CN110672744B

CN110672744B - UPLC fingerprint construction method and detection method of bellyache pills

Info

Publication number: CN110672744B
Application number: CN201910978443.0A
Authority: CN
Inventors: 范春林; 叶文才; 雷婷; 尚晓; 曾瑚瑚; 林瑞珍; 宁娜; 苏碧茹; 张春波; 尹震
Original assignee: Guangzhou Baiyunshan Zhongyi Pharmaceutical Co ltd
Current assignee: Guangzhou Baiyunshan Zhongyi Pharmaceutical Co ltd
Priority date: 2019-10-15
Filing date: 2019-10-15
Publication date: 2022-05-17
Anticipated expiration: 2039-10-15
Also published as: CN110672744A

Abstract

The invention relates to a construction method and a detection method of UPLC fingerprint of a stomach ache pill. The method comprises the following steps: preparation of control solutions: taking magnolol as a reference substance, dissolving with a solvent to obtain a reference substance solution; preparation of a test solution: taking a stomachache pill sample, adding an extraction solvent for extraction, filtering an obtained extracting solution, and taking a subsequent filtrate as a test sample solution; and (3) determination: and injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph, and measuring to obtain the UPLC fingerprint. The method has the advantages of simple operation, short analysis time, strong specificity, good precision, reproducibility and stability, can quickly and comprehensively realize quality monitoring of a plurality of characteristic components in the stomachache pill, provides scientific basis for quality evaluation of the stomachache pill, and ensures the safety and effectiveness of the preparation.

Description

UPLC fingerprint construction method and detection method of stomach ache pill

Technical Field

The invention relates to the technical field of medicines, in particular to a UPLC fingerprint spectrum construction method and a detection method of a stomach ache pill.

Background

The stomachache treating pill is prepared with ten kinds of Chinese medicinal materials including immature bitter orange, dried ginger, aucklandia root, long pepper, magnolia bark, cassia bark, lindera root, amomum fruit, cardamom and poppy shell, and has the functions of warming spleen and stomach, dispelling cold, regulating vital energy and relieving pain. It is clinically used for abdominal cold pain, chest and hypochondrium swelling and stuffiness, vomiting and acid regurgitation caused by cold congealing and qi stagnation.

The stomachache pill consists of ten medicinal materials, has various and complex chemical components, wherein the main components of the immature bitter orange comprise flavonoid, volatile oil and a small amount of alkaloid compounds (Zhang Xiao and the like, China traditional Chinese medicine journal 2015,40(02): 185-190.); the main components of Zingiberis rhizoma include volatile oil, gingerol and diphenyl heptane compounds (Sufengjiao, etc. Chinese wild plant resource 2015,34(03): 34-37); the effective components of costustoot mainly comprise terpenoids, alkaloids, anthraquinones and flavonoids (Weihua and other Chinese herbal medicines 2012,43(03): 613-; the main chemical components of the lindera strychnifolia comprise volatile oil, isoquinoline alkaloid and furan sesquiterpenoids (the natural products are researched and developed by the chenmenmou rain, etc., 2017,29(12): 2147-; the main components of the long pepper comprise alkaloid, amides, volatile oil, lignans, terpenes and sterol compounds (Biying et al, China journal of pharmacy 2011,46(22): 1697-1700.); the main components in the amomum villosum comprise volatile components such as borneol acetate, camphor, borneol and the like, and non-volatile components such as polysaccharide, flavonoid glycoside, organic acid and inorganic component (Lilaili, et al, modern biomedical progress, 2018,18(22): 4390-4396.); the main components of the magnolia officinalis comprise phenols, alkaloids and volatile oil compounds (Zhang Shujie and other Chinese medicinal materials, 2013,36(05):838 and 843); the main components of cinnamon comprise volatile oil, flavanols, terpenes, lignans, phenolic acids and polysaccharide compounds (Chinese herbal medicines, 2018,49(01): 20-34); the main effective components of poppy shell include alkaloid components (Liu Yongli, etc. Chinese medicinal materials 2011,34(09): 1386-1388.); the chemical components of the cardamom mainly comprise volatile oil, flavone and diphenylheptane compounds (Xipeng, etc. the university of Liaoning traditional Chinese medicine, 2017,19(03): 60-63). But currently there is less research on the chemical composition of their entire formulations.

Zhang Xiaolong et al (Zhang Xiaolong et al, Chinese pharmacist, 2018,21(03): 518) and 520) adopt a GC-MS method to carry out content determination on six components of cinnamaldehyde, bornyl acetate, costunolide, dehydrocostus lactone, magnolol and honokiol in the stomachache pill. The belly pain pill is currently collected in volume 6 of the traditional Chinese medicine prescription preparation of the original ministry of health, and the standard only has relevant regulations for inspection under the item of the pill, but has no requirement on content measurement. The reported literature only focuses on certain components in the stomachache pill, and the overall quality of the preparation cannot be fully reflected only by individual indexes in view of the diversity of the traditional Chinese medicine components.

Disclosure of Invention

Based on the above, one of the purposes of the invention is to provide a UPLC fingerprint spectrum construction method of the stomach ache pill. The UPLC fingerprint spectrum constructed by the method marks 10 common peaks and designates 6 chromatographic peaks, can quickly and comprehensively realize quality monitoring of a plurality of characteristic components in the stomachache pill, and provides a scientific basis for quality evaluation of the stomachache pill.

The specific technical scheme is as follows:

a UPLC fingerprint construction method of a stomach ache pill comprises the following steps:

preparation of control solutions: taking magnolol as a reference substance, dissolving with a solvent to obtain a reference substance solution;

preparation of a test solution: taking a stomachache pill sample, adding an extraction solvent for extraction, filtering an obtained extracting solution, and taking a subsequent filtrate as a test sample solution;

and (3) determination: injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph, and measuring to obtain the UPLC fingerprint;

wherein, the elution mode adopted by the ultra-high performance chromatograph is as follows: using 0.05-0.2% formic acid aqueous solution or 0.005-0.02% trifluoroacetic acid aqueous solution as mobile phase A, and methanol or acetonitrile as mobile phase B, to make gradient elution.

The invention also aims to provide a method for detecting the stomachache pill, which comprises the following steps:

preparing a sample solution to be detected: adding an extraction solvent into the stomachache pill to be detected for extraction, filtering the obtained extracting solution, and taking a subsequent filtrate as a sample solution to be detected;

and (3) detection: injecting the sample solution to be detected into an ultra-high performance liquid chromatograph, and measuring;

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts ultra-performance liquid chromatography (UPLC) to construct and obtain the fingerprint of the stomach ache pill. Wherein, magnolol is used as a reference substance, 0.05-0.2 volume percent formic acid water solution or 0.005-0.02 volume percent trifluoroacetic acid water solution is used as a mobile phase A, and methanol or acetonitrile is used as a mobile phase B, within the collection time of 35min, 10 common peaks are calibrated, 6 characteristic peaks are identified, the quality monitoring of a plurality of characteristic components in the stomach ache pill can be rapidly and comprehensively realized, scientific basis is provided for the quality evaluation of the stomach ache pill, and the safety and the effectiveness of the preparation are ensured.

In addition, the method is simple and convenient to operate, short in analysis time, strong in specificity, and good in precision, reproducibility and stability.

Drawings

FIG. 1 is a UPLC spectrum of a 40 batch stomachache pill sample;

FIG. 2 is UPLC standard fingerprint of the stomach ache pill;

FIG. 3 is UPLC fingerprint of stomachache pill with chemical identification;

fig. 4 is a UPLC chromatogram of a sample to be tested.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In some of these embodiments, the method of gradient elution comprises:

the volume percentage of the mobile phase A is reduced from 95% to 65% and the volume percentage of the mobile phase B is increased from 5% to 35% in 0-3 min;

3-12min, the volume percentage of the mobile phase A is reduced from 65% to 47%, and the volume percentage of the mobile phase B is increased from 35% to 53%;

12-13min, the volume percentage of the mobile phase A is reduced from 47% to 35%, and the volume percentage of the mobile phase B is increased from 53% to 65%;

13-24min, the volume percentage of the mobile phase A is reduced from 35% to 20%, and the volume percentage of the mobile phase B is increased from 65% to 80%;

for 24-27min, the volume percent of the mobile phase A is reduced from 20% to 5%, and the volume percent of the mobile phase B is increased from 80% to 95%;

27-35min, the volume percentage of the mobile phase A is kept at 5%, and the volume percentage of the mobile phase B is kept at 95%.

In some embodiments, the chromatographic conditions of the ultra-high performance chromatograph comprise:

stationary phase: a chromatographic column using octadecylsilane chemically bonded silica as a filler;

detection wavelength: 205-280 nm;

column temperature: 30-45 ℃;

flow rate: 0.25-0.5 mL/min.

In some of these embodiments, the column is a Waters acquisition UPLC HSS T3.

In some of these embodiments, the sample solution is prepared by using an extraction solvent selected from methanol, ethanol, 30-70% methanol by volume or 75-98% ethanol by volume.

In some of these embodiments, the extraction is by ultrasonic extraction, cold-dip extraction, or thermal extraction.

A detection method of a stomach ache pill comprises the following steps:

In some of these embodiments, the method of gradient elution comprises:

detection wavelength: 205-280 nm;

column temperature: 30-45 ℃;

flow rate: 0.25-0.5 mL/min.

In some of these embodiments, the column is a Waters acquisition UPLC HSS T3.

In some embodiments, the extraction solvent is selected from methanol, ethanol, 30-70% methanol aqueous solution by volume, or 75-98% ethanol aqueous solution by volume.

In some of these embodiments, the extraction is by ultrasonic extraction, cold-dip extraction, or thermal extraction. The extraction time is 50-70 min. The hot extraction is heating reflux extraction.

The present invention will be described in further detail with reference to specific examples.

The following examples refer to the following reagents:

example 1

1. Reagent, reagent and instrument

1.1 reagent:

methanol and acetonitrile are both chromatographically pure (Merck, Germany); ethanol is analytically pure (Tianjin Kemi Euro Chemicals Co., Ltd.); formic acid, trifluoroacetic acid (alatin reagent (shanghai) ltd.).

1.2 reagent:

reference naringin was purchased from WUDU Biotechnology GmbH; the rest were purchased from VICKQI Biotech, Inc., Sichuan province.

The purity and lot number of the control are shown in table 1:

TABLE 1 purity of control

The belly pain pill: the samples of the stomachache pill tested in this example were 40 batches, and the 40 batches of stomachache pills were all produced by a pharmaceutical company ltd in the white cloud mountain, guangzhou, and the batch numbers are XS0001, XS0002, XS0003, XS0004, XS0005, XS0006, X00007M, X00008M, X00009M, X00010M, X00011M, X00012M, X00014M, X00015M, X00016M, X00017M, YR0006, YN0004, Y00001, Y00002, Y00003, Y00004, Y00005, Y00008, Y00010, Y00013, W00001, W00002, W00004, W00005, W00006, W00007, W00008, W00009, W00010, W11, W00012, W13, W00014, W00015.

1.3 instruments

ACQUITY UPLC H-Class ultra high performance liquid chromatograph (Vout Shanghai science and technology Co., Ltd.); agilent G6540 UPLC tandem quadrupole time-of-flight mass spectrometer (Agilent technologies, Inc.); model BX8200HP ultrasonic apparatus (shanghai new-seedling medical device manufacturing ltd); sartorius BP211D one ten-thousandth electronic analytical balance (Sartorius, germany); ultrapure water system (Millpore corporation, usa).

2. Method of producing a composite material

2.1 construction of fingerprint

2.1.1 preparation of control solutions

Precisely weighing narirutin, hesperidin, vanilloid, piperine, honokiol and magnolol as reference substances, respectively adding methanol to dissolve and dilute into 0.05-1.0mg/mL solution, filtering with microporous membrane, and collecting the filtrate.

2.1.2 preparation of test solutions

Taking 1.0g of fine powder of the stomach ache pill, precisely weighing, adding 50mL of methanol, performing ultrasonic extraction at 30 ℃ for 60min, standing to room temperature, supplementing the weight loss with methanol, shaking uniformly, standing, filtering the supernatant with a microporous filter membrane (0.22 mu m), and taking the subsequent filtrate.

2.1.3 chromatographic conditions and System Adaptation

A chromatographic column: waters ACQUITY UPLC HSS T3 column with specification of 2.1 × 100mm, 1.8-2.1 μm; detection wavelength: 254 nm; flow rate: 0.3 mL/min; column temperature: 40 ℃; sample introduction amount: 2 mu L of the solution;

mobile phase: the mobile phase A is formic acid-water solution with the volume percentage of 0.1 percent, the mobile phase B is methanol, and gradient elution is carried out; the gradient elution procedure is shown in table 2:

TABLE 2 Dutong pill UPLC gradient elution procedure

2.1.4 construction of UPLC fingerprint of stomach ache pill

2.1.4.1 identification of characteristic peak

Taking 40 batches of the stomach-ache pills, preparing a stomach-ache pill test solution according to the method under item 2.1.2, and measuring by adopting the chromatographic condition under item 2.1.3 to obtain the liquid chromatogram of the test solution and the reference solution.

And (3) carrying out online monitoring at the wavelength of 190-400nm by adopting a PDA detector, and comparing the retention time and the ultraviolet absorption of chromatographic peaks of the test solution and each reference substance. Based on the chromatogram retention time, the ultraviolet absorption and the molecular weight control of the control and the test solution, 6 main chromatogram peaks in the UPLC of the stomachache pill can be identified, and the retention time and the relative retention time relative to the reference peak are shown in Table 3 (X00008M).

TABLE 3 identification of main chromatographic peak of test solution of stomachache treating pill

Peak number	Name of Compound	Retention time/min	Relative retention time
				1	Rutachinin	7.330	0.3582
2	Hesperidin	8.117	0.3966
				3	Isochrysin	11.167	0.5457
4	Piperine	17.189	0.8399
				5	Honokiol	18.56	0.9069
6(S)	Magnolol	20.465	1.0000

2.1.4.2 Mass spectrometric identification of characteristic peaks

2.1.4.2.1 liquid phase conditions

A chromatographic column: waters ACQUITY UPLC HSS T3 column (2.1X 100mm,1.8 μm); mobile phase: methanol (B) -0.1% strength by volume formic acid water (a); elution gradient (volume fraction): 0-3min 5% -35% B, 3-12min 35% -53% B, 12-13min 53% -65% B, 13-24min 65% -80% B, 24-27min 80% -95% B, 27-35min 95% B; column temperature: 40 ℃; detection wavelength: 254 nm; flow rate: 0.30 mL/min; sample introduction amount: 2 mu L of the solution; collecting time: and (5) 35 min.

2.1.4.2.2 Mass Spectrometry conditions

An acquisition mode: a positive ion; capillary voltage: 4000V; capillary exit voltage: 175V, taper hole voltage: 65V, eight-level rod voltage: 750V; temperature of the drying gas: 350 ℃, flow rate of drying gas: 8L/min; the pressure of the sprayer is as follows: 35 psig.

An acquisition mode: negative ions; capillary voltage: 3500V; capillary exit voltage: 175V, taper hole voltage: 65V, eight-level rod voltage: 750V; temperature of the drying gas: 350 ℃, flow rate of drying gas: 8L/min; the pressure of the sprayer is as follows: 35 psig.

2.1.4.2.3 results of the experiment

The results of comparing the chromatographic retention time and mass spectrum information of the stomachache pill test solution and the mixed control solution in the negative ion mode and the positive ion mode are shown in table 4. The result shows that 6 main chromatographic peaks identified in the fingerprint of the stomach ache pill are consistent with the retention time of the corresponding reference substance and the molecular weight under different acquisition modes, and the 6 main chromatographic peaks in the fingerprint are further identified by mass spectrometry.

TABLE 4 retention time and molecular weight of corresponding chromatographic peak of mixed control and stomach ache pill

And (3) analyzing the detection data of the UPLC spectra of the 40 batches of the stomachache pills by taking the No. 6 peak (S, magnolol) as a reference peak, and calculating the relative retention time and the relative peak area of each characteristic peak and the reference peak. The results are shown in tables 5 and 6.

TABLE 540 relative retention time test results of UPLC spectra of stomach pain pills

Note: "S" is the reference peak.

Relative peak area detection result of UPLC (ultra Performance liquid chromatography) spectra of 640 stomachache pills in table

Note: "S" is the reference peak.

The average values of the relative retention time and the relative peak area of each characteristic peak in 40 batches of stomachache pill spectra are respectively used as the relative retention time and the relative peak area of each characteristic peak of the UPLC standard fingerprint spectrum of the stomachache pills, and the results are shown in Table 7.

TABLE 7 UPLC standard fingerprint data of stomach ache pill

Note: "S" is the reference peak.

And (3) superposing UPLC (ultra Performance liquid chromatography) spectrums of 40 batches of stomachache pills, analyzing by software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2004 edition, and generating the UPLC standard fingerprint spectrum of the stomachache pills after multipoint correction and automatic matching by adopting a median method and a time window of 0.1 and taking a No. 6 peak as a reference peak. The UPLC standard fingerprint of the stomach ache pill is shown in figure 2, 10 common peaks are determined, and compounds represented by 6 common peaks capable of being chemically identified (in sequence: narirutin (1), hesperidin (2), vanillyl glycoside (3), piperine (4), honokiol (5) and magnolol (6)) are determined by a reference substance, so that the UPLC fingerprint of the stomach ache pill capable of being chemically identified is constructed, and is shown in figure 3.

2.1.5 methodological validation

2.1.5.1 Instrument precision test

Taking the stomachache pill with the batch number of X00008M, preparing a stomachache pill test solution according to the method under item 2.1.2, continuously injecting samples for 6 times, and measuring by adopting the chromatographic conditions under item 2.1.3, wherein the measurement results are shown in tables 8 and 9.

TABLE 8 method precision investigation results (relative retention time of major peaks)

Note: "S" is the reference peak.

TABLE 9 results of precision examination of methods (relative peak area of main peak)

Peak number	1	2	3	4	5	6	RSD％
								1	0.9566	0.9107	0.9071	0.9323	0.9055	0.9081	2.22
2	4.2806	4.4355	4.4766	4.2837	4.4712	4.4219	2.04
								3	0.2098	0.2121	0.2101	0.2098	0.2138	0.2083	0.93
4	1.2130	1.2130	1.2088	1.2067	1.2047	1.2026	0.36
								5	0.9159	0.9248	0.9238	0.9229	0.9229	0.9229	0.34
6(S)	1.0000	1.0000	1.0000	1.0000	1.0000	1.0000	0.00
								7	0.2094	0.2072	0.2108	0.2133	0.2084	0.2087	1.02
8	0.1227	0.1252	0.1256	0.1251	0.1245	0.1251	0.82
								9	0.4791	0.4825	0.4734	0.4740	0.4722	0.4723	0.89
10	0.4147	0.4230	0.4227	0.4229	0.4207	0.4228	0.78

Note: "S" is the reference peak.

The result shows that the relative retention time (RSD < 1%) of all the common peaks in the test solution is basically consistent with the relative peak area (RSD < 3%) of the main peak, and the instrument precision of the method is good. The similarity of the spectra obtained by the first sample injection is taken as a reference, the similarity evaluation system of the Chinese medicine chromatogram fingerprint similarity evaluation system 2004 version is used for calculating the similarity of the spectra obtained by the last 5 sample injections, the result is shown in a table 10, and the similarity meets the technical requirements of the fingerprint.

Table 10 similarity calculation results of instrument precision test (n ═ 6)

Sample introduction number	1	2	3	4	5	6
							Similarity of the two	1.00	1.00	1.00	1.00	1.00	1.00

2.1.5.2 repeatability test

A test solution of 6 stomach-ache pill samples was prepared from the stomach-ache pill of lot number X00008M by the method of item 2.1.2, and the test results were shown in tables 11 and 12 by the chromatographic conditions of item 2.1.3.

TABLE 11 method repeatability test results (relative retention time of major peaks)

Peak number	1	2	3	4	5	6	RSD％
								1	0.3569	0.3572	0.3568	0.3569	0.3567	0.3567	0.06
2	0.3946	0.3950	0.3946	0.3945	0.3943	0.3943	0.06
								3	0.5426	0.5428	0.5424	0.5423	0.5422	0.5422	0.04
4	0.8402	0.8405	0.8401	0.8402	0.8401	0.8401	0.02
								5	0.9070	0.9074	0.9069	0.9071	0.9070	0.9069	0.02
6(S)	1.0000	1.0000	1.0000	1.0000	1.0000	1.0000	0.00
								7	1.1501	1.1498	1.1501	1.1500	1.1499	1.1500	0.01
8	1.3514	1.3515	1.3513	1.3513	1.3518	1.3519	0.02
								9	1.4372	1.4376	1.4373	1.4376	1.4380	1.4382	0.03
10	1.4882	1.4887	1.4888	1.4890	1.4894	1.4897	0.04

Note: "S" is the reference peak.

TABLE 12 methods repeatability test results (relative peak area of major peaks)

Note: "S" is the reference peak.

The result shows that the relative retention time of all the common peaks (RSD < 1%) and the relative peak area of the main peak in the test solution of the stomachache pill are basically consistent (RSD < 3%), which indicates that the repeatability of the method is good. The atlas obtained from the 1 st sample injection is taken as a reference, the similarity of the atlas obtained from the 5 th sample injection is calculated by a similarity evaluation system of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2004 edition, the result is shown in a table 13, and the similarity meets the technical requirements of the fingerprint.

TABLE 13 calculation of similarity for reproducibility test

Numbering	1	2	3	4	5	6
							Degree of similarity	1.00	1.00	1.00	1.00	1.00	1.00

2.1.5.3 stability test

A test solution of the stomach pain pill of the batch number X00008M was prepared according to the method of item 2.1.2, stability was checked for 0, 2,4, 8, 12, and 24 hours, and the test was performed 6 times under the chromatographic conditions of step (3) of item 2.1.3, and the test results are shown in tables 14 and 15.

TABLE 14 method stability test results (relative retention time of major peaks)

Note: "S" is the reference peak.

TABLE 15 methods stability test results (relative peak area of main peak)

Peak number	1(0h)	2(2h)	3(4h)	4(8h)	5(12h)	6(24h)	RSD％
								1	0.9566	0.9323	0.9055	0.9110	0.9074	0.9089	2.21
2	4.2806	4.2837	4.2809	4.3005	4.3054	4.5184	2.17
								3	0.2098	0.2098	0.2088	0.2135	0.2090	0.2084	0.89
4	1.2130	1.2067	1.2011	1.1981	1.1983	1.2006	0.48
								5	0.9159	0.9229	0.9244	0.9259	0.9254	0.9148	0.54
6(S)	1.0000	1.0000	1.0000	1.0000	1.0000	1.0000	0.00
								7	0.2094	0.2133	0.2091	0.2120	0.2105	0.2110	0.76
8	0.1227	0.1251	0.1254	0.1264	0.1266	0.1231	1.32
								9	0.4791	0.4740	0.4728	0.4742	0.4742	0.4709	0.57
10	0.4147	0.4229	0.4229	0.4316	0.4313	0.4166	1.68

Note: "S" is the reference peak.

The result shows that the relative retention time of all the common peaks (RSD < 1%) and the relative peak area of the main peak in the test solution of the stomachache pill are basically consistent (RSD < 3%), which indicates that the method has good stability. The atlas obtained by the 1 st sample injection is taken as a reference, the similarity of the atlas obtained by the 5 th sample injection is calculated by a similarity evaluation system of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2004 edition, the result is shown in a table 16, and the similarity meets the technical requirement of the fingerprint.

TABLE 16 results of similarity calculation for stability test

Numbering	1	2	3	4	5	6
							Degree of similarity	1.00	1.00	1.00	1.00	1.00	1.00

The result of methodology investigation shows that the UPLC fingerprint spectrum construction method of the stomachache pill established by the invention has good instrument precision and method repeatability, the sample solution is stable within 24h, the fingerprint spectrum of the preparation can be accurately measured, and the preparation can be used for quality control of the stomachache pill.

2.1.6 similarity survey

The UPLC standard fingerprint of the stomachache pill established under item 2.1.4 is used as a reference (figure 2), and the similarity of UPLC of each stomachache pill batch is calculated by using the similarity evaluation system 2004 edition of traditional Chinese medicine chromatogram fingerprint, and the result is shown in Table 17.

Table 1740 batch UPLC spectrum similarity investigation result of stomachache pill

The result shows that the similarity between UPLC standard fingerprint of the stomachache pill of 40 batches and UPLC standard fingerprint of the stomachache pill is more than 0.99, the method has good repeatability and high similarity.

3. Method optimization

3.1 optimization of sample extraction conditions

In the experiment, the extraction method (cold soaking, ultrasonic treatment and heating reflux), the extraction solvent comparison (30% methanol, 70% methanol, 100% methanol and 95% ethanol, all volume concentrations) and the extraction time (30min, 60min and 90min) are all considered, and finally the optimal extraction condition is 100% methanol ultrasonic extraction for 60min, so that more chromatographic peaks are obtained and the baseline is stable.

3.2 optimization of chromatographic conditions

This experiment was conducted to examine 4 different systems of mobile phases (methanol-water, acetonitrile-water, methanol-acid water, acetonitrile-acid water), different column temperatures (30 ℃, 35 ℃,40 ℃, 45 ℃), and different flow rates (0.25mL/min, 0.30mL/min, 0.35mL/min, 0.40 mL/min). The results show that at 40 ℃, the mobile phase is methanol-0.1% formic acid water, and the flow rate is 0.30mL/min, the chromatographic peaks are more, the peak shapes and the separation effects of the peaks are better, and the base line is stable. Therefore, a methanol-acid water system is selected as a mobile phase, the temperature of the detection column is 40 ℃, and the flow rate is determined to be 0.30 mL/min.

The experiment compares the chromatograms of the test solution under different detection wavelengths (210nm, 254nm, 280nm and 330nm), and the result shows that when 254nm is taken as the detection wavelength, the base line of the chromatogram is stable, the detected chromatographic peaks are more, the peak shapes and the separation degrees of the various chromatographic peaks are good, the overall appearances of various components in the stomachache pill can be reflected more comprehensively, so 254nm is selected as the detection wavelength for the research of UPLC fingerprint of the stomachache pill.

The results of the experiments compared the chromatographic peak separation conditions on 3 different types of chromatographic columns respectively adopting ACQUITY UPLC HSS T3-C18(2.1 × 100mm,1.8 μm, Waters) column, Eclipse XDB-C18(3.0 × 100mm,3.5 μm, Agilent) column and Eclipse Plus C18 RRHD (2.1 × 100mm,1.8 μm, Agilent) column show that the chromatographic column obtained by the ACQUITY UPLC HSS T3-C18 column has stable baseline chromatogram, more chromatographic peaks and good separation effect, so the experiment selects ACQUITY UPLC HSS T3-C18(2.1 × 100mm,1.8 μm, Waters) column as the chromatographic column for stomachache UPLC fingerprint research.

Example 2

The embodiment is a method for detecting a stomachache pill, and the method comprises the following steps:

1. chromatographic conditions are as follows: reference is made to the "2.1.3 chromatographic conditions and system suitability" term in example 1;

2. preparing a sample solution to be tested: taking 1.0g of the fine powder of the stomachache pill to be detected, precisely weighing, adding 50mL of methanol, performing ultrasonic extraction at 30 ℃ for 60min, standing to room temperature, complementing the loss weight with methanol, shaking uniformly, standing, filtering the supernatant with a microporous filter membrane (0.22 mu m), and taking the subsequent filtrate;

3. precisely sucking 2 mu L of the obtained sample solution to be measured, and measuring according to the chromatographic conditions in the step 1 to obtain the target product.

As shown in fig. 4, when compared with UPLC fingerprint of the stomach-ache pill established in example 1, it can be seen that: the UPLC of the stomachache pill to be detected has high similarity with the UPLC fingerprint of the stomachache pill established in the embodiment 1, and the quality is qualified.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A UPLC fingerprint construction method of a stomach ache pill is characterized by comprising the following steps:

preparation of control solutions: dissolving narirutin, hesperidin, vanilloid, piperine, honokiol and magnolol as reference substances with solvent to obtain reference substance solution;

preparation of a test solution: taking a stomachache pill sample, adding 30-70% methanol water solution or 100% methanol or 75-98% ethanol water solution by volume concentration for extraction, filtering the obtained extract, and taking the subsequent filtrate as a test solution;

wherein, the elution mode adopted by the ultra-high performance liquid chromatograph is as follows: taking 0.05-0.2% by volume of formic acid aqueous solution or 0.005-0.02% by volume of trifluoroacetic acid aqueous solution as a mobile phase A, and taking methanol or acetonitrile as a mobile phase B to carry out gradient elution;

the stationary phase of the ultra-high performance liquid chromatograph: a chromatographic column using octadecylsilane chemically bonded silica as a filler;

the method of gradient elution comprises:

2. The method for constructing the UPLC fingerprint according to claim 1, wherein the chromatographic conditions of the ultra high performance liquid chromatograph comprise:

detection wavelength: 205-280 nm;

column temperature: 30-45 ℃;

flow rate: 0.25-0.5 mL/min.

3. The method for constructing UPLC fingerprints according to any of the claims 1-2, wherein said chromatography column is Waters acquisition UPLC HSS 3.

4. The method for constructing the UPLC fingerprint as claimed in any one of claims 1-2, wherein the extraction mode in the preparation of the test solution is ultrasonic extraction, cold-dipping extraction or hot extraction.

5. The UPLC fingerprint construction method according to any one of claims 1-2, wherein the constructed fingerprint defines 10 common peaks.

6. The method for constructing UPLC fingerprint according to claim 5, wherein said constructed fingerprint identifies compounds represented by 6 chemically identifiable common peaks, including narirutin (1), hesperidin (2), vanilloid (3), piperine (4), honokiol (5) and magnolol (6).

7. The detection method of the stomach ache pill is characterized by comprising the following steps:

preparing a sample solution to be detected: taking the stomachache pill to be detected, adding 30-70% methanol water solution or 100% methanol or 75-98% ethanol water solution to extract, filtering the obtained extract, and taking the subsequent filtrate as a sample solution to be detected;

wherein, the elution mode adopted by the ultra-high performance liquid chromatograph is as follows: using 0.05-0.2% formic acid aqueous solution or 0.005-0.02% trifluoroacetic acid aqueous solution as mobile phase A, using methanol or acetonitrile as mobile phase B, proceeding gradient elution;

the method of gradient elution comprises:

8. The detection method according to claim 7, wherein the chromatographic conditions of the ultra high performance liquid chromatograph include:

detection wavelength: 205-280 nm;

column temperature: 30-45 ℃;

flow rate: 0.25-0.5 mL/min.

9. The method of detection according to any one of claims 7 to 8, wherein the chromatographic column is Waters acquisition UPLC HSS 3.

10. The detection method according to any one of claims 7 to 8, wherein the sample solution to be detected is prepared by ultrasonic extraction, cold-dipping extraction or hot extraction.