liquid chromatography column

[0242]

[0243] (2) Experimental Results

[0244] Experimental results are as follows Figure 14 As shown, by Figure 14 It can be seen that the blank solvent does not interfere with the fingerprint peaks of the test solution, indicating that the specificity and integrity of the fingerprint spectroscopy determination method described in Example 8 meet the requirements.

[0245] 2. Sample injection precision

[0246] (1) Experimental methods

[0247] The test sample, red yeast rice, was tested using the method described in Example 8. The test sample solution was injected five times consecutively, and the sample numbers were recorded as injection precision-1, injection precision-2, injection precision-3, injection precision-4, and injection precision-5, respectively. Chromatograms were recorded for each sample. Using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)," the peak corresponding to the reference peak was designated as the S peak. The relative retention time (RRT), relative peak area (RRA), and relative standard deviation (RSD) of RRT and RRA for the seven fingerprint peaks were calculated.

[0248] (2) Experimental Results

[0249] The results of the injection precision test are shown in Table 8, and the similarity results of the fingerprint chromatograms obtained from the injection precision test are shown in Table 9. The results show that the similarity of each chromatogram is 1.00 (>0.95); the experimental data of the 5 groups of precision experiments show that the maximum value of the relative retention time RSD of each fingerprint peak is 0.2% (≤5%), and the maximum value of the relative peak area RSD is 2.4% (≤10%), indicating that the injection precision of the fingerprint chromatogram determination method described in Example 8 meets the requirements.

[0250] Table 8 Results of the injection precision test

[0251]

[0252]

[0253] Table 9. Sample injection precision similarity results

[0254]

[0255] 3. Repeatability

[0256] (1) Experimental methods

[0257] Six parallel test solutions were prepared, all of which were red yeast rice. The fingerprint determination method described in Example 8 was used. The six test solutions were numbered as Repeatability 1, Repeatability 2, Repeatability 3, Repeatability 4, Repeatability 5, and Repeatability 6, respectively, and their chromatograms were recorded. Using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)," the peak corresponding to the reference peak was designated as the S peak. The relative retention time (RRT), relative peak area (RRA), and relative standard deviation (RSD) of RRT and RRA for the seven fingerprint peaks were calculated.

[0258] (2) Experimental Results

[0259] The repeatability test results are shown in Table 10, and the repeatability similarity results are shown in Table 11. The results show that the repeatability similarity of the six parallel test solutions is 1.00 (>0.95); the experimental data of the six sets of repeatability tests show that the maximum value of the relative retention time RSD of each fingerprint peak is 0.3% (≤5%), and the maximum value of the relative peak area RSD is 3.3% (≤10%), indicating that the repeatability of the fingerprint spectrum determination method described in Example 8 meets the requirements.

[0260] Table 10 Results of Repeatability Tests

[0261]

[0262]

[0263] Table 11 Repeatability Similarity Results

[0264]

[0265] 4. Solution stability

[0266] (1) Experimental methods

[0267] The fingerprint determination method described in Example 8 was used, except that the same test sample (red yeast rice) was used. The sample was placed at room temperature for 0h, 4h, 8h, 12h, 26h, and 36h before injection, and the chromatograms were recorded. Using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)," the peak corresponding to the reference peak was designated as the S peak. The relative retention time (RRT), relative peak area (RRA), and relative standard deviation (RSD) of RRT and RRA for the seven fingerprint peaks were calculated.

[0268] (2) Experimental Results

[0269] The results of the solution stability test are shown in Table 12, and the results of the solution stability similarity test are shown in Table 13. The results show that, using the fingerprint spectrum at 0 h as the reference spectrum, the similarity of the fingerprint spectra obtained after the test solution was placed at room temperature for 4 h, 8 h, 12 h, 26 h, or 36 h was 1.00 (>0.95); the maximum RSD of the relative retention time of each fingerprint peak was 0.5% (≤5%), and the maximum RSD of the relative peak area was 3.6% (≤10%). This indicates that the test solution is stable within 36 h.

[0270] Table 12 Results of the stability test of the test sample solution

[0271]

[0272]

[0273] Table 13 Results of Stability Similarity of Test Sample Solutions

[0274]

[0275] Example 11: Robustness of the Red Yeast Rice Fingerprint Establishment Method

[0276] 1. Effect of different column temperatures on the robustness of the method for establishing red yeast rice fingerprint spectra

[0277] (1) Experimental methods

[0278] Red yeast rice was used as the test sample to investigate the effect of different column temperatures on the robustness of the red yeast rice fingerprinting method. The fingerprinting method described in Example 8 was used, except that experimental groups with different column temperatures were set up:

[0279] The column temperature for experimental group 1 was 28℃; the column temperature for experimental group 2 was 30℃.

[0280] The column temperature in experimental group 3 was 32℃; the effects of different column temperatures on the elution results were compared.

[0281] The peak corresponding to the reference peak is designated as the S peak, and the relative retention time of each fingerprint peak is calculated. Using the method of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)," the peak corresponding to the reference peak is designated as the S peak, and the relative retention time (RRT), relative peak area (RRA), and relative standard deviation (RSD) of RRT and RRA of the 7 fingerprint peaks are calculated.

[0282] (2) Experimental Results

[0283] The results of column temperature durability relative retention time are shown in Table 14, and the results of column temperature durability similarity are shown in Table 15.

[0284] The results showed that under different column temperatures, the fingerprint peaks in the measured spectra were sharp, symmetrical, and well-separated, with a similarity of 1.000 (>0.90). The experimental data from the three groups showed that the maximum relative retention time (RSD) of each fingerprint peak was 1.6% (≤5%), and the maximum relative peak area (RSD) was 4.5% (≤10%). This indicates that the fingerprint spectroscopy determination method described in Example 8 has good robustness under different column temperatures (28℃~32℃).

[0285] Table 14 Results of Column Temperature Durability Test

[0286]

[0287] Table 15 Column Temperature Durability Similarity Results

[0288]

[0289] 2. The effect of different flow rates on the robustness of the red yeast rice fingerprinting method

[0290] (1) Experimental methods

[0291] Red yeast rice was used as the test sample to investigate the effect of different flow rates on the robustness of the method for establishing red yeast rice fingerprint patterns.

[0292] The fingerprint spectrum determination method described in Example 8 was used, except that experimental groups with different flow rates were set up:

[0293] The flow rate for experimental group 1 was 0.9 ml / min; the flow rate for experimental group 2 was 1.0 ml / min.

[0294] Experimental group 3 had a flow rate of 1.2 ml / min; the effects of different flow rates on the elution results were compared.

[0295] The peak corresponding to the reference peak is designated as the S peak, and the relative retention time of each fingerprint peak is calculated. Using the method of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)," the peak corresponding to the reference peak is designated as the S peak, and the relative retention time (RRT), relative peak area (RRA), and relative standard deviation (RSD) of RRT and RRA of the 7 fingerprint peaks are calculated.

[0296] (2) Experimental Results

[0297] The results of flow rate durability relative retention time are shown in Table 16, and the results of flow rate durability similarity are shown in Table 17.

[0298] The results showed that under different flow rates, the fingerprint peaks in the measured spectra were sharp, symmetrical, and well-separated, with a similarity of 1.000 (>0.90). Experimental data from the three groups showed that the maximum relative retention time RSD for each fingerprint peak was 4.5% (≤5%), and the maximum relative peak area RSD was 7.4% (≤10%). This indicates that the method in Example 8 exhibits stable relative retention time and relative peak area under different flow rates (0.9 ml / min to 1.2 ml / min), demonstrating good robustness.

[0299] Table 16 Results of Flow Velocity Durability Test

[0300]

[0301] Table 17 Flow velocity durability similarity results

[0302]

[0303] 3. The effect of different volume percentages of phosphoric acid aqueous solution as mobile phase A on the robustness of the method for establishing red yeast rice fingerprint spectra.

[0304] (1) Experimental methods

[0305] Red yeast rice was used as the test sample, and the effect of different volume percentages of phosphoric acid aqueous solution as mobile phase A on the robustness of the method for establishing red yeast rice fingerprint spectrum was detected.

[0306] The fingerprint spectroscopy determination method described in Example 8 was used, except that experimental groups with different volume percentages of phosphoric acid aqueous solution were set up as mobile phase A:

[0307] Experimental group 1 was a 0.08% (v / v) aqueous solution of phosphoric acid;

[0308] Experimental group 2 consisted of a 0.10% (v / v) aqueous solution of phosphoric acid.

[0309] Experimental group 3 was a 0.12% (v / v) aqueous solution of phosphoric acid;

[0310] The effects of different volume percentages of phosphoric acid aqueous solution as mobile phase A on the elution results were compared.

[0311] The peak corresponding to the reference peak is designated as the S peak, and the relative retention time of each fingerprint peak is calculated. Using the method of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)," the peak corresponding to the reference peak is designated as the S peak, and the relative retention time (RRT), relative peak area (RRA), and relative standard deviation (RSD) of RRT and RRA of the 7 fingerprint peaks are calculated.

[0312] (2) Experimental Results

[0313] The results of relative retention time for the durability of phosphoric acid aqueous solutions with different volume percentages are shown in Table 18, and the results of similarity in durability for phosphoric acid aqueous solutions with different volume percentages are shown in Table 19.

[0314] The results showed that under different volume percentages of phosphoric acid aqueous solution as mobile phase A, the fingerprint peaks in the measured spectra were sharp, symmetrical, and well-separated, with a similarity of 1.000 (>0.90). Experimental data from the three groups showed that the maximum relative retention time RSD for each fingerprint peak was 0.7% (≤5%), and the maximum relative peak area RSD was 5.7% (≤10%). This indicates that the fingerprint determination method described in Example 8 has good robustness under different volume percentages of phosphoric acid aqueous solution (0.08%–0.12%) and meets the requirements.

[0315] Table 18 Results of durability tests on phosphoric acid aqueous solutions with different volume percentages

[0316]

[0317] Table 19. Durability Similarity Results of Phosphoric Aqueous Solutions with Different Volume Percentages

[0318]

[0319] 4. The effect of different chromatographic columns on the robustness of the method for establishing red yeast rice fingerprints

[0320] (1) Experimental methods

[0321] Red yeast rice was used as the test sample to investigate the effect of different chromatographic columns on the robustness of the method for establishing red yeast rice fingerprint chromatograms.

[0322] The fingerprint chromatogram determination method described in Example 8 was used, except that different types of chromatographic columns were used in the experimental groups:

[0323] The chromatographic column for experimental group 1 was an Agilent ZORBAX SB-Aq.

[0324] The chromatographic column for experimental group 2 was Yuexu Ultimate LP-C18;

[0325] The chromatographic column used in experimental group 3 was Shimadzu Inertsil ODS-3;

[0326] The chromatographic column used in experimental group 4 was Waters XSelect HSS T3;

[0327] Compare the effects of different column types on chromatograms.

[0328] The peak corresponding to the reference peak is designated as the S peak, and the relative retention time of each fingerprint peak is calculated. Using the method of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)," the peak corresponding to the reference peak is designated as the S peak, and the number of fingerprint peaks, the relative retention time (RRT) of the fingerprint peaks, and the relative standard deviation (RSD) of the RRT are calculated.

[0329] (2) Experimental Results

[0330] The results of the durability tests on different column types are shown in Table 20. The results show that in the fingerprint chromatograms obtained from the four experimental groups, fingerprint peaks 5 and 7 did not appear in experimental groups 2, 3, and 4, meaning the number of fingerprint peaks was less than in experimental group 1. This indicates that the method in Example 8 has poor durability with different column types, and it is necessary to consistently use an Agilent ZORBAX SB-Aq column.

[0331] Table 20 Results of durability tests on different chromatographic column models

[0332]

[0333] Example 12: Application of the Red Yeast Rice Fingerprint Spectrum Establishment Method in the Detection of Shi Guogong Medicinal Wine

[0334] I. Experimental Methods

[0335] Sample 1 and Sample 2 are Shi Guogong medicinal wines, and the fingerprint spectrum determination method described in Example 8 was used. The difference is that the preparation methods of the test solution of Sample 1 and Sample 2 are different.

[0336] The preparation method of the test solution of Sample 1 is as follows: accurately weigh 6g of Shiguogong medicinal wine, evaporate it to dryness in a water bath to obtain residue, add 50% methanol aqueous solution to 10ml of the residue, dissolve it completely, and then sonicate it at 250W power and 40kHz frequency for 15 minutes. After shaking, centrifuge at 12000 rpm for 10 minutes, take the supernatant and filter it through a 0.45μm filter membrane. Take the filtrate to obtain the test solution of Sample 1.

[0337] The preparation method of the test solution of Sample 2 is the same as that of the test solution of Sample 1, except that 12g of Shi Guogong medicinal wine is accurately weighed.

[0338] II. Experimental Results

[0339] Figure 15 The chromatogram of sample 1 obtained using the method of Example 8; Figure 16 The chromatogram of sample 2 was obtained using the method of Example 8. Figure 15Peaks 2, 3, 4, 6, and 7 in Example 9 can be identified. The peak corresponding to the open-ring lovastatin reference is designated as peak S. The relative retention times of each fingerprint peak and peak S are calculated. The relative retention times of each fingerprint peak are as follows: peak 2 0.535; peak 3 0.854; peak S 1.000; peak 6 1.067; peak 7 1.112. Figure 16 Peaks 2, 3, 4, 6, and 7 in Example 9 can be identified. The peak corresponding to the open-ring lovastatin reference is designated as peak S. The relative retention times of each fingerprint peak and peak S are calculated. The relative retention times of each fingerprint peak are as follows: peak 2 0.535; peak 3 0.852; peak S 1.000; peak 6 1.068; peak 7 1.111.

[0340] The results showed that the fingerprint spectroscopy determination method described in Example 8 could identify peaks 2, 3, 4, 6, and 7 of Example 9 in the Shi Guogong medicinal wine samples obtained by different preparation methods. Furthermore, the relative retention times of the five fingerprint peaks were within ±10% of the common peaks identified in Example 9. This indicates that the method in Example 8 can identify the chemical components of red yeast rice in Shi Guogong medicinal wine, and can identify the main chemical components of red yeast rice, namely, open-ring lovastatin and lovastatin. Figure 15 and Figure 16 Peak 4 in the middle has good separation, good symmetry, and no interfering peaks, which can meet the requirements for calculating the content of open-ring lovastatin by using the peak area of ​​peak 4. It is suitable for the quality testing of Shi Guogong medicinal wine and / or the quality testing of red yeast rice components in Shi Guogong medicinal wine.

[0341] Comparative Example 1: Application of Red Yeast Rice Fingerprint Spectrum Establishment Method in the Detection of Shi Guogong Medicinal Wine

[0342] I. Experimental Methods

[0343] Samples 1 and 2 were Shi Guogong medicinal wine, and sample 3 was red yeast rice. The red yeast rice was detected using the HPLC detection method described in the existing technology (Gao Wenming, Lin Yuesong, Dai Guoliang, Zhao Lingang. Simultaneous determination of the contents of three components, including adenosine, in red yeast rice from different sources by HPLC [J]. Pharmaceutical and Clinical Research, 2022, 30(2):140-142). The preparation methods of the test solutions for samples 1 and 2 were the same as those described in Example 12, and the preparation method of the test solution for sample 3 was the same as the method described in "(1) Preparation of the test solution" in Example 8.

[0344] II. Experimental Results

[0345] Figure 17 The chromatogram of sample 1 obtained by the red yeast rice HPLC detection method disclosed in the prior art; Figure 18 The chromatogram of sample 2 was obtained using the red yeast rice HPLC detection method disclosed in the prior art; Figure 19 S1 in the figure is the chromatogram of red yeast rice obtained using the method in Example 8. Figure 19 S2 in the figure represents the chromatogram of sample 3 obtained using existing technology. However, the total elution time differs between the disclosed HPLC detection method for red yeast rice and the method in Example 8. Figure 19 The peak times of S1 and S2 in the sample are different.

[0346] Depend on Figure 19 It is evident that the peak areas of the open-ring lovastatin peak (peak 4) and lovastatin peak (peak 7) in the red yeast rice fingerprint chromatogram obtained by the method of Example 8 of this invention are significantly higher than those of the red yeast rice HPLC detection method disclosed in the prior art. Furthermore, the chromatogram obtained using the red yeast rice HPLC detection method disclosed in the prior art has an uneven baseline, with a resolution of only 1.1 for peak 7, which is lower than the requirement of at least 1.5 for the resolution between the analyte peak and adjacent peaks in high-performance liquid chromatography. In contrast, the fingerprint chromatogram obtained using the method of Example 8 achieves a resolution of 6.4 for peak 7, exhibiting both higher resolution and a more stable baseline. Therefore, the method of Example 8 not only more sensitively detects the main chemical components in red yeast rice but also yields a more stable baseline and higher resolution, thereby improving the accuracy and reliability of the analysis, and is superior to the prior art.

[0347] The results showed that the chromatograms obtained from the Shi Guogong medicinal wine samples prepared by different methods using the publicly available HPLC detection method for red yeast rice could only identify three fingerprint peaks in the red yeast rice material, which could not accurately and comprehensively reflect the chemical composition relationship between the finished product and the medicinal material; moreover, Figure 17 and Figure 18 The resolution of peak 7 was less than 1.5, indicating peak overlap or indistinguishability. Since peak 7 is lovastatin, one of the main chemical components of red yeast rice, it can be seen that the existing technology has limited ability to identify complex components in Shi Guogong medicinal wine and cannot accurately quantify the main chemical components in red yeast rice. Therefore, using the existing technology to detect the quality of red yeast rice components in Shi Guogong medicinal wine will lead to deviations in efficacy evaluation. The existing technology is not suitable for the quality detection of Shi Guogong medicinal wine and / or the quality detection of red yeast rice components in Shi Guogong medicinal wine.

[0348] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for quality testing of Shi Guogong medicinal wine, characterized in that, Includes the following steps: Reference solution: Lovastatin and acetonitrile were mixed to prepare a lovastatin-acetonitrile solution with a concentration of 0.3-0.7 mg / ml. Sodium hydroxide solution with a concentration of 0.15-0.25 mol / L was added to the lovastatin-acetonitrile solution to obtain a mixed solution. The volume ratio of the lovastatin-acetonitrile solution to the sodium hydroxide solution was 1:10-20. After ultrasonic conversion of the mixture for 30-60 minutes, add phosphoric acid aqueous solution to adjust the pH to 6.5-7.5 to obtain a solution. Dilute the solution with acetonitrile 1-3 times to obtain a reference solution. Test solution: methanol extract of Shi Guogong medicinal wine; HPLC chromatographic conditions: An Agilent ZORBAX SB-Aq column was used. Mobile phase A consisted of 0.08–0.12% (v / v) phosphoric acid aqueous solution, and mobile phase B consisted of acetonitrile. During the gradient elution program, the volume percentage of mobile phase B in the mobile phase system was as follows: From 0 to 26 minutes, mobile phase B increased from 1% to 15%; Over 26–40 minutes, mobile phase B increased from 15% to 50%. Within 40–58 minutes, the mobile phase B increased from 50% to 60%. 58–60 min, mobile phase B decreased from 60% to 1%; The detection wavelength is a variable wavelength detection: The detection wavelength was 257 nm, and the time interval was 0–36 min. 36–60 min, detection wavelength 238 nm; The fingerprint chromatograms of Shi Guogong medicinal wine were obtained by performing HPLC detection on the reference solution and the test solution respectively according to the HPLC chromatographic conditions. The fingerprint spectrum of Shi Guogong medicinal wine contains 5 fingerprint peaks. The corresponding peak of the reference solution is designated as the S peak. The specified values ​​for the relative retention times of the 5 fingerprint peaks are 0.528, 0.853, and for the S peak, 1.000, 1.066, and 1.108, respectively. If the relative retention times of the 5 fingerprint peaks are within ±10% of the specified values, then the quality of the red yeast rice component in the Shi Guogong medicinal wine to be tested is qualified.

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