CN112083092A

CN112083092A - Method for simultaneously measuring multiple index components in gallbladder warming tablets

Info

Publication number: CN112083092A
Application number: CN202010881161.1A
Authority: CN
Inventors: 袁慧君; 于娟; 侴桂新; 华腊; 连赟芳
Original assignee: Zhangzhou Pientzehuang Pharmaceutical Co Ltd
Current assignee: Zhangzhou Pientzehuang Pharmaceutical Co Ltd
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2020-12-15

Abstract

The invention provides a method for simultaneously measuring various index components in a gallbladder warming tablet, which takes the gallbladder warming tablet as a test sample to carry out high performance liquid chromatography detection and simultaneously measures p-hydroxycinnamic acid, rutin, hesperidin and glycyrrhizic acid in the gallbladder warming tablet. The method can also quantitatively determine the content of the 4 components in the gallbladder warming tablet, and the quantitative determination method comprises an external standard method and a one-test-multiple evaluation method. The determination method realizes the qualitative and quantitative determination of a plurality of components (p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid) in the gallbladder warming tablet, wherein the one-determination and multi-evaluation detection method ensures the effective, stable and controllable quality of the preparation, reduces the detection cost of purchasing a reference substance, obtains excellent technical effect and has wide application prospect.

Description

Method for simultaneously measuring multiple index components in gallbladder warming tablets

Technical Field

The invention belongs to the field of quality control of traditional Chinese medicines, and particularly relates to a method for simultaneously measuring multiple index components in a gallbladder warming tablet.

Background

The gallbladder warming tablet is a Chinese medicinal preparation prepared by extracting and processing seven Chinese medicinal materials of rhizoma pinellinae praeparata, caulis bambusae in taeniam, fructus aurantii immaturus, pericarpium citri reticulatae, poria cocos, radix curcumae and radix glycyrrhizae preparata, has the effects of regulating qi, clearing heat, reducing phlegm, warming gallbladder, harmonizing stomach, clearing heart fire and relieving depression, is clinically used for treating generalized anxiety disorder at present, and has definite curative effect. The gallbladder warming tablet is a modern preparation obtained by improving a classic famous prescription gallbladder warming soup, and contains various active ingredients, such as p-hydroxycinnamic acid, narirutin, hesperidin, liquiritin, glycyrrhizic acid and the like. At present, the quality control of the gallbladder-warming tablets is generally carried out by adopting a method for measuring the content of one or more index components in the gallbladder-warming tablets.

CN106323794A discloses a quality control method of a gallbladder warming tablet: the product quality stability of the warming gallbladder tablets is confirmed by measuring the synephrine transfer rate in the warming gallbladder tablets through a liquid chromatography, but the method established by the invention takes synephrine (components in immature bitter orange and dried orange peel) as a single quality control index for measurement, and cannot comprehensively reflect the quality of the warming gallbladder tablets.

The Wanlijuan and the like (research and quality standard research of Wendan granules, Hubei university of traditional Chinese medicine) disclose a quality standard method for determining Wendan granules: and respectively establishing chromatographic conditions for hesperidin, liquiritin and glycyrrhizic acid in the gallbladder warming granules by using a reversed phase liquid chromatography for content determination. However, the method is troublesome in determination, the content of hesperidin in the gallbladder warming granules needs to be determined firstly, and the content of liquiritin and glycyrrhizic acid in the gallbladder warming granules needs to be determined, so that the content cannot be detected simultaneously; in addition, the determination method needs to adopt various reference substances (hesperidin, liquiritin and ammonium glycyrrhizinate reference substances), so that the detection cost is greatly increased.

Therefore, there is a need to develop a quality control method for simultaneously measuring the contents of multiple active ingredients in a gallbladder-warming tablet by using only one cheap and easily available component as a reference substance.

In 2006, a multi-index quality control method for measuring multiple assessments (QAMS) was proposed by the Wang Chin of Chinese academy of sciences, traditional Chinese medicine, and the like, and the method realizes simultaneous measurement of multiple components by using a relatively easily available and cheap reference substance of an internal reference substance, and finally achieves the purpose of controlling the overall quality of the traditional Chinese medicine. QAMS has the advantages of low detection cost, high analysis efficiency and the like, so once the QAMS is provided, huge reverberation is caused at home and abroad, the QAMS is considered to be a multi-index quality control and evaluation mode suitable for the characteristics of traditional Chinese medicines, and the QAMS is applied to the quality control of a plurality of traditional Chinese medicines such as pseudo-ginseng, fructus psoraleae, platycodon grandiflorum, radix puerariae, herba houttuyniae, radix curcumae and the like at present.

At present, no report is found on a method for simultaneously measuring 4 components of hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid in the gallbladder warming tablet, and no report is found on a quality control method for simultaneously measuring the contents of the 4 components in the gallbladder warming tablet by using a one-measurement-multiple-evaluation method.

Disclosure of Invention

The invention aims to provide a method for simultaneously measuring various index components (p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid) in a gallbladder warming tablet, which is used for qualitatively and quantitatively analyzing the p-hydroxycinnamic acid, the narirutin, the hesperidin and the glycyrrhizic acid in the gallbladder warming tablet.

The invention provides a method for simultaneously determining multiple index components in a gallbladder warming tablet, which takes the gallbladder warming tablet as a test sample, carries out high performance liquid chromatography detection, and simultaneously determines p-hydroxycinnamic acid, rutin, hesperidin and glycyrrhizic acid in the gallbladder warming tablet; the method comprises the following steps:

(1) preparation of a test solution: extracting fel warming tablet with organic solvent, and collecting extractive solution to obtain test solution;

(2) injecting into a high performance liquid chromatograph for detection;

the chromatographic conditions were as follows:

a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent;

mobile phase: the phase A consists of acetonitrile and phosphoric acid aqua.

Further, the method is a method for quantitatively measuring hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid; the method is an external standard method or a one-test-multiple evaluation method.

Further, the external standard method takes p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid as reference substances, and the content of p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid in the gallbladder warming tablet is measured; the control solution was prepared as follows: respectively weighing p-hydroxycinnamic acid, rutin, hesperidin and glycyrrhizic acid, and respectively adding organic solvent for dissolving to obtain the final product.

Further, the one-test-multiple evaluation method takes hesperidin as a reference substance to measure the content of the p-hydroxycinnamic acid, the narirutin, the hesperidin and the glycyrrhizic acid in the gallbladder warming tablet.

Further, the one-test-multiple-evaluation method comprises the following steps of:

1) preparation of control solutions: weighing hesperidin, and dissolving in organic solvent to obtain hesperidin solution;

2) preparation of a test solution: weighing fel warming tablet, adding organic solvent for extraction, and collecting extractive solution;

3) respectively sucking the test solution and the reference solution, injecting into a high performance liquid chromatograph, and measuring the content of hesperidin in the fel warming tablet;

4) respectively calculating the content of the component to be detected in the gallbladder warming tablet by using relative correction factors among the component to be detected, namely hydroxycinnamic acid, narirutin, glycyrrhizic acid and hesperidin through the following formula;

f_s/i＝(A_s×C_i)/(A_i×C_s)

wherein f is_s/iIs a relative correction factor between the component to be measured and hesperidin, A_sIs the peak area of hesperidin in the chromatogram of the test solution, C_iContent of component to be measured in Wendan tablet, A_iIs the peak area, C, of the component to be measured in the chromatogram of the sample solution_sIs the content of hesperidin in the gallbladder warming tablet.

Further, in the step 4), the relative correction factor between the p-hydroxycinnamic acid and the hesperidin is 0.92-1.01, preferably 0.93, the relative correction factor between the narirutin and the hesperidin is 0.97-1.05, preferably 1.00, and the relative correction factor between the glycyrrhizic acid and the hesperidin is 1.12-1.26, preferably 1.13.

Further, the octadecylsilane bonded silica is Inertsil ODS-3, Diamonsil C18, CAPCELL PAK ADME, Chrom Core C18 or Thermo Syncronis C18, preferably Inertsil ODS-3.

Further, the chromatographic conditions were gradient elution, the elution conditions are shown in the following table:

the time in the table is minutes,% A represents the volume of phase A in the mobile phase, and% B represents the volume of phase B in the mobile phase;

and/or the mass concentration of the phosphoric acid aqueous solution is 0.05-0.20%, preferably 0.10%;

and/or the detection wavelengths in the chromatographic conditions are: 252-268 nm; the column temperature is 25-35 ℃; the flow rate is 0.8-1.2 ml/min; preferably, the detection wavelength is: 256nm for 0-25 min; 25-42 min, 268 nm; 42-65 min, 252 nm; the column temperature is 30 ℃; the flow rate was 1.0 ml/min.

Further, in the preparation of the test solution, the organic solvent is an alcohol solvent, preferably 60% to 80% ethanol, and more preferably 70% ethanol;

and/or the mass-volume ratio of the warming gallbladder tablets to the organic solvent is 5 mg/mL-20 mg/mL, preferably 10 mg/mL; and/or the extraction mode is ultrasonic extraction, the extraction time is 30-60 min, preferably 45min, the extraction temperature is room temperature, the ultrasonic power is 200-300W, and preferably 250W.

Further, in the preparation of the reference solution, the organic solvent is an alcohol solvent, preferably methanol; and/or the concentration of the control solution is 0.025-0.405 mg/mL, preferably 0.1 mg/mL.

In the present invention, the phosphoric acid concentration of the mobile phase is the mass concentration of the phosphoric acid aqueous solution, and for example, 0.10% phosphoric acid means a phosphoric acid aqueous solution having a mass concentration of 0.10%.

Experiments prove that under the specific chromatographic conditions, the method for determining multiple index components in the gallbladder warming tablet can not only perform qualitative analysis on multiple components (p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid) in the gallbladder warming tablet, but also perform quantitative determination. Under the specific chromatographic conditions of the invention, the detection accuracy of quantitative determination by using a one-test-multiple-evaluation method is equivalent to that of an external standard method.

Particularly, the one-test-multiple-evaluation method for determining the content of various index components in the gallbladder warming tablet takes hesperidin as a reference substance and establishes relative correction factors for hydroxycinnamic acid, narirutin, glycyrrhizic acid and hesperidin, so that the synchronous determination of 4 contents of hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid in the gallbladder warming tablet is realized.

The measuring method of the invention can accurately and comprehensively evaluate the quality of the gallbladder warming tablets, is simple, convenient and quick, has good stability and repeatability, and is beneficial to improving the quality standard of the gallbladder warming tablets. Particularly, the method for evaluating more than one test ensures the effective, stable and controllable quality of the preparation, reduces the detection cost of purchasing a reference substance, obtains excellent technical effect and has wide application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a chromatogram of a test sample of the WENDAN tablet of example 3, wherein 1-p-hydroxycinnamic acid, 2-narirutin, 3-hesperidin, and 4-glycyrrhizic acid.

FIG. 2 is a chromatogram of a mixed standard solution of Experimental example 1, wherein 1-p-hydroxycinnamic acid, 2-narirutin, 3-hesperidin, and 4-glycyrrhizic acid.

Figure 3 is a linear relationship graph for hydroxycinnamic acid.

FIG. 4 is a linear relationship graph of narirutin.

FIG. 5 is a graph of the linear relationship of hesperidin.

FIG. 6 is a linear relationship diagram of glycyrrhizic acid.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Agilent Technologies 1260Infinity II high performance liquid chromatography systems; BSA124S-CW type electron analytical balance (Satorius), BSA 323S type electron analytical balance (Satorius); KQ-250DB type digital control ultrasonic cleaner (Kunshan ultrasonic instruments, Inc., 150W, 40KHz), Zeal Quest ultra pure water instrument; the chromatographic column is Inertsil ODS-3 (4.6X 250mm, 5 μm); acetonitrile is chromatographically pure, phosphoric acid is chromatographically pure, water is ultrapure water, and other reagents are analytically pure;

p-hydroxycinnamic acid (PRF9070423, purity of 98% and purity of 98%, Chenopou technology Co., Ltd.), glycyrrhizic acid (PRF10010203, purity of 98% and purity of 98%, Chenopou technology Co., Ltd.), narirutin (purity of 98%, Shanghai center for standardization of Chinese medicine), hesperidin (110721-; the samples of the warming gallbladder are from Zhangzhou pian zaihuang medicinal industry GmbH.

Example 1 method for qualitative determination of Multi-index Components in Winterepha tablet

Under the following chromatographic conditions, whether the warm gall bladder tablets (purchased from Zhangzhou PIZAIHUANG medicinal industry Co., Ltd.) contain p-hydroxycinnamic acid, rutin, hesperidin and glycyrrhizic acid or not is simultaneously determined.

Preparation of a test solution: grinding fel Ursi tablet into powder, collecting powder about 0.5g, precisely weighing, precisely adding 70% ethanol 50ml, weighing, ultrasonic extracting at room temperature (ultrasonic power of 250W and frequency of 40kHz) for 45min, supplementing weight loss, filtering, and collecting filtrate.

Chromatographic conditions are as follows: a chromatographic column: inertsil ODS-3 (4.6X 250mm, 5 μm); the elution conditions are shown in Table 1; detection wavelength: 256nm for 0-25 min; 25-42 min, 268 nm; 42-65 min, 252 nm; column temperature: 30 ℃; flow rate: 1.0 ml/min. The theoretical plate number is not less than 5000 calculated according to hesperidin.

TABLE 1 gradient elution conditions

And detecting to obtain a chromatogram. Then, carrying out qualitative analysis on the retention time of the hydroxycinnamic acid, the narirutin, the hesperidin and the glycyrrhizic acid under the chromatographic condition, and judging whether the gallbladder warming tablet sample contains the hydroxycinnamic acid, the narirutin, the hesperidin and the glycyrrhizic acid.

Example 2 quantitative determination of contents of various index components in Winterepha tablet by external standard method

The content of p-hydroxycinnamic acid, naringin, hesperidin and glycyrrhizic acid in the warming gallbladder slices (purchased from Zhangzhou PIZAIHUANG pharmaceutical industry GmbH) is measured simultaneously by using an external standard method under the following chromatographic conditions by taking the p-hydroxycinnamic acid, the narirutin, the hesperidin and the glycyrrhizic acid as reference substances.

And detecting to obtain a chromatogram. Then, the contents of the p-hydroxycinnamic acid, the narirutin, the hesperidin and the glycyrrhizic acid in the gallbladder warming tablet sample are calculated according to a standard curve of the p-hydroxycinnamic acid, the narirutin, the hesperidin and the glycyrrhizic acid (the measuring method of the standard curve is described in experimental example 1, namely 'linear relation examination').

Example 3 quantitative determination of the content of various index components in a Winterephora by one-test-multiple-evaluation method

With hesperidin as a reference, the content of four components of p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid in the gallbladder warming tablets (purchased from Zhangzhou Pianzaihuang pharmaceutical industry Co., Ltd.) is simultaneously measured by adopting a one-test-multiple evaluation method (QAMS method) established by the invention, and the specific steps are as follows:

(1) preparation of the solution

Preparation of control solutions: taking a proper amount of hesperidin, precisely weighing, and adding methanol to prepare a solution containing 0.1mg of hesperidin per 1 ml.

Preparation of a test solution: grinding fel Ursi tablet into powder, collecting powder about 0.5g, precisely weighing, precisely adding 70% ethanol 50ml, weighing, ultrasonic extracting at room temperature for 45min, supplementing weight loss, filtering, and collecting filtrate.

(2) Measurement method

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring under the following chromatographic conditions, wherein the chromatogram is shown in FIG. 1.

(3) Calculating the content of the component to be measured

Firstly, calculating to obtain the content C of hesperidin in the gallbladder warming tablet test sample_s。

TABLE 2 relative retention time and correction factor

Then, taking the hesperidin peak area in the chromatogram as a reference, and using the relative retention value (r) of the chromatographic peak of the component to be detected and the hesperidin chromatographic peak shown in Table 2_i/s) Determining the peak positions of p-hydroxycinnamic acid, narirutin and glycyrrhizic acid. Then, the relative correction factors (f) shown in Table 2 were applied_s/i) Substituting the following formula, and calculating the content C of the component to be measured to hydroxycinnamic acid, narirutin and glycyrrhizic acid_i：

f_s/i＝f_s/f_i＝(A_s×C_i)/(A_i×C_s)

Wherein A is_sArea of peak for control (i.e., hesperidin) C_iContent of component to be measured, A_iIs the peak area of the component to be measured, C_sIs the control (i.e. hesperidin) content.

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1, methodological examination

1.1 Linear relationship investigation

Precisely weighing a reference substance: hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid are respectively prepared into mother liquor of 0.8430mg/ml, 2.0280mg/ml, 1.0130mg/ml and 2.0480mg/ml, mother liquor with different volumes is respectively and precisely absorbed and placed into a 10ml volumetric flask to prepare mixed standard solution I of 67.4 mu g/ml, 202.8 mu g/ml, 405.2 mu g/ml and 204.8 mu g/ml, and then mixed standard solutions II (I5/10), III (II 5/10), IV (III 3.5/5), V (IV 3/5) and VI (V3/5) are sequentially diluted to be detected by adopting the chromatographic conditions of the example 2.

Then, the peak area (y) is used as the ordinate, the concentration (x) of the reference substance is used as the abscissa, and a standard curve (see fig. 3 to 6) is drawn to obtain a regression equation (see table 3). The results show that the R value of each control is 0.9999, indicating that each compound has a good linear relationship in the concentration range.

TABLE 3 Standard curves and Linear concentration ranges of 4 ingredients in Wendan tablets

1.2 precision investigation

In-day precision, each mixed standard solution III prepared by 1.1 linear relation investigation is taken, sample introduction is continuously carried out for 6 times in one day by adopting the chromatographic conditions of the example 1, peak areas of the hydroxycinnamic acid, the narirutin, the hesperidin and the glycyrrhizic acid are recorded, and the in-day precision is measured, and the result is shown in a table 4. The results show that the daily precision of the instrument is good.

And (3) daytime precision, namely taking each mixed standard solution III prepared by 1.1 linear relation investigation, continuously sampling for three days by adopting the chromatographic condition of the example 1, continuously sampling for 3 times every day, recording peak areas of the hydroxycinnamic acid, the narirutin, the hesperidin and the glycyrrhizic acid, and measuring the daytime precision. The results are shown in Table 4. The results show that the instrument has good daytime precision.

TABLE 4 precision measurement results of 4 ingredients in Wendan tablet

1.3 repeatability test

6 test solutions were prepared in parallel in accordance with the method of example 1, peak areas of the respective components were measured under the same chromatographic conditions as in example 1, and the contents of p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid were calculated from a standard curve, and the average contents of the respective components and RSD values are shown in Table 5, indicating that the method was excellent in reproducibility.

TABLE 5 repeatability test results of 4 ingredients in Wendan tablet

Composition (I)	Average content (mg/g)	RSD/％,n＝6
			P-hydroxy cinnamic acid	1.2427	0.44
Rutachinin	4.8055	0.75
			Hesperidin	12.8202	0.34
Glycyrrhizic acid	5.0488	0.19

1.4 stability test

The same test solutions were prepared according to the method of example 1, injected under the chromatographic conditions described in example 1 for 0, 4, 8, 12, 24, 36, 48, 60, 72h, respectively, the peak areas at the time points were recorded, and the RSD values thereof were calculated.

The results in Table 6 show that the peak area of p-hydroxycinnamic acid is reduced at 48-72h, and the RSD is 2.96% within 48h, which indicates that the p-hydroxycinnamic acid has good stability within 48 h; the RSD of the narirutin, the hesperidin and the glycyrrhizic acid is below 1.71 percent within 48 hours, which shows that the narirutin, the hesperidin and the glycyrrhizic acid have good stability within 72 hours. Therefore, the test solution of the warming gallbladder tablet has good stability within 48 hours.

Table 6 test results of stability of 4 ingredients in Wendan tablet

Composition (I)	RSD/%, n 6 within 48h	RSD/%, n 6 within 72h
			P-hydroxy cinnamic acid	2.74	4.57
Rutachinin	1.23	1.71
			Hesperidin	0.13	0.19
Glycyrrhizic acid	0.27	0.32

1.5 sample application recovery test

0.25g of a known content of a piece sample of a Wendan tablet (lot: 19032105) was weighed precisely, 3 levels of 50%, 100%, and 150% of the known content of a control were added to the piece sample, and a sample solution was prepared in the same manner as in example 1, and the average recovery rate and RSD calculation results measured under the same chromatographic conditions as in example 2 are shown in Table 7. The accuracy of the determination method is good.

TABLE 7 results of sample recovery test for 4 ingredients in Wendan tablet

Composition (I)	Average recovery (%)	RSD(％)(n＝6)
			P-hydroxy cinnamic acid	101.47	1.28
Rutachinin	101.17	1.50
			Hesperidin	99.75	1.50
Glycyrrhizic acid	98.47	2.75

1.6 detection and quantitation limits

Taking a mixed reference substance (the concentration of p-hydroxycinnamic acid is 13.1 mu g/ml, the concentration of naringin is 50.1 mu g/ml, the concentration of hesperidin is 130.9 mu g/ml and the concentration of glycyrrhizic acid is 50.1 mu g/ml), diluting by proper times, measuring according to the chromatographic condition of the embodiment 2, selecting a noise range of 14-15 min, and calculating according to the signal-to-noise ratio (S/N) of 3 to obtain the detection limit; the quantitative limit is obtained by calculation when the signal-to-noise ratio (S/N) is 10. The detection limits and the quantitative limits for hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid are shown in the following table 8.

TABLE 8 detection and quantitation limits

	P-hydroxy cinnamic acid	Rutachinin	Hesperidin	Glycyrrhizic acid
					Quantitative limit (ng)	3.41	2.00	2.62	2.00
Detection limit (ng)	0.53	0.90	0.79	0.60

1.7 durability test

1.7.1 investigation of different column temperatures

The same batch of samples of the warm glass pieces of example 1 was used to prepare test solutions as described in example 1, and the durability of the samples was examined under the conditions of the column temperature of 25 ℃ 30 ℃ and 35 ℃ with the column temperature changed under the chromatographic conditions described in example 1, and the results are shown in Table 9.

The result shows that under the condition of the column temperature of 25-35 ℃, the separation degree, the symmetry factor and the theoretical plate number of the component to be measured all meet the requirements, and the determination method is applicable to the column temperature of 25-35 ℃.

TABLE 9 investigation results of different column temperatures

1.7.2 investigation of different flow rates

The same batch of samples of the Wendan tablet as in example 1 was used to prepare a test solution as in example 1, and the flow rates of the mobile phase were varied under the chromatographic conditions as in example 1, and the flow rates of 0.8ml/min, 1.0ml/min and 1.2ml/min were examined, and the results are shown in Table 10. The result shows that under the condition of the flow rate of 0.8 ml/min-1.2 ml/min, the separation degree, the symmetry factor and the theoretical plate number of the component to be measured all meet the requirements, and the method is applicable to the flow rate of 0.8 ml/min-1.2 ml/min.

TABLE 10 investigation results of different flow rates

1.7.3 investigation of different phosphoric acid concentrations

The same batch of samples of the Welch tablet of example 1 was sampled to prepare a test solution as described in example 1, and the durability was examined under the conditions of the phosphoric acid concentration of 0.05%, 0.1% and 0.2% by changing the phosphoric acid concentration of the mobile phase under the chromatographic conditions described in example 1, and the results are shown in Table 11. The results show that the concentration of the mobile phase phosphoric acid is in the range of 0.05-0.2%, and the separation degree, the symmetry factor and the theoretical plate number of the component to be measured all meet the requirements, which indicates that the concentration of the mobile phase phosphoric acid adopted by the measuring method is applicable in the range of 0.05-0.2%.

TABLE 11 examination of different phosphoric acid concentrations

1.7.4 investigation of different chromatography columns

A sample of the same batch of Welch tablets as in example 1 was taken to prepare a test solution as described in example 1, and the chromatographic conditions as described in example 1 were changed to examine different columns, Inertsil ODS-3 (4.6X 250mm, 5 μm), Diamonsil C₁₈(4.6×250mm，5μm)、Thermo Syncronis C₁₈(4.6X 250mm, 5 μm), CAPCELL PAK ADME (4.6X 250mm, 5 μm), nanop analysis Chrom Core C₁₈(4.6X 250mm, 5 μm). The results show that Inertsil ODS-3 and Diamonsil C₁₈The separation degree, the symmetry factor and the theoretical plate number under the chromatographic column all meet the requirements.

TABLE 12 investigation results of different chromatography columns

Experimental example 2 calculation of relative correction factor

A batch of gallbladder warming tablets with known contents of p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid are taken as test samples I to VI by taking hesperidin as a reference substance, and the chromatographic conditions are respectively detected according to the embodiment 3.

2.1 calculation of relative Retention values

Calculating the retention time difference (delta t) between each component to be detected and the hesperidin as an internal reference in the samples I to VI_Ris)，Δt_Ris＝t_i-t_s(ii) a Calculating the relative retention value (r) of each component to be detected and the hesperidin as an internal reference substance in the samples I to VI_i/s)，r_i/s＝t_i/t_s。t_iRetention time of the component to be measured, t_sIs the retention time of the internal reference substance, the result isSee table 13.

TABLE 13 retention time differences and relative retention values of chromatographic peaks of the components to be determined

The results showed a relative retention of 0.57 for hydroxycinnamic acid, a retention time differential of-15.74, a relative retention of 0.85 for narirutin, a retention time differential of-5.66, a relative retention of 1.58 for glycyrrhizic acid, a retention time differential of 21.36, and an RSD of less than 1%.

2.2 calculation of relative correction factor

Taking hesperidin as an internal reference, calculating the relative correction factor (f) of each component to be detected in the samples I-VI_s/i)，f_s/i＝f_s/f_i＝(A_s×C_i)/(A_i×C_s)，A_sIs the peak area of the internal reference substance, C_iThe content of the component to be measured (known), A_iIs the peak area of the component to be measured, C_sIs the content of internal reference (known). The calculation results are shown in Table 14.

TABLE 14 relative correction factors for the components to be tested

	f_{P-hydroxycinnamic acid/hesperidin}	f_{Narirutin/hesperidin}	f_{Hesperidin/hesperidin}	f_{Glycyrrhizic acid/hesperidin}
					Ⅰ	0.93	1.00	1.00	1.12
Ⅱ	0.93	1.00	1.00	1.13
					Ⅲ	0.93	0.99	1.00	1.13
Ⅳ	0.93	1.00	1.00	1.12
					Ⅴ	0.92	1.01	1.00	1.13
Ⅵ	0.94	1.01	1.00	1.14
					Mean value of	0.93	1.00	1.00	1.13
RSD(％)	0.68％	0.75％	0.00％	0.67％

The results show that relative correction factors of the hesperidin as an internal reference material to the hydroxycinnamic acid, the narirutin and the glycyrrhizic acid are respectively 0.93, 1.00 and 1.13, and the RSD is less than 1 percent.

Experimental example 3 evaluation of durability by QAMS method

3.1 investigation of different chromatography columns

Referring to the method for calculating the relative correction factors of experimental example 2, the mixed standard solution prepared in experimental example 1 was used as a test sample, and the types of the chromatographic columns were changed under the same chromatographic conditions, and the relative correction factors calculated under different chromatographic columns (Inertsil ODS-3(4.6 × 250mm, 5 μm), CAPCELL PAK ADME (4.6 × 250mm, 5 μm), Napyl analysis Chrom Core C18(4.6 × 250mm, 5 μm), Thermo Syncronis C18(4.6 × 250mm, 5 μm), Diamonsil C18(4.6 × 250mm, 5 μm) were examined, and the results are shown in Table 15, wherein the RSD of the relative correction factors is less than 5%, indicating that the relative correction factors calculated by using rutin as an internal reference substance according to the present invention are all suitable for the above-mentioned different kinds of chromatographic columns.

TABLE 15 different chromatographic column examination results with correction factors

Chromatographic column	f_{P-hydroxycinnamic acid/hesperidin}	f_{Narirutin/hesperidin}	f_{Hesperidin/hesperidin}	f_{Glycyrrhizic acid/hesperidin}
					Diamonsil C₁₈(n＝6)	0.97	1.05	1.00	1.15
Thermo Syncronis C₁₈(n＝6)	0.99	1.00	1.00	1.19
					Inertsil ODS-3(n＝6)	0.93	1.00	1.00	1.13
CAPCELL PAK ADME(n＝6)	0.97	0.97	1.00	1.16
					Mean value of	0.96	1.01	1.00	1.16
RSD(％)	2.32％	3.04％	0.00％	2.22％

3.2 investigation of different instruments

Referring to the method for calculating the relative correction factor of experimental example 2, the mixed standard solution prepared in experimental example 1 was used as a sample, and the relative correction factors calculated by different instruments (Agilent 1260 II, Agilent1200 and Waters e2695 HPLC instruments) were examined under the same chromatographic conditions. The results are shown in table 17, and it can be seen that the RSD relative to the correction factor is less than 5%, with no significant difference. The method is applicable to high performance liquid chromatographs of different brands and models, wherein relative correction factors of the hydroxycinnamic acid, the narirutin and the glycyrrhizic acid are calculated by taking the hesperidin as an internal reference substance.

TABLE 16 correction factor different Instrument findings

Instrument for measuring the position of a moving object	f_{P-hydroxycinnamic acid/hesperidin}	f_{Narirutin/hesperidin}	f_{Hesperidin/hesperidin}	f_{Glycyrrhizic acid/hesperidin}
					Agilent 1200(n＝6)	0.95	0.99	1.00	1.17
Agilent 1260Ⅱ(n＝6)	0.93	1.00	1.00	1.13
					Waters e2695(n＝6)	1.01	0.98	1.00	1.26
Mean value of	0.96	0.99	1	1.18
					RSD(％)	3.60％	1.45％	0.00％	4.67％

Experimental example 4 comparison of measurement results of one-test-multiple-evaluation method (QAMS method) and external standard method (ESM method)

1. Experimental methods

15 batches of the gallbladder-warming tablet samples are respectively detected according to the methods of the embodiment 2 and the embodiment 3, the peak areas of the gallbladder-warming tablet samples to the hydroxycinnamic acid, the narirutin, the hesperidin and the glycyrrhizic acid are calculated according to the obtained chromatograms, and the contents of the hydroxycinnamic acid, the narirutin, the hesperidin and the glycyrrhizic acid are respectively calculated by adopting a QAMS method (as described in the embodiment 3 of the invention) and an ESM method (as described in the embodiment 2 of the invention).

Comparing the difference between the measurement results of the one-test multi-evaluation method and the external standard method by using the formula (1) and the formula (2): percentage difference (PD%), cosine of angle Cos (θ).

Wherein a and b are the contents (%) of the substances to be detected calculated by QAMS and ESM respectively, and n is all batches of the fel warming tablets.

2. Results of the experiment

TABLE 1715 relative retention of samples from Wendan tablets (n ═ 3)

Batches of	r_{P-hydroxycinnamic acid/hesperidin}	r_{Narirutin/hesperidin}	r_{Hesperidin/hesperidin}	r_{Glycyrrhizic acid/hesperidin}
					19032108	0.57	0.85	1.00	1.59
1711002	0.58	0.85	1.00	1.59
					19032104	0.58	0.85	1.00	1.59
19032105	0.58	0.85	1.00	1.59
					19032103	0.57	0.84	1.00	1.58
19032106	0.57	0.84	1.00	1.57
					19032110	0.57	0.84	1.00	1.58
19032107	0.57	0.84	1.00	1.58
					1711003	0.59	0.86	1.00	1.61
1711001	0.57	0.85	1.00	1.58
					1812002	0.59	0.85	1.00	1.62
19032101	0.59	0.85	1.00	1.62
					19032109	0.57	0.85	1.00	1.59
19032102	0.59	0.85	1.00	1.63
					1812001	0.59	0.85	1.00	1.63
Mean value of	0.58	0.85	1.00	1.60
					RSD(％)	1.58％	0.66％	0.00％	1.25％

TABLE 18 QAMS method and ESM method for determining the content of 4 ingredients in the Winternake tablet (n is 3)

"-" indicates that there is impurity interference, the results of the relative retention values of the samples of the QAMS method for content can not be calculated are shown in Table 17, and the results of the content measurement and comparison of 4 components in the gallbladder warming tablets by the ESM method (measured value) and the QAMS method (calculated value) are shown in Table 18.

(1) The average hesperidin content calculated by an external standard method is 11.6427mg/g, the average p-hydroxycinnamic acid content is 1.1479mg/g, the average narirutin content is 4.7781mg/g, and the average glycyrrhizic acid content is 3.9654 mg/g; the average content of p-hydroxycinnamic acid calculated by one-test-multiple evaluation method is 1.1339mg/g, the average content of narirutin is 4.9386mg/g, and the average content of glycyrrhizic acid is 4.0279 mg/g. The results show that the one-test-multiple-evaluation method and the external standard method of 4 components in 15 batches of samples have no obvious difference.

(2) The ratio of the calculated value of the hydroxycinnamic acid to the measured value is 98.17-99.30%, PD is less than 5%, and Cos (theta) is 1.0000; the proportion of the calculated value of the narirutin in the measured value is 99.97-104.98%, PD is less than 5%, and Cos (theta) is 0.9999; the ratio of the calculated glycyrrhizic acid value to the measured value is 99.16-102.76%, PD < 5%, and Cos (theta) ═ 0.9999. The results show that the content results of the components measured by the two methods have no significant difference, and the one-measurement and multi-evaluation method established by the invention is feasible.

The results show that the results of the one-test-multiple-evaluation method and the external standard method for 4 components in 15 batches of samples have no obvious difference. Compared with an external standard method, the established one-test-multiple evaluation method can simultaneously and accurately measure the content of 4 components (p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid) in the gallbladder warming tablet, and is suitable for quality control of the gallbladder warming tablet.

Moreover, compared with 4 control samples (p-hydroxycinnamic acid, rutin, hesperidin and glycyrrhizic acid control samples) required by an external standard method, the one-test-multiple-evaluation method established by the invention only needs to adopt one control sample (hesperidin control sample), thereby effectively reducing the detection cost for purchasing the control samples.

In conclusion, the invention provides a method for simultaneously measuring multiple index components in a gallbladder warming tablet. Under the specific chromatographic conditions, the method can not only carry out qualitative analysis on a plurality of components (p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid) in the gallbladder warming tablet, but also carry out quantitative determination. Under the specific chromatographic conditions of the invention, the detection accuracy of quantitative determination by using a one-test-multiple-evaluation method is equivalent to that of an external standard method.

Particularly, the one-test-multiple-evaluation method for determining the content of various index components in the gallbladder warming tablet takes hesperidin as a reference substance and establishes relative correction factors for hydroxycinnamic acid, narirutin, glycyrrhizic acid and hesperidin, so that the synchronous determination of 4 contents of hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid in the gallbladder warming tablet is realized. The measuring method of the invention can accurately and comprehensively evaluate the quality of the gallbladder warming tablets, is simple, convenient and quick, has good stability and repeatability, and is beneficial to improving the quality standard of the gallbladder warming tablets. Particularly, the method for evaluating more than one test ensures the effective, stable and controllable quality of the preparation, reduces the detection cost of purchasing a reference substance, obtains excellent technical effect and has wide application prospect.

Claims

1. A method for simultaneously measuring multiple index components in a gallbladder warming tablet is characterized in that:

the method comprises the steps of taking a gallbladder warming tablet as a test sample, carrying out high performance liquid chromatography detection, and simultaneously measuring p-hydroxycinnamic acid, rutin, hesperidin and glycyrrhizic acid in the gallbladder warming tablet; the method comprises the following steps:

(2) injecting into a high performance liquid chromatograph for detection;

the chromatographic conditions were as follows:

mobile phase: the phase A consists of acetonitrile and phosphoric acid aqua.

2. The method of claim 1, wherein: the method is a method for quantitatively measuring hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid; the method is an external standard method or a one-test-multiple evaluation method.

3. The method of claim 2, wherein: the external standard method comprises the steps of measuring the content of p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid in the gallbladder warming tablet by taking p-hydroxycinnamic acid, narirutin, hesperidin and glycyrrhizic acid as reference substances; the control solution was prepared as follows: respectively weighing p-hydroxycinnamic acid, rutin, hesperidin and glycyrrhizic acid, and respectively adding organic solvent for dissolving to obtain the final product.

4. The method of claim 2, wherein: the one-test-multiple evaluation method uses hesperidin as a reference substance to measure the contents of p-hydroxycinnamic acid, rutin, hesperidin and glycyrrhizic acid in the gallbladder warming tablet.

5. The method of claim 4, wherein: the method comprises the following steps:

f_s/i＝(A_s×C_i)/(A_i×C_s)

wherein f is_s/iIs a relative correction factor between the component to be measured and hesperidin, A_sIs the peak area of hesperidin in the chromatogram of the test solution,C_icontent of component to be measured in Wendan tablet, A_iIs the peak area, C, of the component to be measured in the chromatogram of the sample solution_sIs the content of hesperidin in the gallbladder warming tablet.

6. The method of claim 5, wherein: in the step 4), the relative correction factor between the p-hydroxycinnamic acid and the hesperidin is 0.92-1.01, preferably 0.93, the relative correction factor between the narirutin and the hesperidin is 0.97-1.05, preferably 1.00, and the relative correction factor between the glycyrrhizic acid and the hesperidin is 1.12-1.26, preferably 1.13.

7. The method according to any one of claims 1 to 6, wherein: the octadecylsilane bonded silica gel is Inertsil ODS-3, Diamonsil C18, CAPCELL PAK ADME, Chrom Core C18 or Thermo Syncronis C18, preferably Inertsil ODS-3.

8. The method according to any one of claims 1 to 6, wherein: the chromatographic conditions were gradient elution, as shown in the following table:

9. The method according to any one of claims 1 to 8, wherein: in the preparation of the test solution, the organic solvent is an alcohol solvent, preferably 60-80% ethanol, and more preferably 70% ethanol;

10. The method of claim 9, wherein: in the preparation of the reference solution, the organic solvent is an alcohol solvent, preferably methanol; and/or the concentration of the control solution is 0.025-0.405 mg/mL, preferably 0.1 mg/mL.