Method for analyzing and determining content of sipeimine, sipeimine glycoside, peimisine, irbesin and irbeine glycoside A in fritillaria pallidiflora

Abstract

The invention discloses a method for analyzing and determining the content of sipeimine, sipeimine, peimisine, irbesin and irbeine glycoside A in fritillaria pallidiflora, and relates to the technical field of medicine detection. According to the technical scheme, the method is characterized in that five main active components including sipeimine, sipeimine, peimisine, irbesin and irbeine glycoside A are quantitatively determined by adopting an ultra-high performance liquid chromatography-mass spectrometry technology, and methodological investigation and tests confirm that the method is simple, convenient and rapid, accurate in result, good in reproducibility and high in sensitivity. The method is an effective means for determining the content of main active components in the fritillaria pallidiflora.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and more specifically, to a method for analyzing and determining the contents of xibei pine, xibei glycoside, beimu xin, ibaexin and ibaexin glycoside A in ibaeculata. Background Art

[0002] Fritillaria walujewii Regel is the dried bulb of Fritillaria walujewii Regel or Fritillaria pallidiflora Schrenk, a plant of the Liliaceae family. It is mainly distributed in Xinjiang and has the effects of clearing heat and moistening the lungs, resolving phlegm and relieving cough, and dispersing nodules. Chemical and modern pharmacological studies have shown that the main active ingredients of Fritillaria walujewii are alkaloids, among which isosteroidal alkaloids, such as fritillary alkaloids, fritillary glycosides, fritillary cin, fritillary cin and fritillary glycoside A, are the main active alkaloids, which have important pharmacological effects on relieving cough and resolving phlegm.

[0003] Due to the weak ultraviolet absorption of Fritillaria steroidal alkaloids, the current methods for determining the content of steroidal alkaloids in Ilex pilosa mainly include capillary electrophoresis, pre-column derivatization gas chromatography, and liquid evaporative light scattering method. However, these methods all have many shortcomings, such as the capillary electrophoresis method has defects such as poor reproducibility, narrow linear range and low sensitivity; the pre-column derivatization gas chromatography method has many pre-treatment steps and difficulties in eliminating interference; the sensitivity of the liquid evaporative light scattering detector method is low, resulting in the inability to detect samples with low content of active ingredients. Therefore, how to establish a method for determining the content of alkaloid components in Ilex pilosa, including xibei alkaloid, xibei alkaloid glycoside, bei xin, yibei xin and yibei glycoside A, with good accuracy, high sensitivity, and convenience, so as to achieve the quality control of Ilex pilosa medicinal materials and the development of new drugs, is a technical problem that technicians in the field urgently need to solve. Summary of the invention

[0004] The invention aims to provide a method for analyzing and determining the contents of xibei pine, xibei glycoside, beimu xin, ibaexin and ibaexin glycoside A in ibaeculata.

[0005] The above technical purpose of the present invention is achieved through the following technical scheme: a method for analyzing and determining the content of xibei pine, xibei glycoside, beimu xin, yibei xin and yibei glycoside A in yibei bupleurum, comprising the following steps:

[0006] S1. Preparation of reference solution;

[0007] S2. Preparation of sample solution;

[0008] S3. Drawing of standard curve;

[0009] S4. Content determination.

[0010] The present invention is further configured as follows: the preparation process of the reference solution in S1 is as follows: accurately weigh the reference substances of xibei pine, xibei pine glycoside, beimucine, ibecine and ibecine glycoside A in centrifuge tubes respectively, add 1 mL of methanol to dissolve, and prepare each into a standard mother solution with a concentration of 10 mM.

[0011] The present invention is further configured as follows: the preparation process of the sample solution in S2 is as follows:

[0012] (1) Weigh 200 g of dried Fritillaria cirrhosa slices and place them in a 3 L electric temperature-controlled ceramic automatic Chinese medicine cooker, add water and boil twice;

[0013] a. Add 7 times the amount of water for the first decoction, soak for 1 hour, heat and decoct for 1 hour, filter with double-layer filter cloth while hot, and filter until no obvious filtrate seeps out;

[0014] b. Add 6 times the amount of water for the second time, heat and boil for 50 minutes, filter with double-layer filter cloth while hot, and filter until no obvious filtrate seeps out;

[0015] (2) The two filtrates were combined, transferred to a round-bottom flask, and concentrated under reduced pressure at 60° C. The resulting concentrate was centrifuged at 7000 r / min and 4° C. for 20 min, and freeze-dried to obtain freeze-dried powder of water decoction of Fritillaria cirrhosa;

[0016] (3) The freeze-dried powder of the water decoction of Fritillaria cirrhosa was re-dissolved in a mixed solution of methanol and water in a volume ratio of 1:1, and filtered through a 0.22 μm organic phase filter membrane into a 2 mL sample bottle to obtain a sample solution for detection on an instrument.

[0017] The present invention is further configured as follows: the drawing process of the standard curve in S3 is as follows: respectively take the standard mother solutions of xibei pine, xibei glycoside, beimucine, ibecine and ibecine glycoside A, dilute them with methanol: water (1:1) to obtain a series of mixed standard working solutions with concentrations of 1, 10, 25, 50, 100, 250, 500, and 1000 nM, respectively, and perform ultra-high performance liquid chromatography-mass spectrometry analysis according to chromatographic conditions; perform regression analysis on peak area using concentration to obtain standard curves of each component, and calculate the regression equation.

[0018] The present invention is further configured as follows: the specific steps of content determination in S4 are as follows: accurately aspirate the sample test liquid, perform ultra-high performance liquid chromatography-mass spectrometry analysis according to chromatographic conditions, and bring the peak area into the standard curve to calculate the contents of xibei pine, xibei glycoside, beimucine, ibecine and ibecine glycoside A respectively.

[0019] The present invention is further configured as follows: the chromatographic column used in the ultra-high performance liquid chromatography in the ultra-high performance liquid chromatography-mass spectrometry analysis is Waters ACQUITY UPLC BEH C18 (100×2.1mm, 1.7μm); the volume percentage of the mobile phase is A-0.1% formic acid aqueous solution, B-acetonitrile, and the flow rate is 0.3mL / min; the column temperature is 40°C; and the injection volume is 1μL.

[0020] The present invention is further configured as follows: the mass spectrometry ion source in the ultra-high performance liquid chromatography-mass spectrometry analysis is an electrospray ion source (ESI); the scanning mode is positive ion scanning; the detection mode is multiple reaction monitoring (MRM); the capillary voltage is 2.8 kV; the ion source temperature is 150°C; the desolvation gas (N2) temperature is 500°C; the desolvation gas flow rate is 1000 L / h; the cone gas flow rate is 50 L / h; and the collision gas (Ar) flow rate is 2.5 L / h.

[0021] In summary, the present invention has the following beneficial effects: the method provided by the present invention for analyzing and determining the contents of xibei pine, xibei glycoside, bei xin, ibei xin and ibei glycoside A in Ilex fusca has good reproducibility, high accuracy, high sensitivity, few pretreatment steps, and convenient operation. It can determine the components of alkaloids in Ilex fusca, and provide a technical basis for the quality control of Ilex fusca medicinal materials and the research and development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the MRM chromatogram in the embodiment of the present invention;

[0023] Figure 2 : It is a standard curve diagram in the embodiment of the present invention, wherein (A) is a standard curve diagram of xibei pine; (B) is a standard curve diagram of xibei pine glycoside; (C) is a standard curve diagram of beimucine; (D) is a standard curve diagram of ibeline glycoside A; (E) is a standard curve diagram of ibecin. DETAILED DESCRIPTION

[0024] The following is combined with Figure 1-2 The present invention is described in further detail.

[0025] Embodiment: A method for analyzing and determining the contents of xibei pine, xibei glycoside, bei xin, yibei xin and yibei glycoside A in yibei bu Figure 1 , Figure 2 As shown, the following steps are included:

[0026] S1. Preparation of reference solution: Accurately weigh the reference substances of xibeiline, xibeiline glycoside, beimucine, ibecine and ibecine glycoside A into centrifuge tubes, add 1 mL of methanol to dissolve, and prepare each into a standard stock solution with a concentration of 10 mM.

[0027] S2. Preparation of sample solution:

[0028] (1) Weigh 200 g of dried Fritillaria cirrhosa slices and place them in a 3 L electric temperature-controlled ceramic automatic Chinese medicine cooker, add water and boil twice;

[0029] a. Add 7 times the amount of water for the first decoction, soak for 1 hour, heat and decoct for 1 hour, filter with double-layer filter cloth while hot, and filter until no obvious filtrate seeps out;

[0030] b. Add 6 times the amount of water for the second time, heat and boil for 50 minutes, filter with double-layer filter cloth while hot, and filter until no obvious filtrate seeps out;

[0031] (2) The two filtrates were combined, transferred to a round-bottom flask, and concentrated under reduced pressure at 60° C. The resulting concentrate was centrifuged at 7000 r / min and 4° C. for 20 min, and freeze-dried to obtain freeze-dried powder of water decoction of Fritillaria cirrhosa;

[0032] (3) The freeze-dried powder of the water decoction of Fritillaria cirrhosa was re-dissolved in a mixed solution of methanol and water in a volume ratio of 1:1, and filtered through a 0.22 μm organic phase filter membrane into a 2 mL sample bottle to obtain a sample solution for detection on an instrument.

[0033] S3. Preparation of standard curve: respectively take the standard mother solutions of xibeiline, xibeiline glycoside, beimucine, ibecine and ibecine glycoside A, dilute them with methanol: water (1:1) to obtain a series of mixed standard working solutions with concentrations of 1, 10, 25, 50, 100, 250, 500 and 1000 nM, respectively, and perform ultra-performance liquid chromatography-mass spectrometry analysis according to chromatographic conditions; perform regression analysis on peak area using concentration to obtain the standard curve of each component, and calculate the regression equation.

[0034] S4. Content determination: Accurately aspirate the sample solution, perform ultra-high performance liquid chromatography-mass spectrometry analysis according to the chromatographic conditions, and bring the peak area into the standard curve to calculate the contents of xibeiline, xibeiline glycoside, beimucine, ibecine and ibecine glycoside A, respectively.

[0035] The chromatographic column used for ultra-high performance liquid chromatography was Waters ACQUITY UPLC BEH C18 (100×2.1 mm, 1.7 μm); the volume percentage of the mobile phase was A-0.1% formic acid aqueous solution, B-acetonitrile, the mobile phase elution gradient was shown in Table 1, the flow rate was 0.3 mL / min; the column temperature was 40° C.; and the injection volume was 1 μL.

[0036] Table 1 Mobile phase elution gradient

[0037]

[0038] The mass spectrometer ion source is an electrospray ion source (ESI); the scanning mode is positive ion scanning; the detection mode is multiple reaction monitoring (MRM); the capillary voltage is 2.8 kV; the ion source temperature is 150 ° C; the desolvation gas (N2) temperature is 500 ° C; the desolvation gas flow rate is 1000 L / h; the cone gas flow rate is 50 L / h; the collision gas (Ar) flow rate is 2.5 L / h. MRM ion pairs and conditions are shown in Table 2.

[0039] Table 2 MRM ion pairs and conditions

[0040]

[0041] It was found through the above steps that the injection volume and peak area of ​​xibeimu alkaloids, xibeimu glycosides, fritillary heart, ibetic acid and ibetic acid glycoside A showed a good linear relationship in the concentration range of 1-1000nM. Under the chromatographic conditions of the method, the retention times of xibeimu alkaloids, xibeimu glycosides, fritillary heart, ibetic acid and ibetic acid glycoside A were 3.72min, 3.26min, 3.87min, 3.23min and 4.31min, respectively. According to the peak area and the standard curve, the contents of xibeimu alkaloids, xibeimu glycosides, fritillary heart, ibetic acid and ibetic acid glycoside A in the water decoction of fritillary heart were calculated to be 77.39μg / g, 84.63μg / g, 20.37μg / g, 2.59μg / g and 54.84μg / g, respectively.

[0042] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for analyzing and determining the contents of xibei pine, xibei glycoside, beimu xin, yibei xin and yibei glycoside A in yibei bupleurum, characterized in that: The following steps are involved: S1. Preparation of reference solution; S2. Preparation of sample solution; S3. Drawing of standard curve; S4. Content determination.

2. The method for analyzing and determining the contents of xibei pine, xibei glycoside, beimu xin, yibei xin and yibei glycoside A in yibei bupleurum according to claim 1, characterized in that: The preparation process of the reference solution in S1 is as follows: accurately weigh the reference substances of xibeiline, xibeiline glycoside, beimucine, ibecine and ibecine glycoside A into centrifuge tubes respectively, add 1 mL of methanol to dissolve, and prepare each into a standard stock solution with a concentration of 10 mM.

3. The method for analyzing and determining the contents of xibei pine, xibei glycoside, beimu xin, yibei xin and yibei glycoside A in yibei bupleurum according to claim 1, characterized in that: The preparation process of the sample solution in S2 is as follows: (1) Weigh 200 g of dried Fritillaria cirrhosa slices and place them in a 3 L electric temperature-controlled ceramic automatic Chinese medicine cooker, add water and boil twice; a. Add 7 times the amount of water for the first decoction, soak for 1 hour, heat and decoct for 1 hour, filter with double-layer filter cloth while hot, and filter until no obvious filtrate seeps out; b. Add 6 times the amount of water for the second time, heat and boil for 50 minutes, filter with double-layer filter cloth while hot, and filter until no obvious filtrate seeps out; (2) The two filtrates were combined, transferred to a round-bottom flask, and concentrated under reduced pressure at 60° C. The resulting concentrate was centrifuged at 7000 r / min and 4° C. for 20 min, and freeze-dried to obtain freeze-dried powder of water decoction of Fritillaria cirrhosa; (3) The freeze-dried powder of the water decoction of Fritillaria cirrhosa was re-dissolved in a mixed solution of methanol and water in a volume ratio of 1:1, and filtered through a 0.22 μm organic phase filter membrane into a 2 mL sample bottle to obtain a sample solution for detection on an instrument.

4. The method for analyzing and determining the contents of xibei pine, xibei glycoside, beimu xin, yibei xin and yibei glycoside A in yibei bupleurum according to claim 1, characterized in that: The process of drawing the standard curve in S3 is as follows: respectively take the standard mother solutions of xibei pine, xibei glycoside, beimucine, ibecine and ibecine glycoside A, dilute them with methanol: water (1:1) to obtain a series of mixed standard working solutions with concentrations of 1, 10, 25, 50, 100, 250, 500, and 1000 nM, respectively, and perform ultra-high performance liquid chromatography-mass spectrometry analysis according to chromatographic conditions; perform regression analysis on peak area using concentration to obtain standard curves of each component, and calculate the regression equation.

5. The method for analyzing and determining the contents of xibei pine, xibei glycoside, beimu xin, yibei xin and yibei glycoside A in yibei bupleurum according to claim 1, characterized in that: The specific steps of content determination in S4 are as follows: accurately aspirate the sample solution to be tested, perform ultra-high performance liquid chromatography-mass spectrometry analysis according to chromatographic conditions, and bring the peak area into the standard curve to calculate the contents of xibei pine, xibei glycoside, beimucine, ibecine and ibecine glycoside A respectively.

6. A method for analyzing and determining the contents of xibei pine, xibei glycoside, beimu xin, yibei xin and yibei glycoside A in yibei bupleurum according to claim 5, characterized in that: The chromatographic column used in the ultra-high performance liquid chromatography-mass spectrometry analysis is Waters ACQUITY UPLC BEH C18 (100×2.1 mm, 1.7 μm); the volume percentage of the mobile phase is A-0.1% formic acid aqueous solution, B-acetonitrile, the flow rate is 0.3 mL / min; the column temperature is 40° C.; and the injection volume is 1 μL.

7. The method for analyzing and determining the contents of xibei pine, xibei glycoside, beimu xin, yibei xin and yibei glycoside A in yibei bupleurum according to claim 5, characterized in that: The mass spectrometry ion source in the ultra-high performance liquid chromatography-mass spectrometry analysis is an electrospray ion source (ESI); the scanning mode is positive ion scanning; the detection mode is multiple reaction monitoring (MRM); the capillary voltage is 2.8 kV; the ion source temperature is 150° C.; the desolvation gas (N2) temperature is 500° C.; the desolvation gas flow rate is 1000 L / h; and the cone gas flow rate is 50 L / h. The collision gas (Ar) flow rate was 2.5 L / h.

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