Detection method of monacolin compound

Abstract

The invention discloses a detection method of a monacolin compound. According to the application, a non-targeted screening thought is adopted, four key daughter ions of the monacolin compound are found for the first time, and non-targeted screening methods of the monacolin compound based on key daughter ion detection are respectively established in a high-resolution mass spectrum and a triple quadrupole mass spectrum by utilizing the property that the compound is easy to crack in a source in a mass spectrum; the distribution and the content of the monacolin compound can be exclusively presented. Compared with a basic analysis method of original substances, the method has the advantages that secondary mass spectrum analysis is not needed, the retention time of the compounds can be quickly locked, and the monacolin compounds are subjected to non-targeted screening; compared with the original analysis method of the monacolin compound, the method can filter out a large part of interference of other compounds, and specifically shows the distribution and content of the monacolin compound. The method can be applied to fermentation products of monascus, aspergillus terreus and other monacolin compound producing strains and products of the fermentation products to detect distribution and relative content of the monacolin compounds, and can also be applied to quality control methods such as establishment of monacolin compound fingerprints, content determination and the like in the matrix.

Description

Technical Field

[0001] This invention relates to the field of analytical detection, and in particular to a method for detecting monacolin-like compounds. Background Technology

[0002] Red yeast rice originated in my country, where it was anciently known as Danqu or red yeast rice. It is a dried rice grain produced by fermenting rice with Monascus spp. Since the 1970s, when Japanese scientist Akira Endo first isolated the lipid-lowering component monacolin K (lovastatin) from Monascus spp., and lovastatin was subsequently developed as a lipid-lowering drug, red yeast rice has remained a hot topic in pharmaceutical research and possesses significant economic value. Literature indicates that red yeast rice contains abundant secondary metabolites, among which monacolin-like compounds are the main source of its lipid-lowering activity. Red yeast rice can produce various monacolin-like compounds, and the distribution of these compounds is directly related to its lipid-lowering effect. Therefore, current research focuses on the comprehensive analysis and detection of monacolin-like compounds in red yeast rice.

[0003]

[0004] Currently, a large number of studies have investigated the monacolin-like compounds in Monascus fermentation products, including the material basis of Monascus fermentation products on solid substrates such as rice, sorghum, and highland barley, liquid fermentation products, and extracts of Monascus fermentation products.

[0005] Li Meng-Ning et al. identified 84 monacolin compounds in red yeast rice using liquid chromatography-quadrupole-time mass spectrometry (LC-quadrupole-time mass spectrometry), which is the most complete analysis of monacolin compounds in red yeast rice to date (Analytica Chimica Acta 982 (2017) 156-157). Liang Jin Xiu et al. identified 39 monacolin compounds in red yeast rice using LC-quadrupole-time mass spectrometry (Journal of Ethnopharmacology 238 (2019) 111879). Chen Jia et al. analyzed the product composition of red yeast rice fermented with rice and barley as solid-state fermentation media (Food and Fusion 13 (2022) 7000).

[0006] Current methods for detecting monacolin compounds in red yeast rice focus on lovastatin and decocyclohexane lovastatin, with limited methods for detecting other monacolin compounds. Existing technologies include a high-performance liquid chromatography (HPLC) method developed by Li Yongguo et al. for the detection of 12 monacolin compounds, and a fingerprinting method for red yeast rice (the raw material for Xuezhikang) as described in the Chinese Pharmacopoeia, used by Wang Mingjuan et al., to detect commercially available red yeast rice samples. High-resolution liquid chromatography-mass spectrometry (HPLC-MS) is used to study the material basis of monocolin compounds, employing a precursor ion and secondary spectrum matching method for identification. However, HPLC-MS relies on researchers identifying monocolin compounds based on literature data, which is time-consuming and requires a high level of experience. Furthermore, HPLC-MS can only be developed for specific monocolin compounds and relies on the physical properties of their UV absorption for detection. Some monocolin compounds do not exhibit UV absorption, making it impossible to perform a comprehensive analysis of the entire monocolin compound class. The discovery and identification of new monocolin compounds requires a significant amount of spectral analysis work, which is both time-consuming and labor-intensive.

[0007] In summary, the methods currently included in standards and literature cannot comprehensively analyze monacolin compounds in red yeast rice, let alone discover new monacolin compounds. There is an urgent need in this field to develop new quality control methods for red yeast rice to comprehensively detect monacolin compounds. Summary of the Invention

[0008] The purpose of this invention is to provide a method for detecting monacolin-like compounds.

[0009] Another objective of this invention is to provide a method for quality control of red yeast rice.

[0010] To address the aforementioned technical problems, a first aspect of the present invention provides a method for detecting monacolin-like compounds, the method comprising the steps of: analyzing a sample to be tested using a liquid chromatography-mass spectrometry (LC-MS) instrument, wherein the mass spectrometry conditions of the LC-MS instrument include: acquiring C... 19 H 23 O2 + C 19 H 25 O2 + C 19 H 27 O2 + and C 19 H 29 O2 + ion.

[0011] In some preferred embodiments, the sample to be tested is a red yeast rice sample.

[0012] In some preferred embodiments, before the sample is injected for testing, a sample pretreatment is also included, which includes the step of dissolving the sample in a mixed solution of acetonitrile and water.

[0013] In some preferred embodiments, the sample pretreatment includes the following steps: dissolving the sample in a mixed solution of acetonitrile and water with a volume ratio of (60-80):(20-40), then centrifuging to obtain the supernatant, and filtering it using a microporous membrane to obtain the final product.

[0014] In some preferred embodiments, the liquid chromatography-mass spectrometry (LC-MS) instrument is selected from either liquid chromatography-tandem high-resolution mass spectrometry (LC-MS / MS) or liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS). In some preferred embodiments, the mass spectrometry conditions for the LC-MS / MS include: extracting ion currents with molecular weights of 283.1693±20ppm, 285.1849±20ppm, 287.2006±20ppm, and 289.2162±20ppm.

[0015] In some preferred embodiments, liquid chromatography-tandem high-resolution mass spectrometry is used to analyze the sample, such as the Agilient Q-Tof6500 mass spectrometer.

[0016] In some preferred embodiments, the mass spectrometry conditions for the liquid chromatography-tandem high-resolution mass spectrometry include:

[0017] Ion source: electrospray;

[0018] Scanning mode: Positive ion scanning mode ESI(+);

[0019] Monitoring methods: Auto MS / MS or Scan;

[0020] Collision gas: N2;

[0021] Collision energies: 10, 20, and 40 eV.

[0022] In some preferred embodiments, the mass spectrometry conditions for the liquid chromatography-tandem high-resolution mass spectrometry include:

[0023] Ion source: electrospray;

[0024] Scanning mode: Positive ion scanning mode ESI(+);

[0025] Monitoring method: Auto MS / MS;

[0026] Atomizer (N2): 35Psi;

[0027] Drying gas (N2): flow rate 15 L / min, temperature 200℃;

[0028] Sheath gas (N2): flow rate 12 L / min, temperature 350℃;

[0029] Capillary forging at 3.5kV;

[0030] The voltage at which the fuse broke was 250V.

[0031] Scan range: m / z 100~1200 Da; and

[0032] Collision gas: N2; Collision energy: 10, 20, 40 eV.

[0033] In some preferred methods, liquid chromatography-tandem triple quadrupole mass spectrometry is used to analyze the sample.

[0034] In some preferred embodiments, the mass spectrometry conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry include: acquiring ions with charge-to-mass ratios of 283.0±1, 285.0±1, 287.0±1, and 289.0±1.

[0035] In some preferred embodiments, the mass spectrometry conditions of the liquid chromatography-tandem triple quadrupole mass spectrometry include: selecting an ion with a charge-to-mass ratio of 283.0±1 as the parent ion, and selecting at least one ion with a charge-to-mass ratio of 223.0±1, 237.0±1, or 265.0±1 as the daughter ion;

[0036] An ion with a charge-to-mass ratio of 285.0±1 is selected as the parent ion, and at least one ion with a charge-to-mass ratio of 225.0±1, 239.0±1, or 267.0±1 is selected as the daughter ion.

[0037] An ion with a charge-to-mass ratio of 287.0±1 was selected as the parent ion, and at least one ion with a charge-to-mass ratio of 227.0±1, 241.0±1, or 269.0±1 was selected as the daughter ion; and

[0038] An ion with a charge-to-mass ratio of 289.0±1 is selected as the parent ion, and at least one ion with a charge-to-mass ratio of 229.0±1, 243.0±1, or 271.0±1 is selected as the daughter ion.

[0039] In some preferred embodiments, the mass spectrometry conditions of the liquid chromatography-tandem triple quadrupole mass spectrometry include: selecting an ion with a charge-to-mass ratio of 283.0±1 as the parent ion and selecting an ion with a charge-to-mass ratio of 223.0±1 as the daughter ion;

[0040] Ions with a charge-to-mass ratio of 285.0±1 were selected as the parent ion, and ions with a charge-to-mass ratio of 225.0±1 were selected as the daughter ion.

[0041] An ion with a charge-to-mass ratio of 287.0 ± 1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 227.0 ± 1 was selected as the daughter ion; and

[0042] An ion with a charge-to-mass ratio of 289.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 229.0±1 was selected as the daughter ion.

[0043] In some preferred embodiments, the collision energy of the liquid chromatography-tandem triple quadrupole mass spectrometer is 22-24 eV.

[0044] In some preferred embodiments, the declustering voltage of the liquid chromatography-tandem triple quadrupole mass spectrometer is 180-200V.

[0045] In some preferred embodiments, the mass spectrometry conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry include:

[0046] At a declustering voltage of 180V and a collision capability of 24eV, an ion with a charge-to-mass ratio of 283.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 223.0±1 was selected as the daughter ion.

[0047] At a declustering voltage of 180V and a collision capability of 24eV, an ion with a charge-to-mass ratio of 285.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 225.0±1 was selected as the daughter ion.

[0048] At a declustering voltage of 200V and a collisional capability of 22eV, an ion with a charge-to-mass ratio of 287.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 227.0±1 was selected as the daughter ion; and

[0049] With a declustering voltage of 200V and a collision capability of 24eV, an ion with a charge-to-mass ratio of 289.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 229.0±1 was selected as the daughter ion.

[0050] In some preferred embodiments, the mass spectrometry conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry include:

[0051] Ion source: Electrospray, positive ion scanning mode ESI(+);

[0052] Monitoring method: MRM mode, ion source temperature 200~600℃ (preferably 450℃);

[0053] At a declustering voltage of 180V and a collision capability of 24eV, an ion with a charge-to-mass ratio of 283.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 223.0±1 was selected as the daughter ion.

[0054] At a declustering voltage of 180V and a collision capability of 24eV, an ion with a charge-to-mass ratio of 285.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 225.0±1 was selected as the daughter ion.

[0055] At a declustering voltage of 200V and a collisional capability of 22eV, an ion with a charge-to-mass ratio of 287.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 227.0±1 was selected as the daughter ion; and

[0056] With a declustering voltage of 200V and a collision capability of 24eV, an ion with a charge-to-mass ratio of 289.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 229.0±1 was selected as the daughter ion.

[0057] In some preferred embodiments, the mass spectrometry conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry further include:

[0058] Drying gas: 50-100 L / min (preferably 50 L / min);

[0059] Air curtain gas: 20-40 L / min (preferably 40 L / min);

[0060] Spray voltage 4500V.

[0061] In some preferred embodiments, the chromatographic conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry and / or liquid chromatography-tandem high-resolution mass spectrometry include:

[0062] Chromatographic column: EC-C18 column;

[0063] Mobile phase: The organic phase is acetonitrile, and the aqueous phase is 0.1% formic acid solution as the mobile phase;

[0064] Elution method: gradient elution.

[0065] In some preferred embodiments, the gradient elution procedure is as follows:

[0066] Time (minutes) Organic phase (%) Aqueous phase (%) 0～10 5→35 95→65 10～18 35 65 18～20 35→40 65→60 20～35 40→45 60→55 35～50 45→55 55→45 50～60 55→75 45→25 60～70 75→95 25→5 70～70.1 95→5 5→95 .

[0067] In some preferred embodiments, the chromatographic conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry and / or liquid chromatography-tandem high-resolution mass spectrometry include:

[0068] Injection volume: 1-5 μL;

[0069] The flow rate was 0.3-0.5 ml / min; and

[0070] The column temperature is 18-22℃.

[0071] In some preferred embodiments, the chromatographic conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry and / or liquid chromatography-tandem high-resolution mass spectrometry include:

[0072] Column: Aglient Poroshell EC-C18 column;

[0073] Mobile phase: The organic phase is acetonitrile, and the aqueous phase is 0.1% formic acid solution as the mobile phase;

[0074] Elution method: gradient elution.

[0075] Injection volume: 1-5 μL;

[0076] The flow rate was 0.4 ml / min; and

[0077] The column temperature is 20℃;

[0078] The gradient elution procedure is as follows:

[0079]

[0080]

[0081] A second aspect of the present invention provides a quality control method for red yeast rice, the method comprising the steps of: detecting monoclonal compounds in red yeast rice, wherein the monoclonal compounds include at least one of the following:

[0082] In some preferred embodiments, the method includes the steps of: pretreating a red yeast rice sample to obtain a test solution, and then using a liquid chromatography-mass spectrometry (LC-MS) instrument to detect the presence of at least one monacolin-like compound in the test solution.

[0083] In some preferred embodiments, the step of using liquid chromatography-mass spectrometry (LC-MS) to detect the presence of at least one monacolin-like compound in the test solution includes the step of: setting the mass spectrometry conditions of the LC-MS, wherein the mass spectrometry conditions of the LC-MS include: collecting C 19 H 23 O2 + C 19 H 25 O2 + C 19 H 27 O2 + and C 19 H 29 O2 + ion.

[0084] In some preferred embodiments, the liquid chromatography-mass spectrometry (LC-MS) instrument is selected from either liquid chromatography-tandem high-resolution mass spectrometry (LC-MS) or liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS).

[0085] In some preferred embodiments, the mass spectrometry conditions for the liquid chromatography-tandem high-resolution mass spectrometry include: extracting ion chromatograms with molecular weights of 283.1693±20ppm, 285.1849±20ppm, 287.2006±20ppm, and 289.2162±20ppm.

[0086] In some preferred embodiments, liquid chromatography-tandem high-resolution mass spectrometry is used to analyze the sample, such as the Agilient Q-Tof6500 mass spectrometer.

[0087] In some preferred embodiments, the mass spectrometry conditions for the liquid chromatography-tandem high-resolution mass spectrometry include:

[0088] Ion source: electrospray;

[0089] Scanning mode: Positive ion scanning mode ESI(+);

[0090] Monitoring method: Auto MS / MS;

[0091] Collision gas: N2;

[0092] Collision energies: 10, 20, and 40 eV.

[0093] In some preferred embodiments, the mass spectrometry conditions for the liquid chromatography-tandem high-resolution mass spectrometry include:

[0094] Ion source: electrospray;

[0095] Scanning mode: Positive ion scanning mode ESI(+);

[0096] Monitoring method: Auto MS / MS;

[0097] Atomizer (N2): 35Psi;

[0098] Drying gas (N2): flow rate 15 L / min, temperature 200℃;

[0099] Sheath gas (N2): flow rate 12 L / min, temperature 350℃;

[0100] Capillary forging at 3.5kV;

[0101] The voltage at which the fuse broke was 250V.

[0102] Scan range: m / z 100~1200 Da; and

[0103] Collision gas: N2; Collision energy: 10, 20, 40 eV.

[0104] In some preferred methods, liquid chromatography-tandem triple quadrupole mass spectrometry is used to analyze the sample.

[0105] In some preferred embodiments, the mass spectrometry conditions of the liquid chromatography-tandem triple quadrupole mass spectrometry include: selecting ions with charge-to-mass ratios of 283.0±1, 285.0±1, 287.0±1, and 289.0±1 as precursor ions.

[0106] In some preferred embodiments, the mass spectrometry conditions of the liquid chromatography-tandem triple quadrupole mass spectrometry include: selecting a parent ion with a charge-to-mass ratio of 283.0±1 and a daughter ion with a charge-to-mass ratio of 223.0±1;

[0107] Select a parent ion with a charge-to-mass ratio of 285.0±1 and a daughter ion with a charge-to-mass ratio of 225.0±1;

[0108] Select a parent ion with a charge-to-mass ratio of 287.0 ± 1 and a daughter ion with a charge-to-mass ratio of 227.0 ± 1; and

[0109] A parent ion with a charge-to-mass ratio of 289.0±1 was selected, and a daughter ion with a charge-to-mass ratio of 229.0±1 was selected.

[0110] In some preferred embodiments, the collision energy of the liquid chromatography-tandem triple quadrupole mass spectrometer is 22-24 eV.

[0111] In some preferred embodiments, the declustering voltage of the liquid chromatography-tandem triple quadrupole mass spectrometer is 180-200V.

[0112] In some preferred embodiments, the mass spectrometry conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry include:

[0113] With a clustering voltage of 180V and a collision capability of 24eV, a parent ion with a charge-to-mass ratio of 283.0±1 and a daughter ion with a charge-to-mass ratio of 223.0±1 are selected.

[0114] With a clustering voltage of 180V and a collision capability of 24eV, a parent ion with a charge-to-mass ratio of 285.0±1 and a daughter ion with a charge-to-mass ratio of 225.0±1 are selected.

[0115] At a declustering voltage of 200V and a collisional capability of 22eV, a parent ion with a charge-to-mass ratio of 287.0±1 and a daughter ion with a charge-to-mass ratio of 227.0±1 were selected; and

[0116] With a declustering voltage of 200V and a collision capability of 24eV, a parent ion with a charge-to-mass ratio of 289.0±1 and a daughter ion with a charge-to-mass ratio of 229.0±1 are selected.

[0117] In some preferred embodiments, when a precursor ion with a mass-to-charge ratio of 283.0±1 and a daughter ion with a mass-to-charge ratio of 223.0±1 are selected from the test sample solution; and / or, a precursor ion with a mass-to-charge ratio of 285.0±1 and a daughter ion with a mass-to-charge ratio of 225.0±1 are selected from the test sample solution; and / or, a precursor ion with a mass-to-charge ratio of 287.0±1 and a daughter ion with a mass-to-charge ratio of 227.0±1 are selected from the test sample solution; and / or, a precursor ion with a mass-to-charge ratio of 289.0±1 and a daughter ion with a mass-to-charge ratio of 229.0±1 are selected from the test sample solution; then it is determined that the test sample solution contains at least one monacolin-like compound.

[0118] Compared with the prior art, the present invention has at least the following advantages:

[0119] (1) This invention employs a non-targeted screening approach, discovering for the first time four key daughter ions of monocorine-like compounds. Utilizing the tendency of these compounds to undergo intra-source fragmentation in mass spectrometry, non-targeted screening methods for monocorine-like compounds were established in both high-resolution mass spectrometry and triple quadrupole mass spectrometry. These methods can specifically reveal the distribution and content of monocorine-like compounds. Compared to the original material-based analytical methods, no secondary mass spectrometry analysis is required, allowing for rapid determination of the retention time of these compounds and enabling non-targeted screening of monocorine-like compounds. Compared to the original analytical methods for monocorine-like compounds, this method can filter out interference from most other compounds, specifically revealing the distribution and content of monocorine-like compounds. This information can be used to establish fingerprint spectra of monocorine-like compounds for overall control.

[0120] (2) The method for detecting monacolin-like compounds provided by the present invention can also be used to discover new monacolin-like compounds.

[0121] (3) The method for detecting monoclonal compounds provided by the present invention has low requirements for the experience of the experimental personnel and is short in time.

[0122] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0123] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0124] Figure 1 The molecular formula of red yeast rice is C 19 H 22O2 (m / z mass number 283.1693) extracted ion chromatogram;

[0125] Figure 2 The molecular formula of red yeast rice is C 19 H 24 O2 (m / z mass number 285.1849) extracted ion chromatogram;

[0126] Figure 3 The molecular formula of red yeast rice is C 19 H 26 O2 (m / z mass number 287.2006) extracted ion chromatogram;

[0127] Figure 4 The molecular formula of red yeast rice is C 19 H 28 O2 (m / z mass number 289.2162) extracted ion chromatogram;

[0128] Figure 5 The molecular formula of red yeast rice is C 19 H 22 O2, C 19 H 24 O2, C 19 H 26 O2 and C 19 H 28 O2 extraction ion flow overlay diagram;

[0129] Figure 6This is a superimposed ion current diagram of monocarboxylic acid compounds extracted from red yeast rice. Peak 1: 3,5-dihydroxy-3,5-dihydromonacolin J; Peak 2: 3-oxo-5-hydroxy-3,5-dihydromonacolin K acid; Peak 3: 3,5-dihydroxy-3,5-dihydrodehydromonacolin J; Peak 4: Monacolin S acid; Peak 5: Dehydromonacolin S-2; Peak 6: Monacolin S; Peak 7: Dehydromonacolin S acid-1; Peak 8: 3-hydroxy-3,5-dihydromonacolin J; Peak 9: 3,5-dihydroxy-3,5-dihydromonacolin L; Peak 10: 3-oxo-5-hydroxy-3,5-dihydromonacolin K; Peak 11: Monacolin R acid; Peak 12: 3-hydroxy-3,5-dihydromonacolin L; Peak 13: 8-(200-methyl) butanoyloxy)monacophenyl; Peak 14: Monacolin J acid; Peak 15: 3-oxo-5-hydroxy-3,5-dihydrodehydromonacolin K acid-2; Peak 16: a,b-hydromonacolin Q acid; Peak 17: 3,5-dihydroxy-3,5-dihydrodehydromonacolinL; Peak 18: Dihydromonacolin J acid Peak 19: Monacolin R; Peak 20: 3-oxo-5-hydroxy-3,5-dihydromonacolin K-1; Peak 21: Monacolin J; Peak 22: Monacolin Qacid; Peak 23: 3-hydroxy-3,5-dihydromonacolin K; Peak 24: Dihydromonacolin J; Peak 25: Monacolin M Peak 26: Monacolin X acid; Peak 27: Dehydromonacolin M acid; Peak 28: M34-3”-hydroxydehydromonacolin K acid; Peak 29: 3-hydroxy-3,5-dihydrodehydromonacolin K Peak 30: Monacolin NT2;Peak 31: Dihydromonacolin N acid; Peak 32: ihydromonacolin M-1; Peak 33: 3”-hydroxymonacolin K; Peak 34: onacolin N; Peak 35: Dehydromonacolin J; Peak 36: MonacolinL acid; Peak 37: a,b-hydromonacolin Q; Peak 38: Compactic acid; Peak 39: Dehydromonacolin Xacid; Peak 40: Monacolin X; Peak 41: Dehydromonacolin N acid; Peak 42: 3”-hydroxydihydromonacolin K; Peak 43: ML-236C; Peak 44: Monacolin NT1; Peak 45: Dihydromonacolin L acid; Peak 46: Dehydromonacolin M; Peak 47: Monacolin K acid; Peak 48: Monacophenyl; Peak 49: M56-Methyl ester of monacolin L acid Peak 50: M55-Dihydromonacolin N; Peak 51: Dihydromonacolin X; Peak 52: Methyl ester of a,bhydromonacolin Q acid; Peak 53: Monacolin L; Peak 54: Compactin; Peak 55: 3”-hydroxydehydromonacolin K; Peak 56: Dehydromonacolin N; Peak 57: DihydromonacolinKacid; Peak 58: Dehydromonacolin L acid; Peak 59: Dehydromonacolin X; Peak 60: Dihydromonacolin L; Peak 61: Monacolin K; Peak 62: Monacolin Q; Peak 63: Methyl ester ofdehydromonacolin J acid; Peak 64: Dihydromonacolin MV; Peak 65: Dihydromonacolin K; Peak 66: Dehydrocompactin; Peak 67: Dehydromonacolin L; Peak 68: ethyl ester ofdehydromonacolin K acid; Peak 69: Dehydromonacolin K; Peak 70: ethyl ester ofdihydromonacolin K acid;Peak 71: Dehydrodihydromonacolin L; Peak 72: ethyl ester ofdehydromonacolin L acid; Peak 73: Dehydrodihydromonacolin K;

[0130] Figure 7 It is an ion flow map of the 283.0→223.0 m / z channel;

[0131] Figure 8 It is an ion flow map of the 285.0→225.0 m / z channel;

[0132] Figure 9 It is an ion flow map of the m / z 287.0→227.0 channel;

[0133] Figure 10 It is an ion flow map of the m / z 289.0→229.0 channel;

[0134] Figure 11 It is the m / z 283.0→223.0 channel;

[0135] Figure 12 This is a magnified diagram of the peak assignments for the m / z 283.0→223.0 channel;

[0136] Figure 13 This is a magnified diagram of the peak assignments for the m / z 283.0→223.0 channel;

[0137] Figure 14 It is the m / z 285.0→225.0 channel;

[0138] Figure 15 This is a magnified diagram of the peak assignments for the m / z 285.0→225.0 channel;

[0139] Figure 16 This is a magnified diagram of the peak assignments for the m / z 285.0→225.0 channel;

[0140] Figure 17 This is a magnified diagram of the peak assignments for the m / z 285.0→225.0 channel;

[0141] Figure 18 This is a magnified diagram of the peak assignments for the m / z 285.0→225.0 channel - 4;

[0142] Figure 19 It is the m / z 287.0→227.0 channel;

[0143] Figure 20 This is a magnified diagram of the peak assignments for the m / z 287.0→227.0 channel - 1;

[0144] Figure 21 This is a magnified diagram of the peak assignments for the m / z 287.0→227.0 channel;

[0145] Figure 22 This is a magnified diagram of the peak assignments for the m / z 287.0→227.0 channel - 3;

[0146] Figure 23 It is the m / z 287.0→227.0 channel;

[0147] Figure 24 This is a high-resolution first-order mass spectrometry spectrum of Monacolin K (lovastatin) at its retention time;

[0148] Figure 25 This is the second-order mass spectrum of Monacolin K (lovastatin) at high resolution (collision energies 1.10 eV, 2.20 eV, 3.30 eV);

[0149] Figure 26 The mass spectrometry fragmentation pattern of Monacolin K (lovastatin);

[0150] Figure 27 The extracted ion chromatogram is for an m / z mass number of 285.1849.

[0151] Figure 28 The extracted ion chromatogram is for an m / z mass number of 303.1955.

[0152] Figure 29 The extracted ion chromatogram is for an m / z mass number of 199.1481.

[0153] Figure 30 The image shows the extracted ion current overlay plots for m / z mass numbers 285.1849, 303.1955, and 199.1481.

[0154] Figure 31 This is a superimposed magnified view of the extracted ion currents at m / z mass numbers 285.1849 and 199.1481;

[0155] Figure 32 It is an ion flow map of the 285.0→225.0 m / z channel;

[0156] Figure 33 It is an ion flow map of the m / z 285.0→239.0 channel;

[0157] Figure 34 It is an ion flow map of the 285.0→267.0 m / z channel;

[0158] Figure 35 It is an ion flow map of the m / z 285.0→199.0 channel;

[0159] Figure 36 It is an ion flow map of the m / z 285.0→169.0 channel;

[0160] Figure 37 It is an ion flow map of the 285.0→155.0 m / z channel;

[0161] Figure 38 The fingerprint spectrum of the m / z 283.0→223.0 channel shows the following peaks: 1: Unknown-1; 2: Unknown-2; 3: Unknown-3; 4: Unknown-4; 5: Unknown-5; 6: 3,5-dihydroxy-3,5-dihydromonacolin J; 7: Unknown-6; 8: 3,5-dihydroxy-3,5-dihydrodehydromonacolin J; 9: Unknown-7; 10: Unknown-8; 11: Monacolin S acid; 12: Unknown-9; 13: Unknown-10; 14: Unknown-11; 15: Unknown-12; 16: Monacolin S; 17: Dehydromonacolin S acid; 18: Unknown-13; 19: Unknown-14; 20: 13-8-(2”-methyl butanoyloxy)monacophenyl; 21: α,β-hydromonacolin Q acid; 22: Monacolin Q. Acid peak 23: α,β-hydromonacolin Q;

[0162] Figure 39The fingerprint spectrum of the m / z 285.0→225.0 channel shows the following peaks: 1: Monacolin J acid; 2: Monacolin J; 3: 3-hydroxy-3,5-dihydromonacolin K; 4: Dihydromonacolin J; 5: Monacolin M; 6: Monacolin X acid and Dehydromonacolin M acid; 7: 3”-hydroxydehydromonacolin Kacid; 8: Monacolin NT2; 9: Monacolin N; 10: Dehydromonacolin X acid; 11: Monacolin X; 12: Monacolin NT1 and Monacolin K acid; 13: Unkwon-1; 14: 3”-hydroxydehydromonacolin K and Dehydromonacolin N; 15: Monacolin K; 16: Dehydromonacolin K.

[0163] Figure 40 The fingerprint spectrum of the m / z 287.0→227.0 channel shows the following peaks: Peak 1: Monacolin R acid; Peak 2: 3-hydroxy-3,5-dihydromonacolin L acid; Peak 3: Monacolin R acid; Peak 4: Dihydromonacolin J acid; Peak 5: Monacolin L acid; Peak 6: Dihydromonacolin N acid; Peak 7: Monacolin L acid; Peak 8: Dehydromonacolin L acid and Dihydromonacolin K acid; Peak 9: Dihydromonacolin K acid; Peak 10: Dehydrodihydromonacolin K acid.

[0164] Figure 41 This is a superimposed fingerprint spectrum of Monacolin-like compounds with a key ion mass number m / z of 283 in red yeast rice;

[0165] Figure 42 This is a superimposed fingerprint spectrum of Monacolin-like compounds with a key ion mass number m / z of 285 in red yeast rice;

[0166] Figure 43 This is a superimposed fingerprint spectrum of Monacolin-like compounds with a key ion mass number m / z of 287 in red yeast rice.

[0167] Figure 44It is a superimposed fingerprint spectrum of monacolin-like compounds with a key ion mass number m / z of 283 in traditional Chinese medicine preparations containing red yeast rice;

[0168] Figure 45 It is a superimposed fingerprint spectrum of monacolin-like compounds with a key ion mass number m / z of 285 in traditional Chinese medicine preparations containing red yeast rice;

[0169] Figure 46 It is a superimposed fingerprint spectrum of monacolin-like compounds with a key ion mass number m / z of 287 in traditional Chinese medicine preparations containing red yeast rice. Detailed Implementation

[0170] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0171] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0172] Unless otherwise specified, the term “or” means the term “and / or” and is used interchangeably with the term “and / or”.

[0173] As used herein, including the appended claims, unless the context clearly indicates otherwise, the singular forms of words such as “an,” “a,” and “the” include their respective plural referents.

[0174] Example 1: Pretreatment Method

[0175] Take approximately 0.5 g of the test sample powder (passed through a No. 3 sieve), accurately weigh it, and place it in a stoppered conical flask. Accurately add 50 mL of acetonitrile-water (volume ratio 70:30) mixed solution, weigh it, sonicate for 30 minutes, cool it, replenish the lost weight with acetonitrile-water (volume ratio 70:30) mixed solution, centrifuge for 5 minutes (centrifugation speed 4000 r / min), take the supernatant, filter it through a microporous membrane (0.22 μm), and take the filtrate.

[0176] Example 2: Chromatographic and Mass Spectrometric Conditions

[0177] (1) Chromatographic conditions

[0178] An Agilent 1290 ultra-high performance liquid chromatograph was used, with an Agilent Poroshell EC-C18 column (150 mm length, 3.0 mm inner diameter, 2.7 μm particle size). Acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1. The flow rate was 0.4 mL / min, and the column temperature was 20 °C. Injection volume: 0.1–5 μL.

[0179] Table 1 Elution gradient of mobile phase

[0180] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 5→35 95→65 10～18 35 65 18～20 35→40 65→60 20～35 40→45 60→55 35～50 45→55 55→45 50～60 55→75 45→25 60～70 75→95 25→5 70～70.1 95→5 5→95

[0181] (2) Mass spectrometry conditions: Agilent Q-Tof 6550 (high resolution mass spectrometer)

[0182] Agilent Q-Tof 6500 mass spectrometer, ion source: electrospray ionization, positive ion scanning mode ESI(+); monitoring mode: Auto MS / MS, nebulizer (N2): 35Psi; dry gas (N2): flow rate 15L / min, temperature 200℃; sheath gas (N2): flow rate 12L / min, temperature 350℃; capillary forging 3.5kV; fragmentation voltage 250V; scanning range: m / z 100~1200Da, collision gas: N2; collision energy: 10, 20, 40eV.

[0183] Ion chromatogram extraction conditions: Extract molecules with the molecular formula C 19 H 22 O2, C 19 H 24 O2, C 19 H 26 O2 and C 19 H 28 Ion chromatograms of O2, or ion chromatograms of precisely extracted molecular weights of 283.1693, 285.1849, 287.2006, and 289.2162, with a mass number deviation of 10 ppm.

[0184] Comparative analysis of the extracted ion maps of monacolin-like compounds in red yeast rice Figures 1-4 ) and the superimposed diagram of the extracted ion currents of the four key daughter ions ( Figure 5 and 6 The high overlap in retention times between the two indicates that the extracted ion chromatograms of the four key daughter ions can filter out interference from other compounds and exclusively present the distribution of monacolin-like compounds.

[0185] (3) Mass spectrometry conditions: triple quadrupole tandem mass spectrometry

[0186] The AB5500 triple quadrupole tandem mass spectrometer uses an electrospray ionization (ESI) source in positive ion scanning mode (ESI+). Monitoring mode is MRM mode, with an ion source temperature of 450℃. Drying gas flow rate is 50 L / min, curtain gas flow rate is 40 L / min, and spray voltage is 4500 V. The mass spectrometry parameters for the four key component ions are shown in Table 2 below.

[0187] Table 2 Key Faction Ion Mass Spectrometry Parameters

[0188] Serial Number Mother ion daughter ions Declustering voltage (V) Collision energy (eV) 1 283.0 223.0 180 24 2 285.0 225.0 180 24 3 287.0 227.0 200 22 4 289.0 229.0 200 24

[0189] The collected red yeast rice samples were analyzed using a triple quadrupole method, and ion chromatograms of each channel were extracted. Figure 7-10 The chromatographic peaks in the sample were assigned based on the material basis results, and the results are as follows: Figure 11-23 As shown, this method can effectively filter out interference from other compounds, specifically reveal the distribution of monacolin-like compounds, and can screen for and discover new monacolin-like compounds.

[0190] Example 3: Key Faction Ion Analysis

[0191] Since the most abundant monacolin compounds in red yeast rice are monacolin K (lovastatin) and monacolin acid, the mass number m / z for these two compounds is 285.1849 (C). 19 H 25 O2 + Key fragment ion analysis. From the primary and secondary spectra of Monacolin K at the high-resolution mass spectrometry retention time, it can be seen that lovastatin fragments were already generated in the ion source, producing secondary ion fragments. Combining these two findings with the secondary fragmentation pattern of Monacolin K, the optimal fragment ions are those with an m / z mass number of 285.1849 (C). 19 H 25 O2 + ), 303.1955 (C 19 H 27 O3 + ), 199.1481(C 15 H 19 + Fragment ions.

[0192] The m / z mass number extracted from the first-order spectrum is 285.1849 (C). 19 H 25 O2 + ), 303.1955 (C 19 H 27 O3 + ), 199.1481(C 15 H 19 +It was found that the chromatographic peaks with an m / z mass number of 285.1849 had the highest responses. Since monacolin K and acid monacolin K are the main components of monacolin-like compounds in red yeast rice, and most monacolin-like compounds are present in small amounts, their mass spectrometric responses are relatively low. The higher the response of the key ion, the more monacolin-like compounds can be detected. Therefore, from the perspective of mass spectrometric response, an m / z mass number of 285.1849 is the optimal choice.

[0193] The fragmentation pattern of lovastatin revealed that the key ion with an m / z mass number of 285.1849 is the smallest component ion characterizing the skeleton of monacolin compounds, while the key ion with an m / z mass number of 303.1955 cannot characterize dehydromonacolin compounds such as Dehydromonacolin K, Dehydromonacolin X, and Dehydromonacolin N. It is evident that the extracted ion chromatogram of m / z mass number 303.1955 shows significantly fewer peaks than that of m / z mass number 285.1849, such as the absence of Dehydromonacolin K with a retention time of 62.2 minutes.

[0194] The key ion fragment with an m / z mass number of 199.1481 is relatively small, and cannot be characterized as having the carbon skeleton structure of monacolin-like compounds. Studies by Zhu Lin et al. have shown that decahydronaphthalene compounds contained in red yeast rice also produce a carbon skeleton with an m / z mass number of 199.1481 (C... 15 H 19 + Fragments of ) (Journal of Chromatography A, 1303(2013)54–61), Chen Jia et al.'s research also showed that pigment compounds in red yeast rice can produce 199.1481 (C 15 H 19 + The fragments of ) (Food and Fusion 13 (2022) 7000). The extracted ion chromatogram with a mass number of 199.1481 m / z is significantly more numerous than that of 285.1849, but almost none of the added chromatographic peaks are monacolin compounds. For example, the chromatographic peak with a retention time of 9.7 min was analyzed to be a decahydronaphthalene compound, Monascusic acid D. Therefore, the specificity of this fragment ion is not high enough.

[0195] In summary, considering both mass spectrometry response and specificity, a mass number of 285.1849 (C) was selected. 19 H 25 O2 + ) is one of the key daughter ions.

[0196] Since triple quadrupole mass spectrometry has lower qualitative capabilities than high-resolution mass spectrometry, the MRM mode is used for triple quadrupole mass spectrometry. The key daughter ion of the monocorine-like compound is used as the parent ion, and the daughter ion of the key daughter ion is used as the daughter ion. This allows us to obtain the distribution of the key daughter ion specific to the monocorine-like compound, and thus obtain the distribution of the monocorine-like compound.

[0197] The mass number m / z assigned to monacolin K (lovastatin) and monacolin acid is 285 (C). 19 H 25 O2 + Taking the key daughter ion as the parent ion as an example, using it as the parent ion, daughter ions with mass numbers m / z of 225, 267, 239, 199, 169, and 155 were obtained through daughter ion scanning. These seven daughter ions, ordered by response, are 225, 199, 267, 169, 239, and 155. According to... Figures 11-23 It can be seen that the mass spectral peaks in the obtained spectrum can indicate the presence of monacolin-like compounds at that retention time, and a high daughter ion mass spectral response can uncover more monacolin-like compounds. Therefore, from the perspective of daughter ion response, the 225 daughter ion is selected.

[0198] Simultaneous comparison of the ion chromatograms of each daughter ion channel reveals that the relative response of the mass spectrum peaks in the ion chromatogram of daughter ion 225 is closest to that in the extracted ion chromatogram of monacolin-like compounds in high-resolution mass spectrometry. In contrast, the relative responses of the mass spectrum peaks in the ion chromatogram of daughter ion 199 differ significantly from the responses of other mass spectrum peaks. Among them, the Dehydromonacolin K at 62.2 minutes has the highest response, rather than the highest response of the most abundant monacolin K and acid monacolin K.

[0199] In summary, considering the high response of each daughter ion and the relatively high similarity to the responses of the parent ions of various monacolin-like compounds in high-resolution mass spectrometry, 225 was selected as the exclusive daughter ion of the key daughter ion with a mass number m / z of 285.

[0200] Example 4

[0201] Based on the method in this patent, a fingerprinting method for monacolin-like compounds in red yeast rice was established using liquid chromatography-tandem triple quadrupole mass spectrometry. Specific non-targeted detection of monacolin-like compounds was performed on 28 batches of collected samples. 48 common peaks from the 28 batches were counted, and the fingerprinting of monacolin-like compounds in red yeast rice was generated by combining the specific spectra of the monacolin-like samples from the 28 batches. The details are as follows:

[0202] For monacolin-like compounds with a key ion mass number m / z of 283, 23 common peaks were identified. The area of ​​these 23 common peaks exceeds 85% of the total peak area and can comprehensively represent this type of monacolin-like compound. A fingerprint spectrum of this type of monacolin-like compound was established based on these 23 common peaks.

[0203] For monacolin-like compounds with a key ion mass number m / z of 285, 15 common peaks were identified. The area of ​​these 15 common peaks exceeds 85% of the total peak area, which can comprehensively represent this type of monacolin-like compound. A fingerprint spectrum of this type of monacolin-like compound was established based on these 15 common peaks.

[0204] For monacolin-like compounds with a key ion mass number m / z of 287, 10 common peaks were identified. The area of ​​these 10 common peaks exceeds 85% of the total peak area, which can comprehensively represent this type of monacolin-like compound. A fingerprint spectrum of this type of monacolin-like compound was established based on these 10 common peaks.

[0205] For monacolin-like compounds with a key ion mass number m / z of 289, fingerprinting was not established for this class of monacolin-like compounds due to their small number and low response.

[0206] The chromatographic conditions, mass spectrometry conditions, and preparation of the test solution are the same as above.

[0207] Preparation of reference solution: Weigh an appropriate amount of lovastatin reference standard accurately, add 70% acetonitrile to prepare a solution containing 1 μg per 1 ml.

[0208] Determination method: ① Accurately pipette 1-5 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine the fingerprint chromatograms of monacolin-like compounds with key ion mass numbers m / z of 287 and 283, respectively. ② Accurately pipette 0.2-1 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine the fingerprint chromatograms of monacolin-like compounds with key ion mass numbers m / z of 285.

[0209] In a fingerprint chromatogram with a key ion mass number m / z of 283, the test sample chromatogram should exhibit 23 characteristic peaks. The similarity between the test sample's characteristic chromatogram and the reference characteristic chromatogram was calculated using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (version 2.0) based on the Mark peaks.

[0210] In a fingerprint chromatogram with a key ion mass number m / z of 285, the test sample chromatogram should exhibit 15 characteristic peaks, of which peak 14 should correspond to the retention time of the reference peak. A similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine was used to calculate the similarity between the characteristic chromatograms of the test sample and the reference chromatogram based on the Mark peak.

[0211] In a fingerprint chromatogram with a key ion mass number m / z of 287, the test sample chromatogram should exhibit 10 characteristic peaks. A similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine was used to calculate the similarity between the characteristic chromatogram of the test sample and the control chromatogram based on the Mark peak.

[0212] (1) Parallelism test of reference solution

[0213] Accurately pipette 1 μl of the reference solution and inject it 6 times consecutively. Record the peak area and calculate the RSD value. The results are shown in Table 4-1 below.

[0214] Table 4-1 Precision Test

[0215] lovastatin f1 2.05E+05 f2 2.04E+05 f3 2.07E+05 f4 2.01E+05 f5 2.02E+05 f6 2.06E+05 average 2.04E+05 RSD 1.1％

[0216] (2) Stability test of the test solution

[0217] Samples were taken and test solutions were prepared according to the prescribed method. The solutions were injected and analyzed at 0, 6, 12, 18, and 24 hours. The results showed that the retention times and peak areas of each compound in the test solutions were basically stable within 0–24 hours (see Tables 4-2 to 4-10).

[0218] Table 4-2 Stability tests (similarity) of Monacolin-like compounds with a key daughter ion mass number m / z of 283.

[0219] time Y1 0h 0.987 6h 0.986 12h 0.981 18h 0.985 24h 0.985 average 0.985 RSD% 0.2

[0220] Table 4-3 Stability tests (retention time / min) of Monacolin-like compounds with a key daughter ion mass number m / z of 283.

[0221]

[0222]

[0223] Table 4-4 Stability tests (peak areas) of monacolin-like compounds with a key daughter ion mass number m / z of 283.

[0224]

[0225] Table 4-5 Stability tests (similarity) of Monacolin-like compounds with a key daughter ion mass number m / z of 285.

[0226] time Y1 0h 1 6h 1 12h 1 18h 1 24h 1 average 1 RSD% 0 Table 4-6 Stability tests (retention time / min) of monacolin-like compounds with a key daughter ion mass number m / z of 285.

[0227]

[0228] Table 4-7 Stability tests (peak areas) for monacolin-like compounds with a key daughter ion mass number m / z of 285.

[0229] time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 0h 1.65E+06 5.47E+06 1.13E+06 1.04E+06 3.67E+05 1.04E+06 4.57E+05 2.35E+06 6h 1.51E+06 5.50E+06 1.11E+06 1.02E+06 3.70E+05 1.08E+06 4.86E+05 2.32E+06 12h 1.58E+06 5.13E+06 1.09E+06 1.02E+06 3.48E+05 1.05E+06 4.85E+05 2.24E+06 18h 1.55E+06 5.12E+06 1.07E+06 9.94E+05 3.52E+05 1.06E+06 4.76E+05 2.20E+06 24h 1.50E+06 5.12E+06 1.10E+06 9.95E+05 3.48E+05 1.02E+06 4.73E+05 2.16E+06 average 1.56E+06 5.27E+06 1.10E+06 1.01E+06 3.57E+05 1.05E+06 4.75E+05 2.25E+06 RSD% 3.8 3.7 2.0 1.9 3.0 2.1 2.4 3.5 time Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 Peak 14 Peak 15 Peak 16 0h 2.78E+06 1.17E+06 1.16E+06 5.22E+07 1.72E+06 6.53E+05 1.30E+08 7.74E+06 6h 2.71E+06 1.20E+06 1.17E+06 5.22E+07 1.70E+06 6.35E+05 1.29E+08 8.41E+06 12h 2.69E+06 1.20E+06 1.15E+06 5.08E+07 1.59E+06 6.35E+05 1.28E+08 8.38E+06 18h 2.59E+06 1.21E+06 1.01E+06 5.08E+07 1.68E+06 6.78E+05 1.30E+08 8.32E+06 24h 2.57E+06 1.14E+06 1.03E+06 4.98E+07 1.52E+06 6.20E+05 1.28E+08 8.19E+06 average 2.67E+06 1.18E+06 1.10E+06 5.11E+07 1.64E+06 6.44E+05 1.29E+08 8.21E+06 RSD% 3.3 2.6 6.8 2.0 5.0 3.4 0.8 3.3

[0230] Table 4-8 Stability tests (similarity) of Monacolin-like compounds with a key daughter ion mass number m / z of 287.

[0231] time Y1 0h 0.999 6h 0.999 12h 0.997 18h 0.997 24h 0.999 average 0.9 RSD% 0.1

[0232] Table 4-9 Stability tests (retention time / min) of Monacolin-like compounds with a key daughter ion mass number m / z of 287.

[0233] time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 0h 14.39 15 21.05 25.45 34.31 42.48 46.18 50.89 59.09 66.45 6h 14.41 15.02 21.08 25.48 34.36 42.52 46.22 50.94 59.12 66.45 12h 14.41 15.03 21.09 25.49 34.36 42.52 46.22 50.94 59.12 66.46 18h 14.38 15.00 21.05 25.45 34.30 42.48 46.17 50.89 59.1 66.45 24h 14.39 15.01 21.05 25.46 34.31 42.48 46.17 50.9 59.1 66.44 average 14.40 15.01 21.06 25.47 34.33 42.50 46.19 50.91 59.11 66.45 RSD% 0 0 0 0 0 0 0 0 0 0

[0234] Table 4-10 Stability tests (peak areas) of Monacolin-like compounds with a key daughter ion mass number m / z of 287.

[0235] time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 0h 3.31E+05 8.38E+06 1.49E+07 4.85E+05 1.61E+06 6h 3.20E+05 8.12E+06 1.45E+07 4.70E+05 1.62E+06 12h 3.27E+05 7.89E+06 1.45E+07 4.51E+05 1.62E+06 18h 3.17E+05 7.96E+06 1.44E+07 4.40E+05 1.71E+06 24h 3.24E+05 7.96E+06 1.42E+07 4.42E+05 1.52E+06 average 3.24E+05 8.06E+06 1.45E+07 4.58E+05 1.62E+06 RSD% 1.6 2.4 1.8 4.2 4.1 time Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 0h 5.94E+05 4.40E+06 4.63E+06 2.60E+07 4.45E+06 6h 5.57E+05 4.49E+06 4.75E+06 2.72E+07 4.69E+06 12h 5.61E+05 4.42E+06 4.57E+06 2.67E+07 4.69E+06 18h 5.41E+05 4.60E+06 4.62E+06 2.67E+07 4.61E+06 24h 5.29E+05 4.33E+06 4.53E+06 2.59E+07 4.50E+06 average 5.56E+05 4.45E+06 4.62E+06 2.65E+07 4.59E+06 RSD% 4.4 2.2 1.7 2 2.3

[0236] (3) Repeatability test

[0237] Take six samples, prepare six test solutions according to the proposed method, and inject them for determination. The results are shown in Table 4-11 below. The results indicate that the method has good repeatability.

[0238] Table 4-11 Repeatability tests (similarity) of Monacolin-like compounds with a key ion mass number m / z of 283.

[0239] Self-numbered Peak 1 Sample 1-1 0.986 Sample 1-2 0.988 Sample 1-3 0.987 Samples 1-4 0.986 Samples 1-5 0.986 Samples 1-6 0.985 average 1.0 RSD% 0.1

[0240] Table 4-12 Repeatability tests (retention time / min) for Monacolin-like compounds with a key daughter ion mass number m / z of 283.

[0241]

[0242]

[0243] Table 4-13 Repeatability tests (peak areas) for Monacolin-like compounds with a key daughter ion mass number m / z of 283.

[0244]

[0245] Table 4-14 Repeatability tests (similarity) of Monacolin-like compounds with a key ion mass number m / z of 285.

[0246]

[0247]

[0248] Table 4-15 Repeatability tests (retention time / min) for Monacolin-like compounds with a key ion mass number m / z of 285.

[0249]

[0250] Table 4-16 Repeatability tests (peak areas) for Monacolin-like compounds with a key daughter ion mass number m / z of 285.

[0251]

[0252] Table 4-17 Repeatability tests (similarity) of Monacolin-like compounds with a key daughter ion mass number m / z of 287.

[0253] Self-numbered Peak 1 Sample 1-1 0.999 Sample 1-2 0.998 Sample 1-3 0.999 Samples 1-4 1.000 Samples 1-5 0.999 Samples 1-6 1.000 average 1.000 RSD% 0.0

[0254] Table 4-18 Repeatability tests (retention time / min) for Monacolin-like compounds with a key ion mass number m / z of 287.

[0255] Self-numbered Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Sample 1-1 14.39 15 21.04 25.45 34.32 42.5 46.19 50.91 59.11 66.46 Sample 1-2 14.4 15.01 21.07 25.48 34.33 42.5 46.19 50.91 59.11 66.46 Sample 1-3 14.4 15.01 21.07 25.48 34.3 42.51 46.2 50.92 59.12 66.46 Samples 1-4 14.36 14.97 21.02 25.44 34.31 42.51 46.19 50.9 59.11 66.46 Samples 1-5 14.4 15.01 21.07 25.48 34.35 42.51 46.21 50.92 59.11 66.46 Samples 1-6 14.4 15.01 21.06 25.47 34.34 42.52 46.22 50.93 59.12 66.46 average 14.40 15.00 21.06 25.47 34.33 42.51 46.20 50.92 59.11 66.46 RSD% 0.1 0.1 0 0 0 0 0 0 0 0

[0256] Table 4-19 Repeatability tests (peak areas) for Monacolin-like compounds with a key ion mass number m / z of 287.

[0257] Self-numbered Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Sample 1-1 3.56E+05 8.55E+06 1.63E+07 5.34E+05 1.57E+06 Sample 1-2 3.48E+05 8.02E+06 1.52E+07 4.93E+05 1.58E+06 Sample 11-3 3.48E+05 8.50E+06 1.50E+07 5.17E+05 1.58E+06 Samples 1-4 3.19E+05 8.56E+06 1.48E+07 4.90E+05 1.64E+06 Samples 1-5 3.37E+05 8.10E+06 1.49E+07 4.91E+05 1.61E+06 Samples 1-6 3.17E+05 8.45E+06 1.49E+07 5.09E+05 1.64E+06 average 3.37E+05 8.36E+06 1.52E+07 5.06E+05 1.60E+06 RSD% 4.8 2.8 3.7 3.5 1.9 Self-numbered Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Sample 1-1 5.68E+05 4.72E+06 4.88E+06 3.02E+07 6.88E+06 Sample 1-2 5.80E+05 4.64E+06 4.85E+06 2.99E+07 6.04E+06 Sample 1-3 5.69E+05 4.71E+06 4.75E+06 2.89E+07 5.95E+06 Samples 1-4 5.60E+05 4.70E+06 4.74E+06 2.84E+07 5.66E+06 Samples 1-5 5.66E+05 4.61E+06 4.71E+06 2.85E+07 5.57E+06 Samples 1-6 5.60E+05 4.48E+06 4.69E+06 2.78E+07 5.43E+06 average 5.67E+05 4.64E+06 4.77E+06 2.90E+07 5.92E+06 RSD% 1.3 1.9 1.6 3.2 8.8

[0258] (4) Sample determination

[0259] Following the proposed method, 28 batches of samples were analyzed, and the results are shown in Table 4-20 below. The similarity was calculated by comparing the fingerprint spectra of the 28 batches of samples with those of the control sample. The fingerprint spectra of monacolin-like compounds with a key ion mass number (m / z) of 283 in 25 batches of samples showed a similarity greater than 0.90; the fingerprint spectra of monacolin-like compounds with a key ion mass number (m / z) of 285 in 27 batches of samples showed a similarity greater than 0.90; and the fingerprint spectra of monacolin-like compounds with a key ion mass number (m / z) of 287 in 25 batches of samples showed a similarity greater than 0.90. This is the first comprehensive analysis of the distribution and relative content of monacolin-like compounds in red yeast rice, indicating that the types and content distribution of monacolin-like compounds in red yeast rice are consistent.

[0260] Table 4-20 Sample Similarity Measurement Results

[0261]

[0262] Example 5

[0263] In this embodiment, the traditional Chinese medicine preparations containing red yeast rice, namely, Lipitor Tablets and Xuezhikang Capsules, were tested according to the proposed method. The results showed that the chromatograms of both preparations contained the 48 chromatographic peaks specified in the method. The similarity results between these peaks and the fingerprint chromatograms generated from 28 batches of red yeast rice samples are shown in Table 5-1 below, indicating that this method can be used to test traditional Chinese medicine preparations containing red yeast rice. However, the similarity between the two preparations and the 28 batches of red yeast rice is relatively small. Therefore, the method described in this invention can be used to establish corresponding reference fingerprint chromatograms based on the distribution of monacolin compounds in different red yeast rice products, and then conduct similarity detection to comprehensively control the distribution and content of monacolin compounds in red yeast rice products.

[0264] Table 5-1 Results of similarity determination of traditional Chinese medicine preparations containing red yeast rice

[0265]

[0266]

[0267] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. A method for detecting monacolin-like compounds, characterized in that, The method includes the steps of: analyzing the sample to be tested using a liquid chromatography-mass spectrometry (LC-MS) instrument, wherein the mass spectrometry conditions of the LC-MS instrument include: acquiring C2... 19 H 23 O2 + C 19 H 25 O2 + C 19 H 27 O2 + and C 19 H 29 O2 + ion.

2. The method according to claim 1, characterized in that, The liquid chromatography-mass spectrometry (LC-MS) instrument is selected from either liquid chromatography-tandem high-resolution mass spectrometry (LC-MS) or liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS).

3. The method according to claim 2, characterized in that, The sample was analyzed using liquid chromatography-tandem high-resolution mass spectrometry (LC-HPLC-HMS). The mass spectrometry conditions for the LC-HPLC-HMS included the extraction of ion currents with molecular weights of 283.1693±20ppm, 285.1849±20ppm, 287.2006±20ppm, and 289.2162±20ppm.

4. The method according to claim 3, characterized in that, The mass spectrometry conditions for the liquid chromatography-tandem high-resolution mass spectrometry include: Ion source: electrospray; Scanning mode: Positive ion scanning mode ESI(+); Monitoring methods: Auto MS / MS or Scan; Collision gas: N2; Collision energies: 10, 20, and 40 eV.

5. The method according to claim 2, characterized in that, The sample was analyzed using liquid chromatography-tandem triple quadrupole mass spectrometry. The mass spectrometry conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry included the acquisition of ions with charge-to-mass ratios of 283.0±1, 285.0±1, 287.0±1, and 289.0±1.

6. The method according to claim 5, characterized in that, The mass spectrometry conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry include: An ion with a charge-to-mass ratio of 283.0±1 is selected as the parent ion, and at least one ion with a charge-to-mass ratio of 223.0±1, 237.0±1, or 265.0±1 is selected as the daughter ion. An ion with a charge-to-mass ratio of 285.0±1 is selected as the parent ion, and at least one ion with a charge-to-mass ratio of 225.0±1, 239.0±1, or 267.0±1 is selected as the daughter ion. An ion with a charge-to-mass ratio of 287.0±1 was selected as the parent ion, and at least one ion with a charge-to-mass ratio of 227.0±1, 241.0±1, or 269.0±1 was selected as the daughter ion; and An ion with a charge-to-mass ratio of 289.0±1 is selected as the parent ion, and at least one ion with a charge-to-mass ratio of 229.0±1, 243.0±1, or 271.0±1 is selected as the daughter ion. Preferably, an ion with a charge-to-mass ratio of 283.0±1 is selected as the parent ion, and an ion with a charge-to-mass ratio of 223.0±1 is selected as the daughter ion; Ions with a charge-to-mass ratio of 285.0±1 were selected as the parent ion, and ions with a charge-to-mass ratio of 225.0±1 were selected as the daughter ion. An ion with a charge-to-mass ratio of 287.0 ± 1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 227.0 ± 1 was selected as the daughter ion; and An ion with a charge-to-mass ratio of 289.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 229.0±1 was selected as the daughter ion.

7. The method according to claim 6, characterized in that, The mass spectrometry conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry include: At a declustering voltage of 180V and a collision capability of 24eV, an ion with a charge-to-mass ratio of 283.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 223.0±1 was selected as the daughter ion. At a declustering voltage of 180V and a collision capability of 24eV, an ion with a charge-to-mass ratio of 285.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 225.0±1 was selected as the daughter ion. At a declustering voltage of 200V and a collisional capability of 22eV, an ion with a charge-to-mass ratio of 287.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 227.0±1 was selected as the daughter ion; and With a declustering voltage of 200V and a collision capability of 24eV, an ion with a charge-to-mass ratio of 289.0±1 was selected as the parent ion, and an ion with a charge-to-mass ratio of 229.0±1 was selected as the daughter ion.

8. The method according to claim 7, characterized in that, The mass spectrometry conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry include: Ion source: Electrospray, positive ion scanning mode ESI(+); Monitoring method: MRM mode, ion source temperature 200~600℃ (preferably 450℃).

9. The method according to any one of claims 1-8, characterized in that, The chromatographic conditions of the liquid chromatography-mass spectrometry (LC-MS) instrument include: Chromatographic column: EC-C18 column; Mobile phase: The organic phase is acetonitrile, and the aqueous phase is 0.1% formic acid solution as the mobile phase; Elution method: gradient elution.

10. The method according to claim 9, characterized in that, The chromatographic conditions for the liquid chromatography-tandem triple quadrupole mass spectrometry include: The gradient elution procedure is as follows: 。

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