A method for constructing a characteristic spectrum of polygonatum and / or wine polygonatum

Abstract

The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a construction method of a characteristic spectrum of rhizoma polygonati and / or jiu rhizoma polygonati. The construction method comprises preparation of a test sample solution, and detection by high performance liquid chromatography; methanol is used as a mobile phase A, water is used as a mobile phase B, and specific gradient elution is adopted. The construction method can provide a quality control basis for rhizoma polygonati, jiu rhizoma polygonati and water-soluble related preparations thereof, and can also explain the component change in the process of processing rhizoma polygonati into jiu rhizoma polygonati. The method can identify the effective components in rhizoma polygonati and / or jiu rhizoma polygonati, and improves the accuracy of detection. The characteristic spectrum of rhizoma polygonati and / or jiu rhizoma polygonati obtained by the method has high accuracy, good separation degree between characteristic peaks and good repeatability, and is suitable for medicinal materials from different producing areas.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a construction method of a characteristic spectrum of Polygonatum sibiricum Red. BACKGROUND

[0002] Polygonatum sibiricum Red. or Polygonatum cyrtonema Hua. The characteristic spectrum established in the prior art has multiple characteristic peaks, but the effective components of the characteristic peaks are not identified, and the Polygonatum sibiricum Red. cannot be controlled in quality. The Polygonatum sibiricum Red. is a processed product of Polygonatum sibiricum Red., and the prior art cannot control the quality of the Polygonatum sibiricum Red. at the same time. SUMMARY

[0003] Therefore, the technical problem to be solved by the application is to overcome the defects that the effective components of Polygonatum sibiricum Red. cannot be identified, and the quality of Polygonatum sibiricum Red. and the Polygonatum sibiricum Red. cannot be accurately controlled in the prior art, so as to provide a construction method of a characteristic spectrum of Polygonatum sibiricum Red.

[0004] To this end, the application provides the following technical scheme.

[0005] The application provides a construction method of a characteristic spectrum of Polygonatum sibiricum Red., comprising the following steps:

[0006] Preparation of a test sample solution: the test sample is prepared into a test sample solution;

[0007] Determination: high performance liquid chromatography is used for detection; methanol is used as a mobile phase A, and water is used as a mobile phase B for gradient elution; the gradient elution program comprises 0-10 min, a volume percentage of the mobile phase A is 0%, and a volume percentage of the mobile phase B is 100%; 10-20 min, the volume percentage of the mobile phase A is 0% to 2%, and the volume percentage of the mobile phase B is 100% to 98%; 20-40 min, the volume percentage of the mobile phase A is 2% to 10%, and the volume percentage of the mobile phase B is 98% to 90%; 40-55 min, the volume percentage of the mobile phase A is 10% to 15%, and the volume percentage of the mobile phase B is 90% to 85%; and 55-65 min, the volume percentage of the mobile phase A is 15%, and the volume percentage of the mobile phase B is 85%.

[0008] The construction method further comprises the following chromatographic conditions: the column temperature is 23-27 DEG C; and / or, the flow rate is 0.9-1.1 ml / min; and / or, octadecylsilane bonded silica gel is used as a filler; and / or, the detection wavelength is 255-285 nm; and / or, the injection amount is 5-20 muL.

[0009] The construction method, the chromatographic conditions further comprise: the column temperature is 25 DEG C; and / or, the flow rate is 1.0ml / min; and / or, octadecylsilane is bonded to silica gel as the filler; and / or, the detection wavelength is 270nm; and / or, the injection amount is 10 muL; and / or, the specification of the chromatographic column is: the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m; and / or, the theoretical plate number calculated according to the adenosine peak should be not less than 5000.

[0010] The characteristic spectrum of the Rhizoma Polygonati includes 10 characteristic peaks, which are peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9 and peak 10;

[0011] Preferably, taking peak 1 as the reference peak 1, the relative retention times of peak 2, peak 3, peak 4, peak 5 and peak 6 are 1.22, 1.42, 1.66, 1.84 and 1.91 respectively;

[0012] Taking peak 9 as the reference peak 2, the relative retention times of peak 7, peak 8 and peak 10 are 0.81, 0.84 and 1.15 respectively;

[0013] The relative retention time is within ±10% of the specified value.

[0014] The characteristic spectrum of the Rhizoma Polygonati includes 10 characteristic peaks, peak 1 is uridine, peak 4 is adenine, peak 5 is 5-hydroxymethylfurfural, peak 6 is guanosine, peak 7 is thymidine, peak 8 is tryptophan, peak 9 is adenosine, and peak 10 is rhizomar A.

[0015] The characteristic spectrum of the Rhizoma Polygonati includes 9 characteristic peaks, which are peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8 and peak 9;

[0016] Preferably, taking peak 1 as the reference peak 1, the relative retention times of peak 2, peak 3, peak 4 and peak 5 are 1.22, 1.43, 1.78 and 1.85 respectively;

[0017] Taking peak 8 as the reference peak 2, the relative retention times of peak 6, peak 7 and peak 9 are 0.81, 0.93 and 1.14 respectively;

[0018] The relative retention time is within ±10% of the specified value.

[0019] The characteristic spectrum of the Rhizoma Polygonati includes 9 characteristic peaks, peak 1 is uridine, peak 5 is 5-hydroxymethylfurfural, peak 6 is 5-(hydroxymethyl)-1H-pyrrol- furfural, peak 8 is adenosine, and peak 9 is rhizomar A.

[0020] The dosage form of the test product is a standard decoction lyophilized powder, a formula granule, a decoction piece or a medicinal material. Specifically, the test product can be a standard decoction lyophilized powder of Huangjing, a formula granule of Huangjing, a decoction piece of Huangjing, a medicinal material of Huangjing, a standard decoction lyophilized powder of Jiu Huangjing, a formula granule of Jiu Huangjing, a decoction piece of Jiu Huangjing or a medicinal material of Jiu Huangjing.

[0021] The extraction solvent includes at least one of ethanol, water and methanol when preparing the test product solution.

[0022] Preferably, the extraction solvent is 5%-30% ethanol.

[0023] Preferably, the ratio of the mass (g) of the test product to the volume (ml) of the extraction solvent is (0.05-0.5):1.

[0024] The control product is uridine and / or adenosine.

[0025] Preferably, 1 ml of the control product solution contains 5-50 μg of at least one of uridine control product and 5-50 μg of adenosine control product.

[0026] The technical scheme of the present application has the following advantages:

[0027] 1. The present application provides a method for constructing a characteristic chromatogram of Huangjing and / or Jiu Huangjing, which comprises preparing a test product solution and detecting by high performance liquid chromatography; methanol is used as mobile phase A and water is used as mobile phase B for gradient elution, and the gradient elution program comprises 0-10 min, 0% of the volume percentage of mobile phase A and 100% of the volume percentage of mobile phase B; 10-20 min, 0%→2% of the volume percentage of mobile phase A and 100%→98% of the volume percentage of mobile phase B; 20-40 min, 2%→10% of the volume percentage of mobile phase A and 98%→90% of the volume percentage of mobile phase B; 40-55 min, 10%→15% of the volume percentage of mobile phase A and 90%→85% of the volume percentage of mobile phase B; 55-65 min, 15% of the volume percentage of mobile phase A and 85% of the volume percentage of mobile phase B. The method can provide quality control basis for Huangjing, Jiu Huangjing and their water-soluble related preparations, and can also explain the component changes in the process of processing Huangjing medicinal material into Jiu Huangjing, the method can identify the effective components in Huangjing and / or Jiu Huangjing, and improves the accuracy of detection. The characteristic chromatogram of Huangjing and / or Jiu Huangjing obtained by the method has high accuracy, good separation degree between characteristic peaks and good repeatability, and is suitable for medicinal materials from different producing areas. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the accompanying drawings required to be used in the specific embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the premise of the accompanying drawings.

[0029] Figure 1 is the control chromatogram of Rhizoma Polygonati in experimental example 1; Figure 2 is the chromatogram of 18 batches of standard decoction pieces of Rhizoma Polygonati in experimental example 1; Figure 3 is the chromatogram of 18 batches of Rhizoma Polygonati in experimental example 1; Figure 4 is the chromatogram of mobile phase investigation in section 2.1.1 of experimental example 2; Figure 5 is the chromatogram of column investigation in section 2.1.2 of experimental example 2; Figure 6 is the chromatogram of different gradient elution program investigation in section 2.1.4 of experimental example 2; Figure 7 is the chromatogram of blank solvent in section 2.2.1 of experimental example 2; Figure 8 is the chromatogram of different flow rate in section 2.2.6 of experimental example 2; Figure 9 is the chromatogram of different column temperature in section 2.2.7 of experimental example 2; Figure 10 is the chromatogram of different instruments in section 2.2.8 of experimental example 2; Figure 11 is the control chromatogram of Rhizoma Polygonati Glycyrhizae in experimental example 3; Figure 12 is the chromatogram of 18 batches of standard decoction pieces of Rhizoma Polygonati Glycyrhizae in experimental example 3; Figure 13 is the chromatogram of 18 batches of Rhizoma Polygonati Glycyrhizae in experimental example 3; Figure 14 is the chromatogram of blank solvent in section 4.1 of experimental example 4; Figure 15 is the chromatogram of different flow rate in section 4.6 of experimental example 4; Figure 16 is the chromatogram of different column temperature in section 4.7 of experimental example 4; Figure 17 is the chromatogram of different instruments in section 4.8 of experimental example 4. DETAILED DESCRIPTION

[0030] The following examples are provided to better further understand the present application, and do not limit the content and protection scope of the present application, and do not constitute a limitation, and any person under the inspiration of the present application or the combination of the present application with other prior art features can obtain any product same or similar to the present application, which falls within the protection scope of the present application.

[0031] The specific experimental steps or conditions not mentioned in the examples can be performed according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments not mentioned by the manufacturer are conventional reagent products that can be obtained by market purchase.

[0032] Instruments: one ten thousandth balance ME104 Mettler-Toledo International Trade (Shanghai) Co., Ltd.; one hundred thousandth balance XPR2 Mettler-Toledo International Trade (Shanghai) Co., Ltd.; digital ultrasonic instrument KQ-500DE Kunshan Ultrasonic Instrument Co., Ltd.; high performance liquid chromatograph Acquity Arc Waters Corporation; high performance liquid chromatograph 1260 Infinity II Agilent Corporation; high performance liquid chromatograph Thermo U3000 Thermo Fisher Corporation; chromatographic column (2.5x250mm, 5μm) Shim pack GIST C18-AQ Shimadzu Corporation; chromatographic column YMC-Pack ODS-AQ (2.5x250mm, 5μm) YMC Corporation; chromatographic column (2.5x250mm, 5μm) Agilent ZORBAX SB-Aq Agilent Corporation.

[0033] Reagents: uridine 99.6% 110887-202104 China Institute for Drug Control and Biological Products; adenosine 99.4% 110807-202204 China Institute for Drug Control and Biological Products; ethanol 99.7% 20230525 Guangdong Guanghua Science and Technology Co., Ltd.; methanol analytical purity 20221201-11 Guangzhou Chemical Reagent Factory; methanol chromatographic purity 20230601 Fisher; acetonitrile chromatographic purity F22MB1201 Fisher.

[0034] 18 batches of standard decoction of Rhizoma Polygonati fummed powder, batch numbers are 001Y~018Y; 18 batches of standard decoction of Rhizoma Polygonati Jiaotang fummed powder, batch numbers are J001Y~J018Y; 18 batches of Rhizoma Polygonati medicinal materials, batch numbers are YC001~YC018. 18 batches of Rhizoma Polygonati Jiaotang medicinal materials, batch numbers are YP001~YP018.

[0035] Example 1

[0036] The present embodiment provides a method for constructing a characteristic map of Rhizoma Polygonati fummed powder, comprising the following steps:

[0037] Preparation of test sample solution: take about 2g of Rhizoma Polygonati fummed powder, put it in a conical flask with a plug, add 10% ethanol 10ml, ultrasonic treatment (power 250W, frequency 40kHz) for 30min, shake well, filter, take the filtrate, and obtain the test sample solution.

[0038] Preparation of reference material of control medicinal material: take 4g of Rhizoma Polygonati control medicinal material, put it in a conical flask with a plug, add water 50ml, heat reflux for 1h, filter, evaporate the filtrate to dryness, cool, add 10% ethanol 10ml to the residue, ultrasonic treatment (power 250W, frequency 40kHz) for 30min, shake well, filter, take the filtrate, and obtain the reference material solution of control medicinal material.

[0039] Preparation of the control solution: Take the uridine control and the adenosine control, add 10% ethanol to prepare a mixed solution containing 20 μg of uridine and 20 μg of adenosine per 1 ml, as the control solution.

[0040] Determination: 10 μl of each was taken and injected into the liquid chromatograph for determination. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel as the filler (specification: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), methanol as the mobile phase A, water as the mobile phase B, gradient elution, gradient elution program: 0-10 min, volume percentage of mobile phase A 0%, volume percentage of mobile phase B 100%; 10-20 min, volume percentage of mobile phase A 0%→2%, volume percentage of mobile phase B 100%→98%; 20-40 min, volume percentage of mobile phase A 2%→10%, volume percentage of mobile phase B 98%→90%; 40-55 min, volume percentage of mobile phase A 10%→15%, volume percentage of mobile phase B 90%→85%; 55-65 min, volume percentage of mobile phase A 15%, volume percentage of mobile phase B 85%; flow rate 1.0 ml / min; column temperature 25°C; detection wavelength 270 nm; the theoretical plate number calculated according to the adenosine peak should not be less than 5000.

[0041] Example 2

[0042] The present embodiment provides a method for constructing a characteristic spectrum of polygonatum sibiricum, comprising the following steps:

[0043] Preparation of the test solution: Take polygonatum sibiricum, grind and pass through a No. 2 sieve, take about 4 g, put into a conical flask with a plug, add water 50 ml, heat and reflux for 1 h, filter, evaporate the filtrate to dryness, cool, add 10% ethanol 10 ml to the residue, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 min, shake well, filter, take the filtrate to obtain the test solution.

[0044] Preparation of the control drug reference: Take 4 g of the polygonatum sibiricum control drug, put into a conical flask with a plug, add water 50 ml, heat and reflux for 1 h, filter, evaporate the filtrate to dryness, cool, add 10% ethanol 10 ml to the residue, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 min, shake well, filter, take the filtrate to obtain the control drug reference solution.

[0045] Preparation of the control solution: Take the uridine control and the adenosine control, add 10% ethanol to prepare a mixed solution containing 20 μg of uridine and 20 μg of adenosine per 1 ml, as the control solution.

[0046] Determination: 10 μl of each was injected into a liquid chromatograph for determination. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel as the filler (specification: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm), methanol as mobile phase A, water as mobile phase B, gradient elution, gradient elution program: 0-10 min, volume percentage of mobile phase A 0%, volume percentage of mobile phase B 100%; 10-20 min, volume percentage of mobile phase A 0%→2%, volume percentage of mobile phase B 100%→98%; 20-40 min, volume percentage of mobile phase A 2%→10%, volume percentage of mobile phase B 98%→90%; 40-55 min, volume percentage of mobile phase A 10%→15%, volume percentage of mobile phase B 90%→85%; 55-65 min, volume percentage of mobile phase A 15%, volume percentage of mobile phase B 85%; flow rate 1.0 ml / min; column temperature 25°C; detection wavelength 270 nm; and the theoretical plate number should not be less than 5000 based on the adenosine peak.

[0047] Example 3

[0048] The present example provides a method for constructing a characteristic chromatogram of wine polygonatum freeze-dried powder, comprising the following steps:

[0049] Preparation of the test sample solution: about 2 g of wine polygonatum freeze-dried powder was placed in a conical flask with a stopper, 10% ethanol 10 ml was added, and ultrasonic treatment (power 250 W, frequency 40 kHz) was performed for 30 min, followed by shaking, filtration, and collection of the filtrate to obtain the test sample solution.

[0050] Preparation of the reference material of the control medicinal material: 4 g of wine polygonatum control decoction pieces was placed in a conical flask with a stopper, water 50 ml was added, and heating reflux was performed for 1 h, followed by filtration, evaporation of the filtrate, cooling, addition of 10% ethanol 10 ml to the residue, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 min, shaking, filtration, and collection of the filtrate to obtain the reference material solution of the control medicinal material.

[0051] Preparation of the control solution: an appropriate amount of uridine control and adenosine control was added to 10% ethanol to prepare a mixed solution containing 20 μg of uridine and 20 μg of adenosine per 1 ml, which was used as the control solution.

[0052] Determination: 10 μl of each was injected into a liquid chromatograph for determination. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel as the filler (specification: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm), methanol as mobile phase A, water as mobile phase B, gradient elution, gradient elution program: 0-10 min, volume percentage of mobile phase A 0%, volume percentage of mobile phase B 100%; 10-20 min, volume percentage of mobile phase A 0%→2%, volume percentage of mobile phase B 100%→98%; 20-40 min, volume percentage of mobile phase A 2%→10%, volume percentage of mobile phase B 98%→90%; 40-55 min, volume percentage of mobile phase A 10%→15%, volume percentage of mobile phase B 90%→85%; 55-65 min, volume percentage of mobile phase A 15%, volume percentage of mobile phase B 85%; flow rate 1.0 ml / min; column temperature 25°C; detection wavelength 270 nm; and the theoretical plate number should not be less than 5000 based on the adenosine peak.

[0053] Example 4

[0054] The present example provides a method for constructing a characteristic chromatogram of wine rhizoma polygonati slices, comprising the following steps:

[0055] Preparation of the test sample solution: wine rhizoma polygonati slices were ground and passed through a No. 2 sieve, about 4 g of which was placed in a conical flask with a stopper, 50 ml of water was added, and heating reflux was carried out for 1 h, then filtration was carried out, the filtrate was evaporated to dryness, and after cooling, 10 ml of 10% ethanol was added to the residue, ultrasonic treatment (power 250 W, frequency 40 kHz) was carried out for 30 min, shaking was carried out, filtration was carried out, and the filtrate was collected to obtain the test sample solution.

[0056] Preparation of the reference material of the control medicinal material: 4 g of wine rhizoma polygonati control slices was placed in a conical flask with a stopper, 50 ml of water was added, and heating reflux was carried out for 1 h, then filtration was carried out, the filtrate was evaporated to dryness, and after cooling, 10 ml of 10% ethanol was added to the residue, ultrasonic treatment (power 250 W, frequency 40 kHz) was carried out for 30 min, shaking was carried out, filtration was carried out, and the filtrate was collected to obtain the reference material solution of the control medicinal material.

[0057] Preparation of the control solution: an appropriate amount of uridine control and adenosine control was added to 10% ethanol to prepare a mixed solution containing 20 μg of uridine and 20 μg of adenosine per 1 ml, which was used as the control solution.

[0058] Determination: each 10 μl was injected into liquid chromatograph for determination. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel as the filler (specification: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), methanol as mobile phase A, water as mobile phase B, gradient elution, gradient elution procedure: 0-10 min, volume percentage of mobile phase A 0%, volume percentage of mobile phase B 100%; 10-20 min, volume percentage of mobile phase A 0%→2%, volume percentage of mobile phase B 100%→98%; 20-40 min, volume percentage of mobile phase A 2%→10%, volume percentage of mobile phase B 98%→90%; 40-55 min, volume percentage of mobile phase A 10%→15%, volume percentage of mobile phase B 90%→85%; 55-65 min, volume percentage of mobile phase A 15%, volume percentage of mobile phase B 85%; flow rate 1.0 ml / min; column temperature 25°C; detection wavelength 270 nm; the theoretical plate number should not be less than 5000 according to the adenosine peak.

[0059] Establishment of the characteristic spectrum of Polygonatum sibiricum

[0060] Preparation of the test solution: about 2 g of the freeze-dried powder of Polygonatum sibiricum was taken into a conical flask with a plug, 10% ethanol 10 ml was added, and ultrasonic treatment (power 250 W, frequency 40 kHz) was carried out for 30 min, followed by shaking and filtration. The filtrate was taken to obtain the test solution.

[0061] Preparation of the control solution: the appropriate amount of uridine and adenosine control samples were added to 10% ethanol to prepare a mixed solution containing 20 μg of uridine and 20 μg of adenosine per 1 ml, which was used as the control solution 1.

[0062] The appropriate amount of adenine, 5-hydroxymethylfurfural and tryptophan control samples were added to 10% ethanol to prepare a mixed solution containing 50 μg of adenine, 50 μg of 5-hydroxymethylfurfural and 50 μg of tryptophan per 1 ml, which was used as the control solution 2.

[0063] The appropriate amount of thymidine and Polygonatum sibiricum alkaloid A control samples were added to 10% ethanol to prepare a mixed solution containing 50 μg of thymidine and 15 μg of Polygonatum sibiricum alkaloid A per 1 ml, which was used as the control solution 3.

[0064] Determination: 10 μl of each was taken and injected into a liquid chromatograph for determination. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel as the filler (specifications: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm), methanol as mobile phase A, water as mobile phase B, gradient elution, gradient elution program: 0-10 min, volume percentage of mobile phase A 0%, volume percentage of mobile phase B 100%; 10-20 min, volume percentage of mobile phase A 0%→2%, volume percentage of mobile phase B 100%→98%; 20-40 min, volume percentage of mobile phase A 2%→10%, volume percentage of mobile phase B 98%→90%; 40-55 min, volume percentage of mobile phase A 10%→15%, volume percentage of mobile phase B 90%→85%; 55-65 min, volume percentage of mobile phase A 15%, volume percentage of mobile phase B 85%; flow rate 1.0 ml / min; column temperature 25°C; detection wavelength 270 nm; and the theoretical plate number calculated according to the adenosine peak should not be less than 5000.

[0065] The characteristic spectrum of Huangjing includes 10 characteristic peaks, namely, Peak No. 1, Peak No. 2, Peak No. 3, Peak No. 4, Peak No. 5, Peak No. 6, Peak No. 7, Peak No. 8, Peak No. 9 and Peak No. 10. Peak No. 1 was used as the reference peak 1, and the relative retention times of Peak No. 2, Peak No. 3, Peak No. 4, Peak No. 5 and Peak No. 6 were 1.22, 1.42, 1.66, 1.84 and 1.91, respectively. Peak No. 9 was used as the reference peak 2, and the relative retention times of Peak No. 7, Peak No. 8 and Peak No. 10 were 0.81, 0.84 and 1.15, respectively. The relative retention times were within ±10% of the specified values. The control spectrum of Huangjing is shown in Figure 1 .

[0066] By comparison with the control, Peak No. 1 was uridine, Peak No. 4 was adenine, Peak No. 5 was 5-hydroxymethylfurfural, Peak No. 6 was guanosine, Peak No. 8 was tryptophan, and Peak No. 9 was adenosine. Peak No. 7 was thymidine and Peak No. 10 was Huangjijin A by mass spectrometry data analysis and control positioning.

[0067] Eighteen batches of Huangjing standard decoction lyophilized powder test sample solutions were prepared according to Example 1 and determined, and the results are shown in the following table and Figure 2 , Figure 2 S1-S18 in Table 1 correspond to batch numbers 001Y-018Y, respectively.

[0068] Table 1 Relative retention times of characteristic spectra of 18 batches of Huangjing standard decoction lyophilized powder

[0069]

[0070]

[0071] Table 2. Relative peak areas of characteristic spectra of freeze-dried powder of Polygonatum sibiricum standard decoction from 18 batches.

[0072]

[0073]

[0074] The relative retention times of the characteristic peaks in the characteristic chromatograms of 18 batches of freeze-dried Polygonatum sibiricum standard decoction powder were all within ±10% of the specified values. The similarity of the characteristic chromatograms were 0.951, 0.989, 0.936, 0.934, 0.923, 0.919, 0.995, 0.999, 0.952, 0.991, 0.927, 0.907, 0.918, 0.903, 0.909, 0.912, 0.914, and 0.918, respectively.

[0075] Eighteen batches of Polygonatum sibiricum medicinal material test solutions were prepared and tested according to Example 2. The results are shown in the table below. Figure 3 . Figure 3 S1 to S18 in the chart correspond to batch numbers YC001 to YC018, respectively.

[0076] Table 3. Relative retention times of 18 batches of Polygonatum sibiricum medicinal materials as determined by chromatogram analysis.

[0077]

[0078]

[0079] Table 4. Relative peak areas of characteristic spectra of 18 batches of Polygonatum sibiricum medicinal materials

[0080]

[0081]

[0082] The relative retention times of the characteristic peaks in the characteristic chromatograms of 18 batches of Polygonatum odoratum medicinal materials were all within ±10% of the specified values. The similarity of the characteristic chromatograms were 0.990, 0.971, 0.999, 0.999, 0.999, 0.996, 0.999, 0.998, 0.990, 0.999, 0.994, 0.995, 0.994, 0.992, 0.973, 0.980, 0.997, and 0.998, respectively.

[0083] Experimental Example 2: Determination of Chromatographic Conditions and Methodological Investigation of Polygonatum

[0084] 2.1 Determination of chromatographic conditions

[0085] 2.1.1 Investigation of the mobile phase

[0086] (1) with acetonitrile as mobile phase A, water as mobile phase B, gradient elution according to the following table, and other preparation of test sample solution and determination according to Example 1, the results are shown in Figure 4 A.

[0087] Time (min) Mobile phase A (%) Mobile phase B (%) 0～60 5→95 95→5

[0088] (2) with methanol as mobile phase A, water as mobile phase B, gradient elution according to the following table, and other preparation of test sample solution and determination according to Example 1, the results are shown in Figure 4 B.

[0089] Time (min) Mobile phase A (%) Mobile phase B (%) 0～60 5→95 95→5

[0090] (3) with methanol as mobile phase A, water as mobile phase B, gradient elution according to the following table, and other preparation of test sample solution and determination according to Example 1, the results are shown in Figure 4 C.

[0091] Time (min) A(％) B(％) 0～10 0 100 10～20 0→2 100→98 20～40 2→10 98→90 40～55 10→15 90→85 55～65 15 85

[0092] From Figure 4 it can be seen that the number of peaks obtained by methanol as mobile phase A is more than that of acetonitrile as mobile phase A, the elution ratio distribution is more extensive, and the separation degree between peaks is better, so methanol is optimized as mobile phase A.

[0093] 2.1.2 Selection of chromatographic column

[0094] With the chromatographic column as the variable, Agilent ZORBAX SB-Aq, YMC-Pack ODS-AQ and Shim pack GIST C18-AQ were used as chromatographic columns A-C respectively, and the test sample solution was prepared and determined according to Example 1, and the results are shown in Figure 5 .

[0095] From the above results, it can be seen that the chromatographic column C has more chromatographic peaks in the spectrum and the time distribution is more uniform, therefore, the chromatographic column C is preferred in the present method.

[0096] 2.1.3 Selection of detection wavelength

[0097] The 3D ultraviolet absorption of each chromatographic peak shows that the maximum absorption wavelength of most chromatographic peaks is distributed at about 270 mm, therefore, the detection wavelength is preferably 270 mm.

[0098] 2.1.4 Selection of gradient elution program

[0099] (1) Preparation of test sample solution and determination according to Example 1, the gradient elution program is the same as Example 1, the results are shown in Figure 6 A.

[0100] (2) methanol as mobile phase A, 0.1% formic acid as mobile phase B, gradient elution according to the following table, flow rate is 1.0 ml / min, column temperature is 30°C, others are the same as in Example 1, results are shown in Figure 6 B.

[0101] Table 5 Gradient elution procedure 2

[0102] Time (min) A(％) B(％) 0～8 0 100 8～20 0→2 100→98 20～85 2→15 98→85 85～95 15→18 85→82 95～110 18→22 82→78

[0103] From the above results, it can be seen that the characteristic peaks of the chromatogram obtained by gradient elution procedure 2 are concentrated in the first 70 min, and when the time is 30-50 min, especially 40-50 min, the separation effect between the chromatographic peaks is poor, the number of chromatographic peaks is less, the separation is poor, and the quality of Huangjing cannot be controlled. By using the gradient elution procedure of the present application, the chromatographic peaks at each time period have good separation effect.

[0104] 2.2 Methodology investigation

[0105] 2.2.1 Verification of chromatographic conditions and system suitability

[0106] Prepare the Huangjing standard decoction lyophilized powder test sample solution and the control sample solution according to Example 1 and measure them to verify the chromatographic conditions and system suitability, and investigate whether the blank solvent causes interference, record the chromatogram, and the blank solvent is shown in Figure 7 From Figure 7 it can be seen that the blank solvent has no interference on the test sample characteristic spectrum, the chromatographic method system suitability and specificity are good, and it can be used as the detection method of Huangjing standard decoction lyophilized powder.

[0107] 2.2.2 Precision

[0108] Prepare the Huangjing standard decoction lyophilized powder test sample solution according to Example 1, repeat the injection for 6 times, and measure the relative retention time and relative peak area of 10 common peaks. Peaks 2-6 take peak 1 (S1 peak) as the reference peak 1, peaks 7, 8 and 10 take peak 9 as the reference peak 2 (S2 peak), the RSD of the relative retention time of each characteristic peak is less than 2%, and the RSD of the relative peak area is less than 5%, which indicates that the precision of the instrument is good.

[0109] Table 6 Precision relative retention time results

[0110]

[0111]

[0112] Table 7 Precision relative peak area results

[0113]

[0114] 2.2.3 Reproducibility

[0115] The same batch of Huangqi standard decoction lyophilized powder was used to prepare 6 test sample solutions according to Example 1 and determine the relative retention times and relative peak areas of the 10 common peaks. The RSD values of the relative retention times of the characteristic peaks were all less than 2.0%, and the RSD values of the relative peak areas were all less than 5.0%, indicating that the reproducibility of the method was good.

[0116] Table 8 Reproducibility experiment relative retention time results

[0117]

[0118]

[0119] Table 9 Reproducibility experiment relative peak area results

[0120]

[0121] 2.2.4 Intermediate precision

[0122] Test sample solutions were prepared by three different analysts at different times according to Example 1, and the relative retention times and relative peak areas of the common peaks were determined using the same equipment. The RSD values of the relative retention times of the characteristic peaks were all less than 2.0%, and the RSD values of the relative peak areas were all less than 5.0%, indicating that the intermediate precision of the method was good.

[0123] Table 10 Intermediate precision relative retention time

[0124]

[0125] Table 11 Intermediate precision relative peak area

[0126]

[0127]

[0128] 2.2.5 Stability investigation

[0129] Test sample solutions were prepared according to Example 1, and the relative retention times and relative peak areas of the 10 common peaks were determined at 0, 4, 8, 12, 16, and 24 h. The RSD values of the relative retention times of the characteristic peaks and the reference peaks were all less than 2%, and the RSD values of the relative peak areas were all less than 5.0%. The results showed that the test sample solutions were stable within 24 hours, meeting the determination requirements.

[0130] Table 12 Stability relative retention time

[0131]

[0132] Table 13 Stability relative peak area results

[0133]

[0134] 2.2.6 Different flow rate durability investigation

[0135] With flow rate as the variable, the flow rates were 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, respectively. The test sample solution was prepared according to Example 1 and determined, and the results are shown in the following table and Figure 8 , S1-S3 correspond to flow rates of 0.9-1.1 ml / min, respectively. The RSD values of the relative retention times of each characteristic peak were all less than 5%, and in combination with the peak area results, the flow rate was preferably 1.0 ml / min.

[0136] Table 14 Relative retention times at different flow rates

[0137]

[0138] Table 15 Relative peak areas at different flow rates

[0139]

[0140] 2.2.7 Different column temperature durability investigation

[0141] With column temperature as the variable, the column temperatures were 23℃, 25℃, and 27℃, respectively. The test sample solution was prepared according to Example 1 and determined, and the results are shown in the following table and Figure 9 , S1-S3 correspond to flow rates of 23-27℃, respectively. The RSD values of the relative retention times of each characteristic peak were all less than 5%, and in combination with the peak area results, the column temperature was preferably 25℃.

[0142] Table 16 Relative retention times at different column temperatures

[0143]

[0144] Table 17 Relative peak areas at different column temperatures

[0145]

[0146]

[0147] 2.2.8 Different instruments

[0148] With high performance liquid chromatograph as the variable, high performance liquid chromatographs 1-3 were Waters Acquity Arc, Thermo U3000, and Agilent 1260 chromatograph, respectively. The test sample solution was prepared according to Example 1 and determined, and the chromatograms obtained by different chromatographs were basically consistent. The results are shown in the following table and Figure 10 .

[0149] Table 18 Relative retention time results of different high performance liquid chromatography characteristic patterns

[0150]

[0151] Table 19 Relative peak area results of different high performance liquid chromatography characteristic patterns

[0152]

[0153] The above results show that the chromatographic conditions have little effect on the characteristic patterns, and the obtained patterns are basically consistent. In order to adapt to the durability, the retention time of each characteristic peak is controlled within ±10% of the specified value range.

[0154] The optimal chromatographic conditions for the standard decoction pieces of Huangjing, formula granules, decoction pieces or medicinal materials are as follows: methanol as mobile phase A, water as mobile phase B, gradient elution, gradient elution program: 0-10 min, volume percentage of mobile phase A 0%, volume percentage of mobile phase B 100%; 10-20 min, volume percentage of mobile phase A 0%→2%, volume percentage of mobile phase B 100%→98%; 20-40 min, volume percentage of mobile phase A 2%→10%, volume percentage of mobile phase B 98%→90%; 40-55 min, volume percentage of mobile phase A 10%→15%, volume percentage of mobile phase B 90%→85%; 55-65 min, volume percentage of mobile phase A 15%, volume percentage of mobile phase B 85%; flow rate 1.0 ml / min; column temperature 25°C; detection wavelength 270 nm; the theoretical plate number calculated according to the adenosine peak should not be less than 5000.

[0155] Experimental Example 3 Establishment of characteristic pattern of Jiu Huangjing

[0156] Preparation of test solution: take about 2 g of Jiu Huangjing freeze-dried powder, put it in a conical flask with a plug, add 10% ethanol 10 ml, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 min, shake well, filter, take the filtrate, and obtain the test solution.

[0157] Preparation of reference material of control medicinal material: take 4 g of Jiu Huangjing control decoction pieces, put it in a conical flask with a plug, add water 50 ml, heat reflux for 1 h, filter, evaporate the filtrate to dryness, cool, add 10% ethanol 10 ml to the residue, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 min, shake well, filter, take the filtrate, and obtain the reference material solution of control medicinal material.

[0158] Preparation of control solution: take appropriate amount of uridine control and adenosine control, add 10% ethanol to prepare a mixed solution containing 20 μg of uridine and 20 μg of adenosine per 1 ml, as control solution 1.

[0159] Take 5-hydroxymethylfurfural control product, add 10% ethanol to make 5-hydroxymethylfurfural 50 μg per 1 ml solution as control solution 2.

[0160] Take 5-(hydroxymethyl)-1H-pyrrole-2-carboxaldehyde control product and Huangjing A control product, add 10% ethanol to make 5-(hydroxymethyl)-1H-pyrrole-2-carboxaldehyde 50 μg, Huangjing A 15 μg per 1 ml mixed solution as control solution 3.

[0161] Determination: respectively each 10 μl, injected into liquid chromatograph, determination, namely. Among them, the chromatographic conditions: octadecylsilane bonded silica gel as the filler (specification: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), with methanol as mobile phase A, with water mobile phase B, gradient elution, gradient elution program: 0-10 min, the volume percentage of mobile phase A 0%, the volume percentage of mobile phase B 100%; 10-20 min, the volume percentage of mobile phase A 0%→2%, the volume percentage of mobile phase B 100%→98%; 20-40 min, the volume percentage of mobile phase A 2%→10%, the volume percentage of mobile phase B 98%→90%; 40-55 min, the volume percentage of mobile phase A 10%→15%, the volume percentage of mobile phase B 90%→85%; 55-65 min, the volume percentage of mobile phase A 15%, the volume percentage of mobile phase B 85%; flow rate 1.0 ml / min; column temperature 25℃; detection wavelength 270 nm; the theoretical plate number calculated by adenosine peak should not be less than 5000.

[0162] Wine Huangjing characteristic spectrum includes 9 characteristic peaks, respectively, peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8 and peak 9; with peak 1 as reference object peak 1, the relative retention time of peak 2, peak 3, peak 4, peak 5 is 1.22, 1.43, 1.78, 1.85 respectively; with peak 8 as reference object peak 2, the relative retention time of peak 6, peak 7 and peak 9 is 0.81, 0.93, 1.14 respectively; the relative retention time is within ±10% of the specified value. Wine Huangjing control spectrum is shown in Figure 11 .

[0163] Compared with the control, peak 1 is uridine, peak 5 is 5-hydroxymethylfurfural, peak 8 is adenosine. By mass spectrometry data analysis and positioning by control, peak 6 is 5-(hydroxymethyl)-1H-pyrrole-2-carboxaldehyde, peak 9 is Huangjing A.

[0164] Prepare 18 batches of wine Huangjing standard decoction freeze-dried powder test solution according to example 3 and determine, the results are as follows: Figure 12 , Figure 12Corresponding to batch number J001Y~J018Y.

[0165] Table 20 Relative retention time results of characteristic chromatogram of 18 batches of standard decoction pieces of Jiu Huang- qin

[0166]

[0167]

[0168] Table 21 Relative peak area results of characteristic chromatogram of 18 batches of standard decoction pieces of Jiu Huang- qin

[0169]

[0170]

[0171] The relative retention time of characteristic peaks of the characteristic chromatogram of 18 batches of standard decoction pieces of Jiu Huang- qin was within ±10% of the specified value, and the similarity of the characteristic chromatogram was 0.949, 0.964, 0.969, 0.972, 0.968, 0.934, 0.934, 0.989, 0.998, 0.934, 0.934, 0.971, 0.971, 0.934, 0.928, 0.931, 0.934, 0.967, respectively.

[0172] Prepare 18 batches of Jiu Huang- qin decoction pieces for testing according to Example 4, and determine the test solution, with the results shown in the following table and Figure 13 , Figure 13 Corresponding to batch number YP001~YP018.

[0173] Table 22 Relative retention time results of characteristic chromatogram of 18 batches of Jiu Huang- qin decoction pieces

[0174]

[0175]

[0176] Table 23 Relative peak area results of characteristic chromatogram of 18 batches of Jiu Huang- qin decoction pieces

[0177]

[0178]

[0179] The relative retention times of the characteristic peaks of the characteristic chromatograms of the 18 batches of Jiuhuang- jiang- xian- ruan- pian were within ±10% of the specified values, and the similarity of the characteristic chromatograms was 0.982, 0.982, 0.982, 0.987, 0.999, 0.936, 0.986, 0.976, 0.997, 0.986, 0.997, 0.994, 0.997, 0.999, 0.995, 0.993, 0.992, 0.998, respectively.

[0180] Experimental Example 4 Methodological investigation of Jiuhuang standard decoction lyophilized powder

[0181] 4.1 Verification of chromatographic conditions and system suitability

[0182] The test sample solution and the reference solution of Jiuhuang standard decoction lyophilized powder were prepared according to Example 3 and determined to verify the chromatographic conditions and system suitability, investigate whether the blank solvent would cause interference, and record the chromatogram, as shown in Figure 14 . The results showed that the blank solvent had no interference with the test sample chromatogram, and the system suitability and specificity of this chromatographic method were good, and it could be used as a detection method for Jiuhuang standard decoction lyophilized powder.

[0183] 4.2 Precision

[0184] The test sample solution of Jiuhuang standard decoction lyophilized powder was prepared according to Example 3, and 6 injections were repeated to determine the relative retention times and relative peak areas of 9 common peaks. Uridine was used as the S1 peak to calculate the relative retention times of peaks 2-5, and adenosine was used as the S2 peak to calculate the relative retention times of peaks 6-7 and 9. The RSDs of the relative retention times of each characteristic peak were all less than 2%, and the RSDs of the relative peak areas were all less than 5.0%, indicating that the precision of the instrument was good.

[0185] Table 24 Results of instrument precision relative retention times

[0186]

[0187] Table 25 Results of instrument precision relative peak areas

[0188]

[0189]

[0190] 4.3 Repeatability

[0191] The same batch of Jiuhuang standard decoction lyophilized powder was taken, and 6 test sample solutions were prepared according to Example 3 and determined to determine the relative retention times and relative peak areas of 9 common peaks. The RSD values of the relative retention times of each characteristic peak were all less than 2.0%, and the RSD values of the relative peak areas were all less than 5.0%, indicating that the repeatability of the method was good.

[0192] Table 26 Method repeatability relative retention time results

[0193]

[0194] Table 27 Method repeatability relative peak area results

[0195]

[0196] 4.4 Intermediate precision

[0197] The relative retention time and relative peak area of common peaks were determined by three different analysts at different times using the same equipment. The RSD values of relative retention time of each characteristic peak were less than 2.0%, and the RSD values of relative peak area were less than 5.0%. The results showed that the intermediate precision of this method was good.

[0198] Table 28 Intermediate precision relative retention time results

[0199]

[0200] Table 29 Intermediate precision relative peak area results

[0201]

[0202] 4.5 Stability study

[0203] The test sample solution was prepared according to Example 3, and the relative retention time and relative peak area of 9 common peaks were determined at 0, 4, 8, 12, 16, 24 h. The RSD values of relative retention time of each characteristic peak were less than 2.0%, and the RSD values of relative peak area were less than 5.0%. The results showed that the test sample solution was stable within 24 hours, which met the determination requirements.

[0204] Table 30 Stability relative retention time results

[0205]

[0206] Table 31 Stability relative peak area results

[0207]

[0208]

[0209] 4.6 Different flow rate robustness study

[0210] The flow rate was used as a variable, and the flow rates were 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, respectively. The test sample solution was prepared according to Example 3 and determined, and the results are shown in the following table and Figure 15S1-S3 correspond to flow rate 0.9-1.1 ml / min. The RSD value of the relative retention time of each characteristic peak is less than 5.0%, and in combination with the peak area result, the flow rate is preferably 1.0 ml / min.

[0211] Table 32 Relative retention time results at different flow rates

[0212]

[0213] Table 33 Relative peak area results at different flow rates

[0214]

[0215] 4.7 Investigation of different column temperature durability

[0216] With column temperature as the variable, the column temperature was 23°C, 25°C, and 27°C, respectively, the test sample solution was prepared and determined according to Example 3, and the results are shown in the following table and Figure 16 S1-S3 correspond to column temperature 23-27°C. The RSD value of the relative retention time of each characteristic peak is less than 2.0%, and in combination with the peak area result, the column temperature is preferably 25°C.

[0217] Table 34 Relative retention time results at different column temperatures

[0218]

[0219]

[0220] Table 35 Relative peak area results at different column temperatures

[0221]

[0222] 4.8 Different instruments

[0223] With high performance liquid chromatograph as the variable, high performance liquid chromatographs 1-3 are Waters Acquity Arc, Thermo U3000, and Agilent 1260 chromatograph, respectively; the test sample solution was prepared and determined according to Example 3, and considering the applicability of different instruments, the relative retention time specification value range is limited to ±10%. The results are shown in the following table and Figure 17 .

[0224] Table 36 Relative retention time determination results of characteristic chromatograms of different high performance liquid chromatographs

[0225]

[0226] Table 37 Relative peak area results of characteristic chromatograms of different high performance liquid chromatographs

[0227]

[0228] The above results show that the chromatographic conditions have little effect on the characteristic map, and the obtained maps are basically consistent. The optimal chromatographic conditions for the standard decoction of Rhizoma Polygonati, formula granules, decoction pieces or medicinal materials are as follows: methanol as mobile phase A, water as mobile phase B, gradient elution, gradient elution program: 0-10 min, volume percentage of mobile phase A 0%, volume percentage of mobile phase B 100%; 10-20 min, volume percentage of mobile phase A 0%→2%, volume percentage of mobile phase B 100%→98%; 20-40 min, volume percentage of mobile phase A 2%→10%, volume percentage of mobile phase B 98%→90%; 40-55 min, volume percentage of mobile phase A 10%→15%, volume percentage of mobile phase B 90%→85%; 55-65 min, volume percentage of mobile phase A 15%, volume percentage of mobile phase B 85%; flow rate 1.0 ml / min; column temperature 25℃; detection wavelength 270 nm; the theoretical plate number should not be less than 5000 according to the adenosine peak.

[0229] Obviously, the above examples are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments cannot be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for constructing a characteristic map of Polygonatum sibiricum and / or wine Polygonatum sibiricum, characterized in that, The method comprises the following steps: Preparation of the test sample solution: the test sample is prepared into a test sample solution; Determination: high performance liquid chromatography is used for detection; methanol is used as the mobile phase A, and water is used as the mobile phase B, gradient elution is adopted, and the gradient elution program comprises 0-10 min, the volume percentage of the mobile phase A is 0%, and the volume percentage of the mobile phase B is 100%; 10-20 min, the volume percentage of the mobile phase A is 0%→2%, the volume percentage of the mobile phase B is 100%→98%; 20-40 min, the volume percentage of the mobile phase A is 2%→10%, the volume percentage of the mobile phase B is 98%→90%; 40-55 min, the volume percentage of the mobile phase A is 10%→15%, the volume percentage of the mobile phase B is 90%→85%; 55-65 min, the volume percentage of the mobile phase A is 15%, and the volume percentage of the mobile phase B is 85%; The chromatographic column is Shim pack GIST C18-AQ; The detection wavelength is 255-285 nm; The test sample is a standard decoction lyophilized powder, a formula granule, a decoction piece or a medicinal material; When the test sample is a standard decoction lyophilized powder or a formula granule, the extraction solvent comprises at least one of ethanol, water and methanol; When the test sample is a medicinal material, the method for preparing the test sample solution is that the medicinal material is taken, ground, sieved, about 4 g is taken, placed in a conical flask with a plug, 50 ml of water is added, heated and refluxed for 1 h, filtered, the filtrate is evaporated to dryness, cooled, 10% ethanol 10 ml is added to the residue, ultrasonic treatment is performed for 30 min, the power of the ultrasonic treatment is 250 W, the frequency is 40 kHz, uniformly shaken, filtered, the filtrate is taken, and the test sample solution is obtained; When the test sample is a decoction piece, the method for preparing the test sample solution is that the decoction piece is ground, sieved, about 4 g is taken, placed in a conical flask with a plug, 50 ml of water is added, heated and refluxed for 1 h, filtered, the filtrate is evaporated to dryness, cooled, 10% ethanol 10 ml is added to the residue, ultrasonic treatment is performed for 30 min, the power of the ultrasonic treatment is 250 W, the frequency is 40 kHz, uniformly shaken, filtered, the filtrate is taken, and the test sample solution is obtained; The control substance in the construction of the characteristic spectrum of Rhizoma Polygonati includes uridine, adenosine, adenine, 5 hydroxymethyl furfural, tryptophan, thymidine, and polygonatin A; The control substances in the construction of the wine Rhizoma Polygonati chart include uridine, adenosine, 5-hydroxymethylfurfural and 5 (hydroxymethyl) 1H pyrrole 2 formaldehyde.

2. The construction method of claim 1, wherein, The chromatographic conditions further comprise that the column temperature is 23-27 ℃; and / or, the flow rate is 0.9-1.1 ml / min; and / or, the injection amount is 5-20 μL.

3. The construction method of claim 2, wherein, The chromatographic conditions further comprise that the column temperature is 25 ℃; and / or, the flow rate is 1.0 ml / min; and / or, the detection wavelength is 270 nm; and / or, the injection amount is 10 μL; and / or, the specification of the chromatographic column is that the column length is 250 mm, the inner diameter is 4.6 mm, the particle size is 5 μm; and / or, the theoretical plate number calculated according to the adenosine peak should be not less than 5000.

4. The construction method of claim 1, wherein, The characteristic spectrum of the Rhizoma Polygonati includes 10 characteristic peaks, namely, peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9 and peak 10; Peak 1 is used as the reference material peak 1, and the relative retention times of peak 2, peak 3, peak 4, peak 5 and peak 6 are 1.22, 1.42, 1.66, 1.84 and 1.91 respectively; Peak 2 is used as the reference material peak 2, and the relative retention times of peak 7, peak 8 and peak 10 are 0.81, 0.84 and 1.15 respectively; The relative retention time is within ±10% of the specified value.

5. The construction method according to claim 4, characterized in that, The characteristic spectrum of the Rhizoma Polygonati includes 10 characteristic peaks, peak 1 is uridine, peak 4 is adenine, peak 5 is 5-hydroxymethylfurfural, peak 6 is guanosine, peak 7 is thymidine, peak 8 is tryptophan, peak 9 is adenosine, and peak 10 is polygonatin A.

6. The construction method of claim 1, wherein, The characteristic spectrum of the Rhizoma Polygonati includes 9 characteristic peaks, peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8 and peak 9; Peak 1 is used as the reference peak 1, and the relative retention times of peak 2, peak 3, peak 4 and peak 5 are 1.22, 1.43, 1.78 and 1.85, respectively; Peak 8 is used as the reference peak 2, and the relative retention times of peak 6, peak 7 and peak 9 are 0.81, 0.93 and 1.14, respectively; The relative retention time is within ±10% of the specified value.

7. The construction method according to claim 6, characterized in that, The characteristic spectrum of the Rhizoma Polygonati includes 9 characteristic peaks, peak 1 is uridine, peak 5 is 5-hydroxymethylfurfural, peak 6 is 5-(hydroxymethyl)-1H-pyrrol-2-furfural, peak 8 is adenosine, and peak 9 is polygonatin A.

8. The construction method according to any one of claims 1 to 7, characterized in that, In the preparation of the test solution, the ratio of the mass of the test sample to the volume of the extraction solvent is (0.05-0.5):1, the unit of mass is g, and the unit of volume is ml.

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