CN115452964B

CN115452964B - UPLC characteristic spectrum construction method and quality control method of calyx seu fructus physalis

Info

Publication number: CN115452964B
Application number: CN202210524863.3A
Authority: CN
Inventors: 魏家保; 赵伟志; 郑晓英; 游庭活; 罗丽娜
Original assignee: China Resources Sanjiu Modern Traditional Chinese Medicine Pharmaceutical Co ltd
Current assignee: China Resources Sanjiu Modern Traditional Chinese Medicine Pharmaceutical Co ltd
Priority date: 2022-05-13
Filing date: 2022-05-13
Publication date: 2023-07-25
Anticipated expiration: 2042-05-13
Also published as: CN115452964A

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a UPLC characteristic spectrum construction method and a quality control method of calyx seu fructus physalis. The construction method of the invention uses octadecylsilane chemically bonded silica as a filler, uses acetonitrile-0.2% phosphoric acid aqueous solution as a mobile phase to carry out gradient elution of a specific program, thus obtaining the characteristic spectrum with uniform distribution of chromatographic peak retention time, good peak symmetry, satisfactory separation degree, stable base line and small noise interference. Meanwhile, 5 common peaks such as luteolin, physalin A, physalin L, 4, 7-didehydro-neophysalin B and the like are identified, each peak has good response, the relative retention time of each characteristic peak is regulated, the characteristic peak is easy to locate, and the relative peak area of the peak 4 is regulated by combining a plurality of batches of research results so as to control the quality. The invention effectively solves the defect that the quality of the calyx seu fructus physalis is inaccurate in the prior art, and the construction method of the invention has good reproduction, accuracy and reliability.

Description

UPLC characteristic spectrum construction method and quality control method of calyx seu fructus physalis

Technical Field

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a UPLC characteristic spectrum construction method and a quality control method of calyx seu fructus physalis.

Background

Calyx seu fructus Physalis is dried calyx of Physalis alkekengi (Physalis alkekengi L. Var. Franketii (Mast.) Makino) of Solanaceae, or fruit-bearing calyx, originally in Shennong Ben Cao Jing. Calyx seu fructus physalis is sweet, sour and cold in nature, has the effects of cooling, detumescence, relieving cough, resolving phlegm, promoting urination, strengthening heart and relieving fever, and is a common traditional Chinese medicine for clearing heat and detoxicating.

For traditional Chinese medicinal materials, the complexity of the traditional Chinese medicinal materials in application is caused by various reasons such as complex natural environment, social condition, humane factors and the like, for example, the traditional Chinese medicinal materials with the same name can come from plants with different basic sources, and the chemical compositions of the traditional Chinese medicinal materials with the same basic source can be different due to different production places, harvesting seasons, growth years and the like, so that the quality and clinical curative effects of the traditional Chinese medicines are necessarily affected. Therefore, the establishment of an accurate and effective method for identifying Chinese medicinal materials, namely a Chinese medicinal characteristic spectrum, is one of the main technical means for solving the problems at present.

The prior art discloses a fingerprint construction method for different fruits of Euphorbia humifusa, which adopts an Agilent TC-18 (4.6 mm multiplied by 1.5mm,5 μm) chromatographic column, uses 0.2% phosphoric acid water (A) -acetonitrile (B) as mobile phase for gradient elution, the column temperature is 25 ℃, the detection wavelength is 220nm, and the flow rate is 1.0 ml.min ^-1 HPLC chromatograms of calyx seu fructus Physalis fruits of different production areas are obtained, and finally a control map is obtained, see figure 105. Although the fingerprint provided by the prior art establishes 11 common peaks, the chromatographic peaks are mainly concentrated in the first 20 minutes, wherein the peak-out time of the peaks 1-5 is close to that of a solvent peak, the area of the peaks 8-11 is too small, the symmetry of all peaks is poor, the separation degree is poor, only trifolitin and physalin A are identified, the defects of difficult positioning of the chromatographic peaks and large integral deviation exist in the actual operation, and in addition, the fingerprint is evaluated by the result of similarity and has little significance. More importantly, the research object of the prior art is calyx seu fructus physalis fruits (without calyx), and is not in Chinese pharmacopoeiaThe fingerprint spectrum established by the calyx seu fructus physalis medicinal material can only be used for evaluating the quality of calyx seu fructus physalis fruits at most, but cannot be used for evaluating the quality of calyx seu fructus physalis.

At present, the qualitative identification of the calyx seu fructus physalis by the Chinese pharmacopoeia only adopts a thin-layer chromatography method, and the quality of the calyx seu fructus physalis is difficult to evaluate integrally, so that a brand-new method for detecting the quality of the calyx seu fructus physalis needs to be established, and the quality of the calyx seu fructus physalis can be controlled more reliably and accurately.

Disclosure of Invention

In view of the above, the invention aims to establish a method capable of rapidly and accurately identifying and detecting calyx seu fructus physalis, thereby providing a UPLC characteristic spectrum construction method and a quality control method of the calyx seu fructus physalis.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a UPLC characteristic spectrum construction method of a calyx seu fructus physalis, which comprises the following steps:

(1) Preparing a sample solution from calyx seu fructus physalis;

(2) The method comprises the steps of detecting a sample solution by adopting an ultra-high performance liquid chromatography, taking octadecylsilane chemically bonded silica as a filler, and taking acetonitrile-0.2% phosphoric acid aqueous solution as a mobile phase for gradient elution, wherein the gradient elution procedure comprises: 0- > 7 min- > 15 min- > 30 min- > 32 min- > 40 min, the volume percentage of acetonitrile in the mobile phase is 15% → 17% → 25% → 28% → 38% → 42%.

Optionally, the step (1) comprises the steps of sequentially carrying out water extraction, ethyl acetate extraction and methanol extraction on the calyx seu fructus physalis by adopting a solvent, wherein the calyx seu fructus physalis is prepared from calyx seu fructus physalis medicinal materials, calyx seu fructus physalis decoction pieces, calyx seu fructus physalis standard decoction freeze-dried powder or calyx seu fructus physalis formula particles.

Optionally, step (1) satisfies any one or more of the following a-E:

A. When the calyx seu fructus physalis is a calyx seu fructus physalis medicinal material or calyx seu fructus physalis decoction pieces, the water extraction is reflux extraction; or when the calyx seu fructus physalis is calyx seu fructus physalis standard decoction lyophilized powder or calyx seu fructus physalis formula granule, the water extraction is dissolved by adding water;

B. the volume ratio of the mass of the calyx seu fructus physalis to the volume of water is 1:25, and the proportional relationship is g/mL;

C. extracting the water extract with ethyl acetate for at least 2 times, each time with at least 10mL, mixing the ethyl acetate extracts, evaporating to dryness, and extracting the residue with methanol;

D. extracting the ethyl acetate extract by adopting a methanol aqueous solution or pure methanol with the volume percentage of more than 30 percent;

E. the methanol extraction mode is ultrasonic extraction, preferably ultrasonic treatment power is 250W, frequency is 40kHz, and time is 30min.

Optionally, the chromatographic conditions of the ultra performance liquid chromatography in step (2) further comprise: the detection wavelength is 220nm, the flow rate is 0.3mL/min, the column temperature is 25 ℃, the sample injection amount is 3 mu L, and the theoretical plate number is not less than 5000 according to luteolin calculation.

Optionally, the construction method further comprises a step of preparing a reference substance solution by adopting luteolin as a reference substance, and a step of replacing the sample solution in the step (2) with the reference substance solution to obtain a reference substance characteristic map.

Optionally, the construction method further comprises a step of preparing a reference substance solution of the reference medicinal material by using calyx seu fructus physalis as the reference medicinal material, and a step of replacing the sample solution in the step (2) with the reference substance solution of the reference medicinal material to obtain a characteristic map of the reference medicinal material.

The UPLC characteristic spectrum of the calyx seu fructus physalis constructed by the construction method disclosed by the invention.

A UPLC control profile of calyx seu fructus physalis selected from any one of the following (1) - (4):

(1) The luteolin peak has 11 common characteristic peaks, the luteolin peak is taken as a reference peak, the relative retention time of each characteristic peak and the reference peak is within +/-10% of a specified value, and the specified value is: 2.23 (peak 2), 2.75 (peak 3), 3.52 (peak 4), 3.69 (peak 5), 4.07 (peak 6), 4.26 (peak 7), 4.46 (peak 8), 4.61 (peak 9), 5.45 (peak 10), 6.32 (peak 11);

(2) The luteolin peak has 11 common characteristic peaks, the luteolin peak is taken as a reference peak, the relative retention time of each characteristic peak and the reference peak is within +/-10% of a specified value, and the specified value is: 2.23 (peak 2), 2.75 (peak 3), 3.52 (peak 4), 3.69 (peak 5), 4.07 (peak 6), 4.26 (peak 7), 4.46 (peak 8), 4.61 (peak 9), 5.45 (peak 10), 6.32 (peak 11); and the relative peak area of peak 4 and reference peak is not less than 0.16;

(3) The characteristic map obtained by using single-batch or multi-batch calyx seu fructus physalis according to the construction method is adopted;

(4) The characteristic spectrum obtained by using the multi-batch calyx seu fructus physalis according to the construction method is prepared into the characteristic spectrum by an average value or a median method.

On the other hand, the invention also provides a method for identifying the calyx seu fructus physalis, which comprises the step of comparing the characteristic spectrum of the product to be detected with the characteristic spectrum of the calyx seu fructus physalis contrast; the characteristic spectrum of the product to be detected is obtained by using the product to be detected according to the construction method of the invention, and the calyx seu fructus physalis control characteristic spectrum is the calyx seu fructus physalis UPLC control characteristic spectrum of the invention.

The invention also provides a quality control method of the calyx seu fructus physalis, which comprises the step of comparing the characteristic spectrum of the calyx seu fructus physalis product to be detected with the characteristic spectrum of the calyx seu fructus physalis contrast; the characteristic spectrum of the calyx seu fructus physalis product to be detected is obtained by using the product to be detected according to the construction method of the invention, and the calyx seu fructus physalis control characteristic spectrum is the calyx seu fructus physalis UPLC control characteristic spectrum of the invention.

In the present invention, peak 1 represents peak No. 1, peak 2 represents peak No. 2, and so on. The reference peak is the S peak.

In the invention, the product to be detected can be a calyx seu fructus physalis product, and can also be other products except the calyx seu fructus physalis product. If the characteristic spectrum of the product to be detected is consistent with the characteristic spectrum of the calyx seu fructus physalis contrast, the product to be detected is calyx seu fructus physalis; if the characteristic spectrum of the product to be detected is inconsistent with the characteristic spectrum of the calyx seu fructus physalis contrast, the product to be detected is not calyx seu fructus physalis.

In the invention, the calyx seu fructus physalis product to be detected can be medicinal materials, decoction pieces or preparations, for example, at least one selected from calyx seu fructus physalis medicinal materials, calyx seu fructus physalis decoction pieces, calyx seu fructus physalis standard decoction freeze-dried powder and calyx seu fructus physalis formula particles. If the characteristic spectrum of the calyx seu fructus physalis product to be detected cannot be matched with the calyx seu fructus physalis contrast characteristic spectrum, namely the relative peak area of the peak 4 and the peak S is lower than 0.16, the quality is considered to be unqualified.

In the invention, at least 2 batches of calyx seu fructus physalis are adopted to obtain a comparison characteristic spectrogram, for example, 3 batches of calyx seu fructus physalis formula particles, 15 batches of calyx seu fructus physalis standard decoction freeze-dried powder, 15 batches of calyx seu fructus physalis medicinal materials and 15 batches of calyx seu fructus physalis decoction pieces are adopted.

The calyx seu fructus physalis mainly contains steroid, flavonoid, phenylpropanoid, alkaloid and polysaccharide compounds, wherein the steroid, flavonoid are used as main components, and have antibacterial, antiinflammatory, antioxidant and antitumor effects. Steroid compounds are the most commonly found chemical components in calyx seu fructus physalis plants in recent decades, and mainly comprise physalis alkekengi bitter elements, neophysalis alkekengi bitter elements and sterol compounds. By combining characteristic spectrum peak identification information, peak 1 is luteolin, peak 3 is luteolin, and both are flavonoid compounds, and have anti-diabetes, anti-inflammatory and anti-tumor activities; peak 4 is Physalin A, peak 6 is Physalin L, physalin compounds in calyx seu fructus Physalis are most, and are characteristic chemical components, and have anti-diabetes, antiinflammatory and antitumor activities. Therefore, the relative peak area data of the peak 1, the peak 3, the peak 4 and the peak 6 are analyzed, the peak 1 is luteolin, and the luteolin is used as a content measurement index of the calyx seu fructus physalis under the item of Chinese pharmacopoeia, so that the luteolin is more suitable for being used as an S peak to calculate and control the relative peak areas of other characteristic peaks; the relative peak areas of the peak 3, the peak 6 and the reference peak have wider batch ranges and have no obvious change rule, if the range is formulated by showing or averaging + -30% of actual measurement of the relative peak areas of the batches, the relative peak area ratio is too low, so that the meaning of the relative peak area ratio in the final finished product standard is not great; the peak area of the peak 4 and the reference peak is moderate in the multi-batch range and can be transferred in an overstable manner, so that the range is established by the measured minimum value of the multi-batch and the relative peak area of-30%, namely, the prescribed range is not lower than 0.16.

The calyx seu fructus physalis medicinal material is prepared into calyx seu fructus physalis decoction pieces after simple purification, and the processing process has no influence on the number and peak area of characteristic peaks, so that the relative peak area value of the calyx seu fructus physalis decoction pieces is executed by referring to the medicinal material. The calyx seu fructus physalis decoction pieces are prepared into calyx seu fructus physalis formula particles by extraction, concentration and drying, and the basic properties of the calyx seu fructus physalis formula particles are basically consistent with that of calyx seu fructus physalis standard decoction, so that the relative peak area value of the calyx seu fructus physalis formula particles is executed by referring to the standard decoction lyophilized powder, namely, the relative peak area of the specified peak 4 and the specified peak S is not less than 0.16.

Compared with the prior art, the technical scheme of the invention has the following advantages:

1. according to the construction method of the UPLC characteristic spectrum of the calyx seu fructus physalis, octadecylsilane chemically bonded silica is used as a filling agent, acetonitrile-0.2% phosphoric acid aqueous solution is used as a mobile phase for gradient elution, an elution program comprises 0-7 min-15 min-30 min-32 min-40 min, the volume percentage of acetonitrile in the mobile phase is 15-17-25-28-38-42%, and therefore chromatographic peaks in the obtained calyx seu fructus physalis characteristic spectrum are uniformly distributed, the peak symmetry is good, the separation degree meets the requirements, the base line is stable, and the noise interference is small. Meanwhile, 5 common peaks such as luteolin, physalin A, physalin L, 4,7 didehydro neophysalin B and the like are identified, each peak has good response, the relative retention time of each characteristic peak is regulated, the characteristic peak is easy to locate, and the relative peak area of peak 4 (physalin A) is regulated by combining the multi-batch research result so as to carry out quality control. The invention effectively solves the defect that the quality of the calyx seu fructus physalis is inaccurate in the prior art, and the construction method of the invention has good reproduction, accuracy and reliability.

2. The invention provides a method for constructing a UPLC characteristic spectrum of a calyx seu fructus physalis, the calyx seu fructus physalis with fruits is taken as a research object, meets the requirements of the Chinese pharmacopoeia on the carrying medicinal materials, therefore, the established characteristic spectrum can be used for solving the quality evaluation of calyx seu fructus physalis medicinal materials, calyx seu fructus physalis decoction pieces, calyx seu fructus physalis standard decoction freeze-dried powder, calyx seu fructus physalis formula particles or calyx seu fructus physalis related preparations in Chinese pharmacopoeia.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a chromatogram under gradient 1 conditions in example 1 of the present invention; FIG. 2 is a chromatogram under gradient 2 conditions in example 1 of the present invention; FIG. 3 is a chromatogram under gradient 3 conditions in example 1 of the present invention; FIG. 4 is a chromatogram under gradient 4 conditions in example 1 of the present invention; FIG. 5 is a chromatogram under gradient 5 conditions in example 1 of the present invention; FIG. 6 is a chromatogram under gradient 6 conditions in example 1 of the present invention; FIG. 7 is a chromatogram under gradient 7 conditions in example 1 of the present invention; FIG. 8 is a chromatogram of an Agilent 1290 II instrument used in example 1 of the present invention; FIG. 9 is a chromatogram of example 1 of the present invention using a Thermo Fisher instrument; FIG. 10 is a chromatogram of example 1 of the present invention using water extraction; FIG. 11 is a chromatogram of example 1 of the present invention using 30% methanol extraction; FIG. 12 is a chromatogram of example 1 of the present invention using 50% methanol extraction; FIG. 13 is a chromatogram of example 1 of the present invention using 70% methanol extraction; FIG. 14 is a chromatogram of example 1 of the present invention using pure methanol extraction; FIG. 15 is a chromatogram of example 1 of the present invention using 50% ethanol extraction; FIG. 16 is a chromatogram of example 1 of the present invention extracted 2 times with ethyl acetate; FIG. 17 is a chromatogram of 2 extractions with n-butanol in example 1 of this invention; FIG. 18 is a chromatogram of example 1 of the present invention extracted 2 times with chloroform; FIG. 19 is a chromatogram of 2 extractions with diethyl ether in example 1 of the present invention; FIG. 20 is a chromatogram of example 1 of the present invention extracted 3 times with ethyl acetate; FIG. 21 is a chromatogram of 0.2g of the sample in example 1 of the present invention; FIG. 22 is a chromatogram of 0.4g of the sample in example 1 of the present invention; FIG. 23 is a chromatogram of 0.6g of the sample in example 1 of the present invention; FIG. 24 is a chromatogram of 0.8g of the sample in example 1 of the present invention; FIG. 25 is a chromatogram of sample injection amount of 3. Mu.L in example 1 of the present invention; FIG. 26 is a chromatogram of sample injection amount of 5. Mu.L in example 1 of the present invention; FIG. 27 is a chromatogram of sample injection amount of 7. Mu.L in example 1 of the present invention; FIG. 28 is a characteristic spectrum of the calyx seu fructus physalis granule in example 1 of the present invention; FIG. 29 is a reference substance positioning chromatogram 1 of example 1 of the present invention; FIG. 30 is a reference substance positioning chromatogram 2 of example 1 of the present invention; FIG. 31 is a chromatogram of the luteolin control in example 1 of the present invention; FIG. 32 is a graph showing the ultraviolet absorption spectrum of the luteolin control in example 1 of the present invention; FIG. 33 is a graph showing the ultraviolet absorption spectrum of luteolin in the sample of example 1 of the present invention; FIG. 34 is a chromatogram of the luteolin control of example 1 of the present invention; FIG. 35 is a graph showing the ultraviolet absorption spectrum of the luteolin control in example 1 of the present invention; FIG. 36 is a graph showing the ultraviolet absorption spectrum of luteolin in the sample of example 1 of the present invention; FIG. 37 is a chromatogram of a control of acid plasma picrin A in example 1 of the present invention; FIG. 38 is a chart showing the ultraviolet absorption spectrum of the control of acid pulp picrin A in example 1 of the present invention; FIG. 39 is a graph showing the ultraviolet absorption spectrum of acid pulp picrin A in the sample of example 1 of the present invention; FIG. 40 is a chromatogram of a control of acid slurry bitter principle L in example 1 of the present invention; FIG. 41 is a graph showing the ultraviolet absorption spectrum of the control substance of acid pulp picrin L in example 1 of the present invention; FIG. 42 is a graph showing the ultraviolet absorption spectrum of the acid pulp picrin L in the sample of example 1 of the present invention; FIG. 43 is a graph showing the ultraviolet absorption spectrum of the 4,7 didehydro-physalin B control in example 1 of the present invention; FIG. 44 is a graph showing the ultraviolet absorption spectrum of 4, 7-didehydro-physalin B in the sample of example 1 of the present invention; FIG. 45 is a characteristic spectrum of a test sample of the calyx seu fructus physalis formula particle in example 1 of the present invention; FIG. 46 is a graph showing the precision sharing mode of the instrument in example 1 (precision 1-6 from top to bottom); FIG. 47 is a graph of the repetitive consensus pattern of example 1 (1-6 repetitive from top to bottom) according to the present invention; FIG. 48 is a graph showing the middle precision sharing mode (first, second, and third) of different persons according to example 1 of the present invention; FIG. 49 is a chromatogram of example 1 of the present invention using a CORTECS UPLC T3 column 01213131419407 lot; FIG. 50 is a chromatogram of example 1 of the present invention using a CORTECS UPLC T3 column 01183110315331 lot; FIG. 51 is a chromatogram of example 1 of the present invention using a CORTECS UPLC T3 column 01213130815310 lot; FIG. 52 is a characteristic spectrum of a negative blank sample in example 1 of the present invention; FIG. 53 is a characteristic spectrum of a test sample of the calyx seu fructus physalis formula particle in example 1 of the present invention; FIG. 54 is a graph showing a stability consensus pattern in example 1 of the present invention; FIG. 55 is a chromatogram of example 1 of the present invention at a flow rate of 0.28 mL/min; FIG. 56 is a chromatogram of example 1 of the present invention at a flow rate of 0.3 mL/min; FIG. 57 is a chromatogram at a flow rate of 0.32mL/min for example 1 of the present invention; FIG. 58 is a chromatogram of example 1 of the present invention at a column temperature of 23 ℃; FIG. 59 is a chromatogram of example 1 of the present invention at a column temperature of 25 ℃; FIG. 60 is a chromatogram of example 1 of the present invention at a column temperature of 28 ℃; FIG. 61 is a graph showing a common pattern of characteristic patterns of 3 batches of calyx seu fructus physalis formula particles in example 2 of the present invention; FIG. 62 is a graph showing the comparative characteristics of the particles of the formulation of calyx seu fructus physalis in example 2 of the present invention; FIG. 63 is a chromatogram of the luteolin control in example 3 of the present invention; FIG. 64 is a chromatogram of a test sample of the freeze-dried powder of calyx seu fructus physalis in example 3 of the present invention; FIG. 65 is a graph showing the precision sharing mode of the instrument according to example 3 of the present invention (precision 1-6 from top to bottom); FIG. 66 is a graph of the repetitive consensus pattern of example 3 (1-6 repetitive from top to bottom) according to the present invention; FIG. 67 is a graph showing the middle precision sharing pattern of different persons (person A, person B, and person C from top to bottom) in example 3 of the present invention; FIG. 68 is a chromatogram of example 3 of the present invention using a CORTECS UPLC T3 column 01213131419407 lot; FIG. 69 is a chromatogram of example 3 of the present invention using a CORTECS UPLC T3 column 01183110315331 lot; FIG. 70 is a chromatogram of example 3 of the present invention using a CORTECS UPLC T3 column 01213130815310 lot; FIG. 71 is a characteristic spectrum of a negative blank sample in example 3 of the present invention; FIG. 72 is a characteristic map of a test sample of the calyx seu fructus physalis lyophilized powder in example 3 of the present invention; FIG. 73 is a graph showing a common mode of stability in example 3 of the present invention; FIG. 74 is a chromatogram of example 3 of the present invention at a flow rate of 0.28 mL/min; FIG. 75 is a chromatogram of example 3 of the present invention at a flow rate of 0.3 mL/min; FIG. 76 is a chromatogram of example 3 of the present invention at a flow rate of 0.32 mL/min; FIG. 77 is a chromatogram at a column temperature of 23℃in example 3 according to the present invention; FIG. 78 is a chromatogram of example 3 of the present invention at a column temperature of 25 ℃; FIG. 79 is a chromatogram of example 3 of the present invention at a column temperature of 28 ℃; FIG. 80 is a graph showing a common model of characteristic maps of 15 batches of calyx seu fructus physalis freeze-dried powder in example 4 of the present invention; FIG. 81 is a graph showing the comparative profile of the freeze-dried powder of calyx seu fructus physalis in example 4 of the present invention; FIG. 82 is a chromatogram of the luteolin control in example 5 of the present invention; FIG. 83 is a chromatogram of a test sample of calyx seu fructus Physalis in example 5 of the present invention; FIG. 84 is a graph showing the precision sharing mode of the instrument according to example 5 of the present invention (precision 1-6 from top to bottom); FIG. 85 is a graph of the repetitive consensus pattern of example 5 (1-6 repetitive from top to bottom) according to the present invention; FIG. 86 is a graph showing the middle precision sharing pattern of different individuals (personnel A, B and C from top to bottom) in example 5 of the present invention; FIG. 87 is a chromatogram of example 5 of the present invention using a CORTECS UPLC T3 chromatography column 01213131419407 lot; FIG. 88 is a chromatogram of example 3 of the present invention using a CORTECS UPLC T3 column 01183110315331 lot; FIG. 89 is a chromatogram of example 5 of the present invention using a CORTECS UPLC T3 column 01213130815310 lot; FIG. 90 is a characteristic spectrum of a negative blank test sample in example 5 of the present invention; FIG. 91 is a characteristic spectrum of a test sample of calyx seu fructus physalis in example 5 of the present invention; FIG. 92 is a graph showing a common mode of stability in example 5 of the present invention; FIG. 93 is a chromatogram at a flow rate of 0.28mL/min in example 5 of the present invention; FIG. 94 is a chromatogram of example 5 of the present invention at a flow rate of 0.3 mL/min; FIG. 95 is a chromatogram of example 5 of the present invention at a flow rate of 0.32 mL/min; FIG. 96 is a chromatogram at a column temperature of 23℃in example 5 of the present invention; FIG. 97 is a chromatogram at a column temperature of 25℃in example 5 of the present invention; FIG. 98 is a chromatogram of example 5 of the present invention at a column temperature of 28 ℃; FIG. 99 is a common pattern diagram of characteristic maps of 15 batches of calyx seu fructus physalis in example 6 of the invention; FIG. 100 is a graph showing a comparative characteristic of calyx seu fructus physalis in example 6 of the present invention; FIG. 101 is a chromatogram of the luteolin control in example 7 of the present invention; FIG. 102 is a chromatogram of a sample of calyx seu fructus physalis decoction pieces in example 7 of the present invention; FIG. 103 is a common pattern diagram of characteristic maps of 15 batches of calyx seu fructus physalis decoction pieces in example 8 of the invention; FIG. 104 is a graph showing the comparative characteristic of the calyx seu fructus physalis decoction pieces of example 8 of the present invention; fig. 105 is a control fingerprint of calyx seu fructus physalis fruit in the prior art.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Example 1: construction method of UPLC characteristic spectrum of calyx seu fructus physalis formula particles

1.1 instruments

Ultra performance liquid chromatograph 1: thermo Fisher Vanquish UPLC chromatography systems, including quaternary solvent manager (Pμmp), autosampler (Autosampler), as-built inlet column incubator (Colμmn component), diode array ultraviolet Detector (Detector), chromeleon chromatography management system;

ultra performance liquid chromatograph 2: agilent 1290 Infinicity II, comprising a G7104A quaternary pump, a 132 bit autosampler, a G7116B column incubator, and a diode array detector;

METTER TOLEDO (METHOD. Switzerland) ME36S, XS, XS205, XSE205 (parts per million);

SK5200H Shanghai Uygur instrument Co., ltd

Chromatographic column: waters CORTECS UPLC T3 2.1X100 nm 1.6 μm

Agilent SB RRHD C18 2.1×100nm 1.8μm

Waters ACQUITY BEH Shield RP18 2.1×100nm 1.7μm

Waters CORTECS UPLC T3 2.1×150nm 1.7μm

Reagent: acetonitrile is chromatographic pure, and water is ultrapure water; other reagents such as phosphoric acid and methanol are all analytically pure.

1.2 reagent reagents

Oleacosides control (lot number 111720-202111, purchased from China food and drug inspection institute)

Luteolin reference substance (lot number 111520-202006, purchased from China food and drug inspection institute)

A control of acid pulp picrin A (lot number CFS202002, available from Chemfacies)

Physalin L reference substance (lot number: 111831-201001, purchased from China food and drug inspection institute)

4,7 didehydro New physalin B control (lot number CFS202102, available from Chemfaces)

1.3 chromatographic Condition optimization

Preparation of reference solution about 1g of calyx seu fructus Physalis reference material is taken, put into a conical flask with a plug, added with 25mL of water, heated and refluxed for 40 minutes, taken out, filtered, evaporated to dryness, cooled, and the residue is added with 20mL of 70% methanol, treated by ultrasound (power 250W, frequency 40 kHz) for 30 minutes, filtered, and the subsequent filtrate is taken to obtain reference solution of reference material. And (3) taking a proper amount of luteolin reference substance, precisely weighing, placing into a brown measuring flask, and adding 70% methanol to prepare a solution containing 20 mug per 1mL serving as the reference substance solution of the reference substance.

Preparation of test solution about 0.2g of calyx seu fructus physalis formula granule was taken, 20mL of 70% methanol was added thereto, and the mixture was subjected to ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, and filtered to obtain a test solution.

Chromatographic conditions: acetonitrile was mobile phase a and 0.2% phosphoric acid solution was mobile phase B.

1.3.1 gradient optimization

The column was first screened and the gradient was optimally examined, as shown in tables 1-8 and figures 1-7.

TABLE 1 chromatographic conditions

TABLE 2 gradient 1

TABLE 3 gradient 2

TABLE 4 gradient 3

TABLE 5 gradient 4

TABLE 6 gradient 5

TABLE 7 gradient 6

TABLE 8 gradient 7

Through the investigation, a Agilent SB RRHD C (2.1 mm multiplied by 100mm;1.8 mu m) chromatographic column is used for gradient 1, the flow rate is 0.3mL/min, the column temperature is 25 ℃ for separation, and the result shows that the main peak has a relatively late peak-to-peak time, the peak 5 to peak 8 separation effect from 18 minutes to 20 minutes later is poor, the base line is uneven, and the continuous optimization is carried out; the gradient 2 makes the peak 1 and 2 peak-out time forward on the basis of the gradient 1, the effect is not greatly different from the gradient 1, and the separation effect from the peak 5 to the peak 8 is still not good; gradient 3 was isolated using CORTECS UPLC T3 (2.1 mm. Times.150 mm;1.7 μm), with uneven baseline; the gradients 4 and 5 are separated by a CORTECS UPLC T3 (2.1 mm multiplied by 100mm;1.6 mu m), and the peak time from the peak 5 to the peak 8 is relatively later and relatively compact; the gradient 6 is inspected by using a ACQUITY BEH Shield RP (2.1 mm multiplied by 100mm;1.7 mu m) chromatographic column, the main peak separation effect is poor, the middle impurity peaks are more, and the chromatographic column has larger influence on the peak separation degree; gradient 7 was isolated using a CORTECS UPLC T3 (2.1 mm. Times.100 mm;1.6 μm) column with good peak 5 to peak 8 separation, and therefore, gradient 7 was the best, and the tentative feature map method was gradient 7.

1.3.2 investigation of durability of different instruments

According to the method, samples are respectively injected under different brands of chromatographs (Thermo Fisher Vanquish UPLC and Agilent 1290 II), the durability of different instruments is examined, the results are shown in figures 8-9, and the results show that the durability of different instruments is good.

From the above investigation, the tentative chromatographic conditions were: octadecylsilane chemically bonded silica is used as a filler; gradient elution was performed as specified in table 9 with acetonitrile as mobile phase a and 0.2% phosphoric acid solution as mobile phase B; the wavelength is 220nm; the column temperature is 25 ℃; the flow rate was 0.3mL per minute; the theoretical plate number should be not less than 5000 calculated by luteolin.

TABLE 9 elution gradient

1.4 examination of sample treatment method

1.4.1 investigation of extraction solvent species

Through the optimized gradient, each characteristic peak is better in separation, but the chromatographic peak response is found to be smaller, and the baseline is unstable, so that the investigation on different extraction solvents of the sample is continued, and the response can be increased under the condition of keeping the main characteristic peak, and the baseline fluctuation is slowed down.

The extraction effect of water, 30% methanol, 50% methanol, 70% methanol, methanol and 50% ethanol on the standard decoction freeze-dried powder is examined. The results are shown in tables 10-15 and FIGS. 10-15. The results show that the baseline fluctuation of the ethanol extraction chromatogram is larger, the chromatographic peak separation effect and the system adaptability parameters of the methanol extraction solvent with different proportions are relatively better, but the characteristic peak response value does not show the lifting trend, the condition of larger baseline fluctuation is not improved, and the tentative extraction solvent is 70% methanol.

Table 10 water extraction system adaptation parameters

TABLE 1130% methanol extraction System Adaptation parameters

TABLE 12 50% methanol extraction System Adaptation parameters

Table 1370% methanol extraction System Adaptation parameters

TABLE 14 pure methanol extraction System Adaptation parameters

TABLE 15 50% ethanol extraction System Adaptation parameters

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1.4.2 investigation of extraction solvent types and times

From the above, by adopting different types and proportions of extraction solvents, each characteristic peak is basically consistent and better in separation, but the characteristic peak response is still lower, and the problem of uneven base line is not solved. Next, the effect on the response of the characteristic peaks after extraction with organic solvents of different polarities was examined, and the results are shown in tables 16 to 21 and FIGS. 16 to 20. The results show that when the extraction solvent is n-butanol, the condition of uneven baseline still exists, and when the extraction solvent is chloroform or diethyl ether, the baseline is stable, but the whole peak area is too small, and the number of peaks is reduced; when the extraction solvent is ethyl acetate, the base line is stable, the number of target peaks is increased by 1, and the peak areas of all characteristic peaks are proper, so that the extraction solvent is ethyl acetate temporarily, and the extraction is examined for 2 times and 3 times, and the result shows that the characteristic peaks are not obviously influenced. Thus, the extraction solvent was determined to be ethyl acetate, the number of times being 2.

Table 16 extraction solvent investigation test sample treatment method

Table 17 ethyl acetate extraction 2 system adaptation parameters

TABLE 18 System Adaptation parameters for n-butanol extraction 2 times

TABLE 19 System adaptive parameters for chloroform extraction 2 times

TABLE 20 System Adaptation parameters for diethyl ether extraction 2 times

TABLE 21 System Adaptation parameters for extraction of ethyl acetate 3 times

1.4.3 sample weighing investigation

After the baseline problem was solved, the symmetrical sample amounts were continuously examined, and the influence of the weighed sample amounts of 0.2g, 0.4g, 0.6g and 0.8g on the chromatographic peak was examined, and the results are shown in tables 22 to 25 and FIGS. 21 to 24. From the results, the peak area of the target peak tended to increase as a whole with increasing sample weighing, so that the baseline, peak shape and degree of separation were combined, and finally the sample weighing was determined to be 0.4g.

TABLE 22 sample weighing 0.2g System adaptive parameters

TABLE 23 sample weighing 0.4g System adaptive parameters

TABLE 24 sample weighing 0.6g System adaptive parameters

Table 25 sample weighing 0.8g System adaptability parameters

1.4.4 sample injection volume investigation

The effect of different sample amounts of 3. Mu.L, 5. Mu.L, 7. Mu.L on the chromatographic peaks was examined according to the method determined above, and the results are shown in tables 26 to 28 and FIGS. 25 to 27. As is clear from the above, when the sample injection volume was 5. Mu.L and 7. Mu.L, peak 1 was a split peak, peak 1 was luteolin, the response in the whole spectrum was highest, and the column used was a 1.6 μm column with a smaller particle size, so that it was assumed that the split peak was caused by overload of the sample amount, and thus the tentative sample injection amount was 3. Mu.L.

TABLE 26 sample size 3. Mu.L System Adaptation parameters

Table 27 sample size 5. Mu.L System Adaptation parameters

Table 28 sample size 7. Mu.L System Adaptation parameters

To sum up, the tentative test article processing method comprises the steps of: taking a proper amount of the product, grinding, taking about 0.4g, adding 10mL of water for dissolution, extracting twice with 10mL of ethyl acetate each time, combining ethyl acetate solutions, evaporating to dryness, adding 10mL of 70% methanol into residues, performing ultrasonic treatment (power is 250W, frequency is 40 kHz) for 30 minutes, and filtering to obtain a sample solution.

1.5 further identification of characteristic peaks in a characteristic Spectrum

Through the determined characteristic spectrum method, the characteristic peaks are reselected by combining a plurality of batches of measurement results, the identification of the characteristic peaks is deeply studied, the molecular structure analysis is carried out through LC/MS/MS, and the reference substance positioning and the spectral study are adopted, so that the peak 1 is luteolin, the peak 3 is luteolin, the peak 4 is physalin A, the peak 6 is physalin L, and the peak 10 is 4, 7-didehydro-physalin B. Since the reference substances were not purchased for the peaks 2, 7, 8 and 9, they were not specified in the present invention. The LC/MS/MS analysis structure is shown in Table 29 and FIGS. 28-44.

Table 29 LC/MS/MS analysis and detection results of calyx seu fructus physalis formula particles

1.6 determination of chromatographic conditions

Through optimizing chromatographic conditions, the tentative UPLC characteristic spectrum chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler; gradient elution was performed as specified in table 30 with acetonitrile as mobile phase a and 0.2% phosphoric acid solution as mobile phase B; the detection wavelength is 220nm; the flow rate was 0.3mL per minute and the column temperature was 25 ℃. The theoretical plate number should be not less than 5000 calculated by luteolin. The results are shown in FIG. 45 and Table 31.

Table 30

Table 31 sample line system adaptation parameters

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1.7 methodological validation

1.7.1 precision

0.4g of calyx seu fructus physalis formula particles is taken, precisely weighed, a test sample is prepared according to the test sample treatment method in 1.4.4, sample injection is continuously carried out for 6 times, a chromatogram is recorded, the relative retention time of each characteristic peak and RSD of the relative peak area are calculated, and the results are shown in tables 32-33.

Table 32 results of instrument precision versus retention time test

Table 33 results of instrument precision versus peak area test

The results show that: the relative retention time RSD% of the 11 characteristic peaks is less than 2% and the relative peak area RSD% is less than 3%. The method has good precision and meets the requirements of characteristic patterns.

1.7.2 method repeatability test

0.4g of the same batch of calyx seu fructus physalis formula particles are taken, 6 parts are weighed in parallel, precisely weighed, a test sample is prepared according to the test sample treatment method in 1.4.4, sample injection analysis is carried out, a chromatogram is recorded, the relative retention time of each characteristic peak and the RSD of the relative peak area are calculated, and the results are shown in tables 34-35 and FIG. 47.

Table 34 method repeatability vs. retention time test results (n=6)

Table 35 method repeatability versus peak area test results (n=6)

The results show that: the relative retention time RSD% of the 11 characteristic peaks is less than 2%, the relative peak area RSD% is less than 2%, and the comprehensive judgment is that the method is good in repeatability and meets the requirements.

1.7.3 intermediate precision (different operators)

The same batch of calyx seu fructus physalis formula particles is taken, the first, second and third laboratory workers are separately and independently operated, a test product is prepared according to the preparation method of the test product, sample injection analysis is carried out, a chromatogram is recorded, the relative retention time of each characteristic peak and the RSD of the relative peak area are calculated, and the results are shown in tables 36-37 and fig. 48.

Table 36 results of intermediate precision versus retention time test (different operators)

Table 37 results of intermediate precision relative peak area test (different operators)

The results show that: the relative retention time RSD% of the 11 characteristic peaks is less than 2% and the relative peak area RSD% is less than 3%. The comprehensive judgment is carried out, the intermediate precision (different personnel) of the method is good, and the requirements of the characteristic spectrum are met.

1.7.4 intermediate precision (different column lot numbers)

According to the chromatographic condition optimization and the chromatographic column selection result, the chromatographic condition reappearance needs to fix CORTECS UPLC T3, 100 x 2.1mm;1.6 μm column, thus, the same sample solution was taken and tested with different production lot (lot 012131419407; lot 01183110315331; lot 01213130815310) columns, the chromatograms were recorded, and the relative retention time of each characteristic peak and RSD of the relative peak area were calculated, and the results are shown in tables 38-39 and fig. 49-51.

Table 38 results of intermediate precision versus retention time experiments (different batch chromatographic columns)

Table 39 results of intermediate precision relative peak area test (different batch chromatographic columns)

The results show that: the relative retention time RSD% of the 11 characteristic peaks is less than 2% and the relative peak area RSD is less than 3.5%. The method has better intermediate precision (chromatographic columns in different batches) and meets the requirements of characteristic patterns.

1.7.5 speciality

Preparing a test solution according to the preparation method of the test solution, and examining whether a calyx seu fructus physalis formula particle negative sample can cause interference. HPLC analysis was performed as described above and chromatograms were recorded as shown in FIGS. 52-53. The result shows that the method has no interference and good specificity.

1.7.6 durability

1.7.6.1 stability investigation

Taking the same sample solution, and injecting samples at 0, 4, 8, 12, 16 and 24 hours after preparation. The chromatogram was recorded and the relative retention time and RSD of the relative peak area for each characteristic peak was calculated to investigate the stability of the test sample solution. The results are shown in tables 40-41 and FIG. 52.

Table 40 stability versus retention time test results

Table 41 stability versus peak area test results

The results show that: the relative retention time of the 11 characteristic peaks is less than 2% and the relative peak area RSD% is less than 3%. The test solution was stable over 24 hours.

Investigation of different flow Rate of 1.7.6.2

The same sample solution was taken and tested at different flow rates of 0.28mL/min, 0.30mL/min and 0.32mL/min, chromatograms were recorded, and the relative retention time and RSD of the relative peak area of each characteristic peak were calculated, with the results shown in tables 42-43 and FIGS. 55-57.

Table 42 relative retention time results for different flow rates

TABLE 43 relative peak area results for different flow rates

The result shows that the relative retention time RSD% of 11 characteristic peaks is 0% -5%, the relative peak area RSD% of 11 characteristic peaks is 0% -8%, and the graph shows that different flow rates have certain influence on the separation degree of the characteristic peaks, and 0.30mL/min is selected as the flow rate of the calyx seu fructus physalis formula particle characteristic spectrum measuring method for guaranteeing the separation reproduction of the characteristic spectrum.

Investigation of 1.7.6.3 different column temperatures

The same sample solution was tested at 23℃25℃and 28℃respectively, chromatograms were recorded, and the relative retention time and RSD of the relative peak area of each characteristic peak were calculated, and the results are shown in tables 44 to 45 and FIGS. 58 to 60.

Table 44 results of different column temperature determinations relative retention time

Table 45 different columns Wen Xiangdui peak area results

The result shows that the relative retention time RSD% of 11 characteristic peaks is less than 5%, the relative peak area RSD% of 7 characteristic peaks is between 0% and 25%, and the graph shows that the separation degree of the characteristic peaks is affected to a certain extent under different column temperature conditions, so as to ensure the separation and reproduction of the characteristic spectrum, the fixed column temperature is suggested to be 25 ℃.

As shown by the result of the methodology investigation, among 11 common peaks of the established characteristic spectrum of the calyx seu fructus physalis formula particle, each color spectrum peak is influenced by different color spectrum columns, different column temperatures and different flow rates to a certain extent, and the influence of other color spectrum conditions is not greatly changed. The relative retention time value is within + -10%, and to accommodate its durability, it is recommended to control the specified value range within + -10%.

Example 2: construction method of UPLC (unified power control language) contrast characteristic spectrum of calyx seu fructus physalis formula particles

3 batches of calyx seu fructus physalis formula particles (batch numbers: 1902001Y, 1902002Y, 1902003Y; manufacturer: huarun Sanjiu medicine Co., ltd.) were taken and operated according to the established characteristic spectrum measuring method of the calyx seu fructus physalis formula particles, and the results are shown in tables 46-47 and FIG. 61. The measurement result shows that the relative retention time specified by each characteristic peak of the characteristic spectrum of the 3 batches of calyx seu fructus physalis formula particles is within +/-10% of a specified value, and the relative peak area meets the requirement.

And (3) referring to technical guidelines (trial runs) of fingerprint research of traditional Chinese medicine injection, fitting the obtained fingerprints by using software of a 'Chinese medicine chromatographic fingerprint similarity evaluation system 2012 edition' recommended by the Chinese pharmacopoeia Committee. And analyzing the common peak information of 3 batches of calyx seu fructus physalis formula particles shown in fig. 61, selecting the common peak with good separation degree and purer chromatographic peak as the common peak of the characteristic spectrum of the calyx seu fructus physalis formula particles, and establishing a UPLC comparison characteristic spectrum of the calyx seu fructus physalis formula particles, as shown in fig. 62.

TABLE 46 relative retention time of batch calyx seu fructus physalis formula particles

TABLE 47 relative peak area of batch calyx seu fructus physalis formula particles

Relative retention time specification: determining 11 characteristic peaks in the chromatogram of the sample to be tested according to the characteristic spectrum measurement result of 3 batches of calyx seu fructus physalis formula particles, and corresponding to 11 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material, wherein the retention time of the peak 1 corresponds to the retention time of the corresponding reference peak of the reference substance; the peak corresponding to the luteolin reference peak is an S peak, the relative retention time of the characteristic peaks 2-11 and the S peak is calculated to be within +/-10% of a specified value, and the specified value is: 2.23 (Peak 2), 2.75 (Peak 3), 3.52 (Peak 4), 3.69 (Peak 5), 4.07 (Peak 6), 4.26 (Peak 7), 4.46 (Peak 8), 4.61 (Peak 9), 5.45 (Peak 10), 6.32 (Peak 11).

Relative peak area specification: from the results, the relative peak areas of the 3 batches of test production formula particles are in the range of the multi-batch standard decoction provided in the embodiment 4, the transmissibility is good, but the multi-batch ranges of the relative peak areas of the peaks 3, 6 and the reference peak are wider, no obvious change rule exists, if the range is established by showing or averaging + -30% of the actual measurement of the relative peak areas of the multi-batch standard decoction, the relative peak area ratio is too low, so that the meaning of the multi-batch standard decoction is not great in the final finished product standard; the peak area of the peak 4 and the reference peak is moderate in the multi-batch range, so that the range is established by the practical lower limit of the peak area of the multi-batch standard decoction, namely the specified range is not lower than 0.16. The calyx seu fructus physalis decoction pieces are prepared into calyx seu fructus physalis formula particles by extraction, concentration and drying, and the basic properties of the calyx seu fructus physalis formula particles are basically consistent with that of the calyx seu fructus physalis standard decoction, so that the relative peak area value of the calyx seu fructus physalis formula particles is carried out by referring to the standard decoction lyophilized powder in the embodiment 4. That is, the relative peak area of the prescribed peak 4 and the S peak is not less than 0.16.

Example 3: UPLC characteristic spectrum construction method of calyx seu fructus physalis standard decoction freeze-dried powder

1. Chromatographic condition and System applicability test

Octadecylsilane chemically bonded silica is used as a filler; gradient elution was performed as specified in table 48 with acetonitrile as mobile phase a and 0.2% phosphoric acid solution as mobile phase B; the wavelength is 220nm; the column temperature is 25 ℃; the flow rate was 0.3mL per minute; the theoretical plate number should be not less than 5000 calculated by luteolin.

Table 48

Preparation of reference solution about 1g of calyx seu fructus Physalis reference material is taken, put into a conical flask with a plug, added with 25mL of water, heated and refluxed for 40 minutes, taken out, filtered, extracted twice with ethyl acetate, 25mL each time, combined with ethyl acetate solution, evaporated to dryness, cooled, the residue is precisely added with 10mL of 70% methanol, subjected to ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, filtered, and the subsequent filtrate is taken, thus obtaining the reference solution of reference material. And (3) taking a proper amount of luteolin reference substance, precisely weighing, placing into a brown measuring flask, and adding 70% methanol to prepare a solution containing 20 mug per 1mL serving as the reference substance solution of the reference substance.

Preparation of sample solution about 0.4g of the sample was taken, dissolved by adding 10mL of water, extracted twice with 10mL of ethyl acetate each time, combined with ethyl acetate solution, evaporated to dryness, and the residue was added with 10mL of 70% methanol, sonicated (power 250W, frequency 40 kHz) for 30 minutes, and filtered to give a sample solution.

3 mu L of each of the reference solution and the sample solution was precisely aspirated, and the mixture was injected into a liquid chromatograph for measurement, and chromatograms were recorded, as shown in FIGS. 63 to 64 and Table 49.

Table 49 System Adaptation parameters

2. Methodological verification

2.1 precision

0.4g of calyx seu fructus physalis freeze-dried powder is taken, precisely weighed, a test product is prepared according to the method of preparing a test product solution, sample injection is continuously carried out for 6 times, a chromatogram is recorded, the relative retention time of each characteristic peak and the RSD of the relative peak area are calculated, and the results are shown in tables 50-51 and fig. 65.

Table 50 results of instrument precision versus retention time test

Table 51 shows results of the instrument precision relative to peak area test

The results show that: the relative retention time RSD% of the 11 characteristic peaks is less than 2% and the relative peak area RSD% is less than 2%. The method has good precision and meets the requirements of characteristic patterns.

2.2 method repeatability test

0.4g of the same batch of calyx seu fructus physalis freeze-dried powder is taken, 6 parts are weighed in parallel, precisely weighed, a test sample is prepared according to the method of preparing test sample solution, sample injection analysis is carried out, a chromatogram is recorded, the relative retention time of each characteristic peak and the RSD of the relative peak area are calculated, and the results are shown in tables 52-53 and fig. 66.

Table 52 method repeatability vs. retention time test results (n=6)

Table 53 method repeatability versus peak area test results (n=6)

The results show that: the relative retention time RSD% of the 11 characteristic peaks is less than 2%, the relative peak area RSD% is less than 3%, and the comprehensive judgment is that the method is good in repeatability and meets the requirements.

2.3 intermediate precision (different operators)

The same batch of calyx seu fructus physalis lyophilized powder is taken, and is separately and independently operated by an experiment personnel A, an experiment personnel B and an experiment personnel C, a sample is prepared according to a sample preparation method, sample injection analysis is carried out, a chromatogram is recorded, the relative retention time of each characteristic peak and the RSD of the relative peak area are calculated, and the results are shown in tables 54-55 and figure 67.

Table 54 results of intermediate precision versus retention time test (different operators)

Table 55 intermediate precision relative peak area test results (different operators)

2.4 intermediate precision (different chromatography column batch numbers)

According to the chromatographic condition optimization and the chromatographic column selection result, the chromatographic condition reappearance needs to fix CORTECS UPLC T3, 100 x 2.1mm;1.6 μm column, thus, the same sample solution was taken and tested with different production lot (lot 012131419407; lot 01183110315331; lot 01213130815310) columns, respectively, and chromatograms were recorded to calculate the relative retention time of each characteristic peak and RSD of the relative peak area, and the results are shown in tables 56-57 and fig. 68-70.

Table 56 results of intermediate precision relative retention time test (different batch chromatographic columns)

Table 57 results of intermediate precision relative peak area test (different batch chromatographic columns)

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The results show that: the relative retention time RSD% of the 11 characteristic peaks is less than 2% and the relative peak area RSD is less than 4%. The method has better intermediate precision (chromatographic columns in different batches) and meets the requirements of characteristic patterns.

2.5 specificity

Preparing a test solution according to the preparation method of the test solution, and examining whether a calyx seu fructus physalis freeze-dried powder negative sample can cause interference. HPLC analysis was performed according to the chromatographic conditions described under item [ feature map ], and chromatograms were recorded, see FIGS. 71-72. The result shows that the method has no interference and good specificity.

2.6 durability

2.6.1 stability investigation

Taking the same sample solution, and injecting samples at 0, 4, 8, 12, 16 and 24 hours after preparation. The chromatogram was recorded and the relative retention time and RSD of the relative peak area for each characteristic peak was calculated to investigate the stability of the test sample solution. The results are shown in tables 58-59 and FIG. 73.

Table 58 stability versus retention time test results

Table 59 stability vs peak area test results

The results show that: the relative retention time of the 11 characteristic peaks is less than 2% and the relative peak area RSD% is less than 4%. The test solution was stable over 24 hours.

2.6.2 investigation of different flow rates

The same sample solution was taken and tested at different flow rates of 0.28mL/min, 0.30mL/min and 0.32mL/min, chromatograms were recorded, and the relative retention time and RSD of the relative peak area of each characteristic peak were calculated, with the results shown in tables 60-61 and FIGS. 74-76.

Table 60 relative retention time results for different flow rates

TABLE 61 relative peak area results for different flow rates

The result shows that the relative retention time RSD% of 11 characteristic peaks is 0% -5%, the relative peak area RSD% of 11 characteristic peaks is 0% -23%, and the graph shows that different flow rates have certain influence on the separation degree of the characteristic peaks, and 0.30mL/min is selected as the flow rate of the calyx seu fructus physalis freeze-dried powder characteristic spectrum measuring method for guaranteeing the separation reproduction of the characteristic spectrum.

Investigation of 2.6.3 different column temperatures

The same sample solution was tested at 23℃25℃and 28℃respectively, chromatograms were recorded, and the relative retention time of each characteristic peak and RSD of the relative peak area were calculated, and the results are shown in tables 62 to 63 and FIGS. 77 to 79.

Table 62 results of different column temperature measurements relative retention time

Table 63 results of different columns Wen Xiangdui peak areas

The result shows that the relative retention time RSD% of 11 characteristic peaks is less than 7%, the relative peak area RSD% of 7 characteristic peaks is between 0% and 60%, and the graph shows that the separation degree of the characteristic peaks is influenced under different column temperature conditions, so that the separation reproduction of the characteristic spectrum is ensured, and the fixed column temperature is recommended to be 25 ℃.

According to the research results of the methodology, among 11 common peaks of the established characteristic spectrum of the calyx seu fructus physalis freeze-dried powder, each chromatographic peak is influenced by different chromatographic columns, different column temperatures and different flow rates to a certain extent, and the influence changes of other chromatographic conditions are small. To accommodate its durability, it is recommended to control the specified value range within ±10%.

Example 4: construction method of UPLC (unified power control) contrast characteristic spectrum of standard calyx seu fructus physalis decoction freeze-dried powder

15 batches of standard calyx seu fructus physalis lyophilized powder (batch numbers: 1808001Y, 1808002Y, 1808003Y, 1808004Y, 1808005Y, 1808006Y, 1808007Y, 1808008Y, 1808009Y, 1808010Y, 1808011Y, 1808012Y, 1808013Y, 1808014Y, 1808015Y; self-developed) were taken, and the operation was performed according to the characteristic spectrum determination method of the calyx seu fructus physalis lyophilized powder provided in example 3, and the results are shown in tables 64-65 and FIG. 80. The measurement result shows that the relative retention time of each characteristic peak of the characteristic spectrum of 15 batches of calyx seu fructus physalis freeze-dried powder is within +/-10% of the specified value of the calyx seu fructus physalis freeze-dried powder control spectrum.

Referring to technical guidelines (trial runs) of Chinese medicinal injection fingerprint research, the obtained fingerprints are fitted by using software of a Chinese pharmacopoeia Committee recommended "Chinese medicinal chromatographic fingerprint similarity evaluation system 2012 edition", common peak information of 15 batches of calyx seu fructus physalis lyophilized powder shown in figure 80 is analyzed, common peaks with good separation degree and purer chromatographic peaks are selected as common peaks of calyx seu fructus physalis formula particle characteristic fingerprints, and a calyx seu fructus physalis formula particle UPLC control characteristic spectrum is established, as shown in figure 81.

Table 6415 relative retention time of standard decoction lyophilized powder of calyx seu fructus physalis

Table 6515 relative peak area of standard decoction lyophilized powder of calyx seu fructus Physalis

Relative retention time specification: according to the characteristic spectrum measurement result of the freeze-dried powder of 15 batches of calyx seu fructus physalis standard decoction, determining: 11 characteristic peaks should be displayed in the sample chromatogram and correspond to 11 characteristic peaks in the reference chromatogram of the reference medicinal material, wherein the retention time of peak 1 corresponds to the retention time of the corresponding reference peak of the reference medicinal material; the peak corresponding to the luteolin reference peak is an S peak, the relative retention time of the characteristic peaks 2-11 and the S peak is calculated to be within +/-10% of a specified value, and the specified value is: 2.23 (Peak 2), 2.75 (Peak 3), 3.52 (Peak 4), 3.69 (Peak 5), 4.07 (Peak 6), 4.26 (Peak 7), 4.46 (Peak 8), 4.61 (Peak 9), 5.45 (Peak 10), 6.32 (Peak 11).

Relative peak area ratio specified value: the calyx seu fructus physalis mainly contains steroid, flavonoid, phenylpropanoid, alkaloid and polysaccharide compounds, etc., wherein the steroid, flavonoid are the main components, and have antibacterial, antiinflammatory, antioxidant and antitumor pharmacological effects. Steroid compounds are the most chemical components found in calyx seu fructus physalis plants in recent decades, and mainly comprise physalis alkekengi bitter elements, neophysalis alkekengi bitter elements and sterol compounds; flavonoids are also one of the components found in calyx seu fructus physalis. By combining characteristic spectrum peak identification information, peak 1 is luteolin, peak 3 is luteolin, and both are flavonoid compounds, and have anti-diabetes, anti-inflammatory and anti-tumor activities; peak 4 is Physalin A, peak 6 is Physalin L, physalin compounds in calyx seu fructus Physalis are most, and are characteristic chemical components, and have anti-diabetes, antiinflammatory and antitumor activities. Therefore, the relative peak area data of the peak 1, the peak 3, the peak 4 and the peak 6 are analyzed, the peak 1 is luteolin, and the luteolin is used as a content measurement index, so that the luteolin is suitable for being used as an S peak to calculate the relative peak areas of other characteristic peaks and control. Analyzing the relative peak area range of 0.07-1.21 of peak 3, the relative peak area range of 0.23-1.18 of peak 4 and the relative peak area range of 0.04-0.58 of peak 6, wherein the relative peak areas of peak 3, peak 6 and reference peak are wider in a plurality of batches, and have no obvious change rule, if the range is established by showing or averaging + -30% of the actual measurement of the relative peak areas of a plurality of batches of standard decoction, the relative peak area ratio is too low, so that the meaning of the relative peak area ratio in the final finished product standard is not great; the relative peak areas of the peak 4 and the reference peak are moderate in a plurality of batches and can be transferred in an overstable manner, so that the range is established by using the measured minimum value of the relative peak areas of the medicinal materials in a plurality of batches of-30%, namely, the specified range is as follows: not less than 0.16.

In order to determine the magnitude transfer conditions throughout the entire study, the analytical method validation and multi-batch testing of the raw materials were re-performed as follows, with reference to the UPLC profile determination methods provided in examples 1-4. The verification result shows that the method is simple and convenient to operate, accurate in result and good in repeatability.

Example 5: construction method of UPLC characteristic spectrum of calyx seu fructus physalis medicinal material

1. Chromatographic condition and System applicability test

Octadecylsilane chemically bonded silica is used as a filler; gradient elution was performed as specified in table 66 with acetonitrile as mobile phase a and 0.2% phosphoric acid solution as mobile phase B; the wavelength is 220nm; the column temperature is 25 ℃; the flow rate was 0.3mL per minute; the theoretical plate number should be not less than 5000 calculated by luteolin.

Table 66

Preparation of sample solution 1.0g of sample powder (sieving with a third sieve), precisely weighing, placing into a conical flask with a plug, adding 25mL of water, heating and refluxing for 40 minutes, taking out, filtering, extracting the filtrate with ethyl acetate twice, 25mL each time, combining ethyl acetate solutions, evaporating to dryness, cooling, precisely adding 70% methanol into residues, 10mL, performing ultrasonic treatment for 30 minutes, filtering, and taking the subsequent filtrate to obtain the sample solution.

The measurement method comprises precisely sucking 3 μl of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and recording chromatograms, as shown in fig. 82-83 and table 67.

Table 67 system adaptation parameters

2. Methodological verification

2.1 precision

1.0g of calyx seu fructus physalis medicinal material is precisely weighed, a test sample is prepared according to the method of preparing a test sample solution, sample injection is continuously carried out for 6 times, a chromatogram is recorded, the relative retention time of each characteristic peak and the RSD of the relative peak area are calculated, and the results are shown in tables 68-69 and figure 84.

Table 68 results of instrument precision versus retention time test

Table 69 instrument precision relative peak area test results

2.2 method repeatability test

1.0g of the same batch of calyx seu fructus physalis medicinal material is taken, 6 parts are weighed in parallel, precisely weighed, a test sample is prepared according to the method of preparing test sample solution, sample injection analysis is carried out, a chromatogram is recorded, the relative retention time of each characteristic peak and the RSD of the relative peak area are calculated, and the results are shown in tables 70-71 and figure 85.

Table 70 method repeatability vs. retention time test results (n=6)

Table 71 method repeatability versus peak area test results (n=6)

The results show that: the relative retention time RSD% of the 11 characteristic peaks is less than 2%, the relative peak area RSD% is less than 6%, and the comprehensive judgment is that the method is good in repeatability and meets the requirements.

2.3 intermediate precision (different operators)

The same batch of calyx seu fructus physalis medicinal materials is taken, the first, second and third laboratory workers are separated and independently operated, a sample is prepared according to the preparation method of the sample, the sample is injected and analyzed, a chromatogram is recorded, the relative retention time of each characteristic peak and the RSD of the relative peak area are calculated, and the results are shown in tables 72-73 and fig. 86.

Table 72 results of intermediate precision versus retention time test (different operators)

Table 73 results of intermediate precision relative peak area test (different operators)

The results show that: the relative retention time RSD% of the 11 characteristic peaks is less than 2% and the relative peak area RSD% is less than 12%. The comprehensive judgment is carried out, the intermediate precision (different personnel) of the method is good, and the requirement of the characteristic spectrum is met.

2.4 intermediate precision (different chromatography column batch numbers)

According to the chromatographic condition optimization and the chromatographic column selection result, the chromatographic condition reappearance needs to fix CORTECS UPLC T3, 100 x 2.1mm;1.6 μm column, thus, the same sample solution was taken and tested with different production lot (lot 01213131419407; lot 01183110315331; lot 01213130815310) columns, respectively, and chromatograms were recorded to calculate the relative retention time of each characteristic peak and RSD of the relative peak area, and the results are shown in tables 74-75 and fig. 87-89.

Table 74 results of intermediate precision relative retention time test (different batch chromatographic columns)

Table 75 results of intermediate precision relative peak area test (different batch chromatographic columns)

The results show that: the relative retention time RSD% of the 11 characteristic peaks is less than 2% and the relative peak area RSD is less than 12%. The method has better intermediate precision (chromatographic columns in different batches) and meets the requirements of characteristic patterns.

2.5 specificity

Preparing a test solution according to the preparation method of the test solution, and examining whether a negative sample of the calyx seu fructus physalis medicinal material can cause interference. HPLC analysis was performed under the aforementioned chromatographic conditions, and chromatograms were recorded, see FIGS. 90-91. The result shows that the method has no interference and good specificity.

2.6 durability

2.6.1 stability investigation

Taking the same sample solution, and injecting samples at 0, 4, 8, 12, 16 and 24 hours after preparation. The chromatogram was recorded and the relative retention time and RSD of the relative peak area for each characteristic peak was calculated to investigate the stability of the test sample solution. The results are shown in tables 76-77 and FIG. 92.

Table 76 stability versus retention time test results

Table 77 stability vs peak area test results

The results show that: the relative retention time of the 11 characteristic peaks is less than 2% and the relative peak area RSD% is less than 20%. The test solution was stable over 24 hours.

2.6.2 investigation of different flow rates

The same sample solution was taken and tested at different flow rates of 0.28mL/min, 0.30mL/min and 0.32mL/min, chromatograms were recorded, and the relative retention time and RSD of the relative peak area of each characteristic peak were calculated, with the results shown in tables 78-79 and FIGS. 93-95.

Table 78 results of different flow rates versus retention time

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TABLE 79 relative peak area results for different flow rates

The result shows that the relative retention time RSD% of 11 characteristic peaks is 0% -3.1%, the relative peak area RSD% of 11 characteristic peaks is 0% -14.2%, and the graph shows that different flow rates have certain influence on the separation degree of the characteristic peaks, and 0.30mL/min is selected as the flow rate of the calyx seu fructus physalis medicinal material characteristic map measuring method for guaranteeing the separation reproduction of the characteristic map.

Investigation of 2.6.3 different column temperatures

The same sample solution was tested at 23℃25℃and 28℃respectively, chromatograms were recorded, and the relative retention time of each characteristic peak and the RSD of the relative peak area were calculated, and the results are shown in tables 80 to 81 and FIGS. 96 to 98.

Table 80 results of relative retention time for different column temperature determinations

Table 81 Peak area results for different columns Wen Xiangdui

The result shows that the relative retention time RSD% of 11 characteristic peaks is less than 2%, the relative peak area RSD% of 11 characteristic peaks is between 0% and 26%, and the graph shows that the separation degree of the characteristic peaks is influenced under different column temperature conditions, so that the separation reproduction of the characteristic spectrum is ensured, and the fixed column temperature is recommended to be 25 ℃.

According to the result of the methodology investigation, among 11 common peaks of the established characteristic spectrum of the calyx seu fructus physalis medicinal material, each chromatographic peak is influenced by different chromatographic columns, different column temperatures and different flow rates to a certain extent, and the influence of other chromatographic conditions is not greatly changed. The relative retention time value is within + -10%, and to accommodate its durability, it is recommended to control the specified value range within + -10%.

Example 6: construction method of UPLC (unified power control) contrast characteristic spectrum of calyx seu fructus physalis medicinal material

15 batches of calyx seu fructus physalis medicinal materials (batch numbers: 1806001, 1806002, 1806003, 1806004, 1806005, 1806006, 1806007, 1806008, 1806009, 1806012, 1806016, 1806017, 1806018, 1806019 and 1806020) were taken and operated according to the characteristic spectrum measuring method of the calyx seu fructus physalis medicinal materials provided in example 5, and the results are shown in tables 82-83 and fig. 99. The measurement result shows that the relative retention time specified by each characteristic peak of the characteristic spectrum of 15 batches of calyx seu fructus physalis is within +/-10% of the specified value of the control spectrum of the calyx seu fructus physalis.

Referring to technical guidelines (trial runs) of Chinese medicinal injection fingerprint research, fitting the obtained patterns by using software of a Chinese pharmacopoeia Committee recommended "Chinese medicinal chromatographic fingerprint similarity evaluation system 2012 edition", analyzing the common peak information of 15 batches of calyx seu fructus physalis medicinal materials shown in figure 99, selecting the common peak with good separation degree and purer chromatographic peak as the common peak of the characteristic patterns of the calyx seu fructus physalis medicinal materials, and establishing a calyx seu fructus physalis medicinal material UPLC comparison characteristic pattern, see figure 100.

Table 82 relative retention time of calyx seu fructus physalis

Surface 83 relative peak area of calyx seu fructus Physalis

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Relative retention time specification: according to the characteristic spectrum measurement result of 15 batches of calyx seu fructus physalis medicinal materials, determining: 11 characteristic peaks should be displayed in the sample chromatogram and correspond to 11 characteristic peaks in the reference chromatogram of the reference medicinal material, wherein the retention time of peak 1 corresponds to the retention time of the corresponding reference peak of the reference medicinal material; the peak corresponding to the luteolin reference peak is an S peak, the relative retention time of the characteristic peaks 2-11 and the S peak is calculated to be within +/-10% of a specified value, and the specified value is: 2.23 (Peak 2), 2.75 (Peak 3), 3.52 (Peak 4), 3.69 (Peak 5), 4.07 (Peak 6), 4.26 (Peak 7), 4.46 (Peak 8), 4.61 (Peak 9), 5.45 (Peak 10), 6.32 (Peak 11).

Relative peak area ratio specified value: the calyx seu fructus physalis mainly contains steroid, flavonoid, phenylpropanoid, alkaloid and polysaccharide compounds, etc., wherein the steroid, flavonoid are the main components, and have antibacterial, antiinflammatory, antioxidant and antitumor pharmacological effects. Steroid compounds are the most chemical components found in calyx seu fructus physalis plants in recent decades, and mainly comprise physalis alkekengi bitter elements, neophysalis alkekengi bitter elements and sterol compounds; flavonoids are also one of the components found in calyx seu fructus physalis. By combining characteristic spectrum peak identification information, peak 1 is luteolin, peak 3 is luteolin, and both are flavonoid compounds, and have anti-diabetes, anti-inflammatory and anti-tumor activities; peak 4 is Physalin A, peak 6 is Physalin L, physalin compounds in calyx seu fructus Physalis are most, and are characteristic chemical components, and have anti-diabetes, antiinflammatory and antitumor activities. Therefore, the relative peak area data of the peak 1, the peak 3, the peak 4 and the peak 6 are analyzed, the peak 1 is luteolin, and the luteolin is used as a content measurement index, so that the luteolin is suitable for being used as an S peak to calculate the relative peak areas of other characteristic peaks and control. Analyzing the relative peak area range of 0.07-1.15 of peak 3, the relative peak area range of 0.24-1.13 of peak 4 and the relative peak area range of 0.04-0.58 of peak 6, wherein the relative peak areas of peak 3, peak 6 and reference peak are wider in a plurality of batches, and have no obvious change rule, if the range is established by showing or averaging + -30% of the actual measurement of the relative peak areas of a plurality of batches of standard decoction, the relative peak area ratio is too low, so that the meaning of the relative peak area ratio in the final finished product standard is not great; the relative peak areas of the peak 4 and the reference peak are moderate in a plurality of batches and can be transferred in an overstable manner, so that the range is established by using the measured minimum value of the relative peak areas of the medicinal materials in a plurality of batches of-30%, namely, the specified range is as follows: not less than 0.16.

Example 7: construction method of UPLC characteristic spectrum of calyx seu fructus physalis decoction pieces

1. Chromatographic condition and System applicability test

Octadecylsilane chemically bonded silica is used as a filler; gradient elution was performed as specified in table 84 with acetonitrile as mobile phase a and 0.2% phosphoric acid solution as mobile phase B; the wavelength is 220nm; the column temperature is 25 ℃; the flow rate was 0.3mL per minute; the theoretical plate number should be not less than 5000 calculated by luteolin.

Table 84

The measurement method comprises precisely sucking 3 μl of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and recording chromatograms, as shown in fig. 101-102.

Table 85 System Adaptation parameters

Example 8: construction method of UPLC (unified power line control) contrast characteristic spectrum of calyx seu fructus physalis decoction pieces

15 batches of calyx seu fructus physalis decoction pieces (batch numbers: 1808001Y, 1808002Y, 1808003Y, 1808004Y, 1808005Y, 1808006Y, 1808007Y, 1808008Y, 1808009Y, 1808010Y, 1808011Y, 1808012Y, 1808013Y, 1808014Y and 1808015Y; calyx seu fructus physalis decoction pieces are self-collected in situ and self-processed) are taken, and the operation is carried out according to the characteristic spectrum measuring method of the calyx seu fructus physalis decoction pieces provided in example 7, and the results are shown in tables 86-87 and figure 103. The measurement result shows that the relative retention time specified by each characteristic peak of the characteristic spectrum of 15 batches of calyx seu fructus physalis decoction pieces is within +/-10% of the specified value of the control spectrum of the calyx seu fructus physalis decoction pieces.

Referring to technical guidelines (trial runs) of Chinese medicinal injection fingerprint research, fitting the obtained patterns by using software of a Chinese pharmacopoeia Committee recommended "Chinese medicinal chromatographic fingerprint similarity evaluation system 2012 edition", analyzing the common peak information of 15 batches of calyx seu fructus physalis decoction pieces shown in figure 103, selecting the common peak with good separation degree and purer chromatographic peak as the common peak of the calyx seu fructus physalis decoction piece characteristic patterns, and establishing a calyx seu fructus physalis decoction piece UPLC comparison characteristic pattern, see figure 104.

Table 8615 results of determination of relative retention time by characteristic spectrum of batch calyx seu fructus physalis decoction pieces

Table 8715 results of measuring relative peak area by characteristic spectrum of calyx seu fructus physalis decoction pieces

Relative retention time specification: according to the characteristic spectrum measurement result of 15 batches of calyx seu fructus physalis decoction pieces, determining: 11 characteristic peaks should be displayed in the sample chromatogram and correspond to 11 characteristic peaks in the reference chromatogram of the reference medicinal material, wherein the retention time of peak 1 corresponds to the retention time of the corresponding reference peak of the reference medicinal material; the peak corresponding to the luteolin reference peak is an S peak, the relative retention time of the characteristic peaks 2-11 and the S peak is calculated to be within +/-10% of a specified value, and the specified value is: 2.23 (Peak 2), 2.75 (Peak 3), 3.52 (Peak 4), 3.69 (Peak 5), 4.07 (Peak 6), 4.26 (Peak 7), 4.46 (Peak 8), 4.61 (Peak 9), 5.45 (Peak 10), 6.32 (Peak 11).

Relative peak area specification: the calyx seu fructus physalis medicinal material is prepared into calyx seu fructus physalis decoction pieces after simple purification, and the processing process has no influence on the number and peak area of characteristic peaks, so that the relative peak area value of the calyx seu fructus physalis decoction pieces is executed by referring to the medicinal material. That is, the relative peak area of the prescribed peak 4 and the S peak is not less than 0.16.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. The UPLC characteristic spectrum construction method of the calyx seu fructus physalis is characterized by comprising the following steps:

(1) Preparing a sample solution from calyx seu fructus physalis;

taking luteolin, physalin A, physalin L and 4,7 didehydro-neophysalin B as reference substances, and respectively preparing a luteolin reference substance solution, a physalin A reference substance solution, a physalin L reference substance solution and a 4,7 didehydro-neophysalin B reference substance solution;

(2) The method comprises the steps of detecting a sample solution by adopting an ultra-high performance liquid chromatography, taking octadecylsilane chemically bonded silica as a filler, and taking acetonitrile-0.2% phosphoric acid aqueous solution as a mobile phase for gradient elution, wherein the gradient elution procedure comprises: 0- > 7 min- > 15 min- > 30 min- > 32 min- > 40 min, the volume percentage of acetonitrile in the mobile phase is 15% → 17% → 25% → 28% → 38% → 42%;

the chromatographic column used is selected from any one of the following:

waters CORTECS UPLC T32.1.1X100 nm 1.6 μm; or (b)

Agilent SB RRHD C182.1.1X100 nm 1.8 μm; or (b)

Waters ACQUITY BEH Shield RP 182.1.1X100 nm 1.7 μm; or (b)

Waters CORTECS UPLC T32.1×150nm 1.7μm。

2. The construction method according to claim 1, wherein the step (1) comprises the steps of sequentially extracting calyx seu fructus physalis with a solvent, extracting calyx seu fructus physalis with ethyl acetate, and extracting calyx seu fructus physalis with methanol, wherein the calyx seu fructus physalis is a calyx seu fructus physalis medicinal material, calyx seu fructus physalis decoction pieces, calyx seu fructus physalis standard decoction lyophilized powder or calyx seu fructus physalis prescription granule.

3. The method of claim 2, wherein step (1) satisfies any one or more of the following a-E:

E. the methanol extraction mode is ultrasonic extraction, the power of ultrasonic treatment is 250W, the frequency is 40kHz, and the time is 30min.

4. A construction method according to any one of claims 1 to 3, wherein the chromatographic conditions of the ultra performance liquid chromatography in step (2) further comprise: the detection wavelength is 220nm, the flow rate is 0.3mL/min, the column temperature is 25 ℃, the sample injection amount is 3 mu L, and the theoretical plate number is not less than 5000 according to luteolin calculation.

5. The construction method according to claim 4, further comprising the step of replacing the sample solution in the step (2) with a reference solution to obtain a reference characteristic map.

6. The method according to claim 4, further comprising the steps of preparing a reference solution of the reference material by using calyx seu fructus physalis as the reference material, and replacing the sample solution in step (2) with the reference solution of the reference material to obtain the characteristic map of the reference material.

7. The identification method of the calyx seu fructus physalis is characterized by comprising the step of comparing the characteristic spectrum of a product to be detected with the characteristic spectrum of calyx seu fructus physalis contrast; the characteristic spectrum of the product to be detected is obtained by using the product to be detected according to the construction method of any one of claims 1-6, and the calyx seu fructus physalis contrast characteristic spectrum is selected from any one of the following (1) - (4):

(1) The luteolin peak has 11 common characteristic peaks, the luteolin peak is taken as a reference peak, the relative retention time of each characteristic peak and the reference peak is within +/-10% of a specified value, and the specified value is:

peak 2:2.23, peak 3:2.75, peak 4:3.52, peak 5:3.69, peak 6:4.07, peak 7:4.26, peak 8:4.46, peak 9:4.61, peak 10:5.45, peak 11:6.32;

(2) The luteolin peak has 11 common characteristic peaks, the luteolin peak is taken as a reference peak, the relative retention time of each characteristic peak and the reference peak is within +/-10% of a specified value, and the specified value is:

peak 2:2.23, peak 3:2.75, peak 4:3.52, peak 5:3.69, peak 6:4.07, peak 7:4.26, peak 8:4.46, peak 9:4.61, peak 10:5.45, peak 11:6.32; and the relative peak area of peak 4 and reference peak is not less than 0.16;

(3) A characteristic map obtained by using single-batch or multi-batch calyx seu fructus physalis according to the construction method of any one of claims 1-4;

(4) The characteristic map obtained by using the multi-batch calyx seu fructus physalis according to the construction method of any one of claims 1-4 is obtained by an average or median method to obtain a pair characteristic map.

8. The quality control method of the calyx seu fructus physalis is characterized by comprising the step of comparing the characteristic spectrum of the calyx seu fructus physalis product to be detected with the characteristic spectrum of the calyx seu fructus physalis contrast; the characteristic spectrum of the calyx seu fructus physalis product to be detected is obtained by using the product to be detected according to the construction method of any one of claims 1-6, and the calyx seu fructus physalis contrast characteristic spectrum is selected from any one of the following (1) - (4):