A method for simultaneously determining multiple index components in Wendan Tablets

Abstract

The present invention provides a method for simultaneously determining multiple index components in Wendan Tablets. In this method, Wendan Tablets are used as test samples for high-performance liquid chromatography detection to simultaneously determine p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid in Wendan Tablets. The method of the present invention can also quantitatively determine the contents of the above four components in Wendan Tablets, and the quantitative determination methods include the external standard method and the multiple components by single marker method. The determination method of the present invention realizes the qualitative and quantitative determination of multiple components (p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid) in Wendan Tablets. Among them, the multiple components by single marker quantitative detection method not only ensures the quality, stability, and controllability of the preparation, but also reduces the detection cost of purchasing reference substances, achieving excellent technical effects and having broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of quality control of traditional Chinese medicines, and particularly relates to a method for simultaneously determining multiple index components in Wendan tablets. Background Art

[0002] Wendan tablets are a traditional Chinese medicine formulated preparation made from seven traditional Chinese medicinal materials, namely Rhizoma Pinelliae Praeparatum, Caulis Bambusae in Taeniam, Fructus Aurantii Immaturus, Pericarpium Citri Reticulatae, Poria, Radix Curcumae, and Radix Glycyrrhizae Preparata, through extraction and processing. It has the effects of regulating qi, clearing heat and resolving phlegm, warming the gallbladder and harmonizing the stomach, and clearing the heart and relieving depression. Currently, it is commonly used clinically to treat generalized anxiety disorder with definite curative effects. Wendan tablets are a modern preparation improved from the classic famous prescription Wendan Decoction, which contains a variety of active ingredients, such as p-hydroxycinnamic acid, naringin, hesperidin, liquiritin, glycyrrhizic acid, etc. At present, the quality control of Wendan tablets is usually carried out by measuring the content of one or more index components in Wendan tablets.

[0003] CN106323794A discloses a quality control method for Wendan tablets: determining the transfer rate of synephrine in Wendan tablets by liquid chromatography to confirm the product quality stability of Wendan tablets. However, the method established in this invention measures with synephrine (the component in Fructus Aurantii Immaturus and Pericarpium Citri Reticulatae) as a single quality control index, and cannot comprehensively reflect the quality of Wendan tablets.

[0004] Wan Lijuan et al. (Research on the development and quality standard of Wendan granules, Hubei University of Chinese Medicine) disclosed a method for determining the quality standard of Wendan granules: establishing chromatographic conditions for the determination of hesperidin, liquiritin, and glycyrrhizic acid in Wendan granules by reverse-phase liquid chromatography. However, this method is relatively troublesome to measure. It is necessary to first measure the content of hesperidin in Wendan granules, and then measure the content of liquiritin and glycyrrhizic acid in Wendan granules, and cannot be detected simultaneously; moreover, its measurement method requires the use of multiple reference substances (hesperidin, liquiritin, and ammonium glycyrrhizinate reference substances), which greatly increases the detection cost.

[0005] Therefore, it is urgent to develop a quality control method that can use only one cheap and easily available component as a reference substance to simultaneously determine the content of multiple active components in Wendan tablets.

[0006] In 2006, Wang Zhimin et al. from the China Academy of Chinese Medical Sciences took the lead in proposing a multi-index quality control method of quantitative analysis of multi-components by single-marker (QAMS). This method uses a relatively easily available and inexpensive internal reference substance reference standard to achieve the simultaneous determination of multiple components, ultimately achieving the purpose of controlling the overall quality of traditional Chinese medicine. QAMS has the advantages of low detection cost and high analysis efficiency. Therefore, once it was proposed, it caused a huge response at home and abroad and was considered a multi-index quality control and evaluation model suitable for the characteristics of traditional Chinese medicine. Currently, it has been applied in the quality control of many traditional Chinese medicines such as Panax notoginseng, Psoralea corylifolia, Platycodon grandiflorum, Pueraria lobata, Houttuynia cordata, and Curcuma wenyujin.

[0007] Currently, there is no reported method for the simultaneous determination of 4 components, namely p-hydroxycinnamic acid, narirutin, hesperidin, and glycyrrhizic acid, in Wendan Tablets. There is even less reported quality control method for the simultaneous determination of the above 4 components in Wendan Tablets by the QAMS method. Summary of the Invention

[0008] The purpose of the present invention is to provide a method capable of simultaneously determining multiple index components (p-hydroxycinnamic acid, narirutin, hesperidin, and glycyrrhizic acid) in Wendan Tablets, and to conduct qualitative and quantitative analysis of p-hydroxycinnamic acid, narirutin, hesperidin, and glycyrrhizic acid in Wendan Tablets.

[0009] The present invention provides a method for simultaneously determining multiple index components in Wendan Tablets. The method uses Wendan Tablets as the test sample for high performance liquid chromatography detection to simultaneously determine p-hydroxycinnamic acid, narirutin, hesperidin, and glycyrrhizic acid in Wendan Tablets, and includes the following steps:

[0010] (1) Preparation of the test sample solution: Take Wendan Tablets, extract with an organic solvent, and take the extract to obtain the test sample solution;

[0011] (2) Inject into a high performance liquid chromatograph for detection;

[0012] The chromatographic conditions are as follows:

[0013] Chromatographic column: Packed with octadecylsilane chemically bonded silica gel;

[0014] Mobile phase: Composed of phase A and phase B. Phase A is acetonitrile, and phase B is an aqueous phosphoric acid solution.

[0015] Furthermore, the method is a method for quantitatively determining p-hydroxycinnamic acid, narirutin, hesperidin, and glycyrrhizic acid; the method is the external standard method or the QAMS method.

[0016] Further, in the external standard method, p-coumaric acid, narirutin, hesperidin and glycyrrhizic acid are used as reference substances to determine the contents of p-coumaric acid, narirutin, hesperidin and glycyrrhizic acid in Wendan tablets; the preparation method of the reference substance solution is as follows: Weigh p-coumaric acid, narirutin, hesperidin and glycyrrhizic acid respectively, and dissolve them with an organic solvent to obtain the solution.

[0017] Further, in the multi-component assay by single internal standard method, hesperidin is used as the reference substance to determine the contents of p-coumaric acid, narirutin, hesperidin and glycyrrhizic acid in Wendan tablets.

[0018] Further, the multi-component assay by single internal standard method includes the following steps:

[0019] 1) Preparation of the reference substance solution: Weigh hesperidin and dissolve it with an organic solvent to obtain the solution;

[0020] 2) Preparation of the test sample solution: Weigh Wendan tablets, extract them with an organic solvent, and take the extract to obtain the solution;

[0021] 3) Respectively absorb the test sample solution and the reference substance solution and inject them into a high performance liquid chromatograph to measure the content of hesperidin in Wendan tablets;

[0022] 4) Use the relative correction factors between the components to be measured, p-coumaric acid, narirutin and glycyrrhizic acid, and hesperidin, and calculate the contents of the components to be measured in Wendan tablets respectively through the following formula;

[0023] f s / i =(A s ×C i ) / (A i ×C s )

[0024] wherein, f s / i is the relative correction factor between the component to be measured and hesperidin, A s is the peak area of hesperidin in the chromatogram of the test sample solution, C i is the content of the component to be measured in Wendan tablets, A i is the peak area of the component to be measured in the chromatogram of the test sample solution, and C s is the content of hesperidin in Wendan tablets.

[0025] Further, in step 4), the relative correction factor between p-coumaric acid and hesperidin is 0.92 - 1.01, preferably 0.93; the relative correction factor between narirutin and hesperidin is 0.97 - 1.05, preferably 1.00; the relative correction factor between glycyrrhizic acid and hesperidin is 1.12 - 1.26, preferably 1.13.

[0026] Further, the octadecylsilyl-bonded silica gel is Inertsil ODS-3, Diamonsil C18, CAPCELL PAK ADME, Chrom Core C18 or Thermo Syncronis C18, preferably Inertsil ODS-3.

[0027] Further, the chromatographic conditions are gradient elution, and the elution conditions are shown in the following table:

[0028]

[0029]

[0030] In the table, the unit of time 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;

[0031] And / or, the mass concentration of the phosphoric acid aqueous solution is 0.05% to 0.20%, preferably 0.10%;

[0032] And / or, the detection wavelength in the chromatographic conditions is: 252 - 268 nm; the column temperature is 25 - 35 °C; the flow rate is 0.8 - 1.2 ml / min; preferably, the detection wavelength is: 0 - 25 min, 256 nm; 25 - 42 min, 268 nm; 42 - 65 min, 252 nm; the column temperature is 30 °C; the flow rate is 1.0 ml / min.

[0033] Further, in the preparation of the test solution, the organic solvent is an alcohol solvent, preferably 60% - 80% ethanol, more preferably 70% ethanol;

[0034] And / or, the mass-to-volume ratio of the Wendan tablets to the organic solvent is 5 mg / mL - 20 mg / mL, preferably 10 mg / mL; and / or, the extraction method is ultrasonic extraction, the extraction time is 30 - 60 min, preferably 45 min, the extraction temperature is room temperature, and the ultrasonic power is 200 - 300 W, preferably 250 W.

[0035] Further, in the preparation of the reference solution, the organic solvent is an alcohol solvent, preferably methanol; and / or, the concentration of the reference solution is 0.025 - 0.405 mg / mL, preferably 0.1 mg / mL.

[0036] In the present invention, the phosphoric acid concentration of the mobile phase is the mass concentration of the phosphoric acid aqueous solution. For example, 0.10% phosphoric acid refers to a phosphoric acid aqueous solution with a mass concentration of 0.10%.

[0037] Experimental results have shown that, under the specific chromatographic conditions of the present invention, the method for determining multiple index components in Wendan Tablets can not only qualitatively analyze multiple components in Wendan Tablets (p-hydroxycinnamic acid, narirutin, hesperidin, and glycyrrhizic acid), but also quantitatively determine them. Under the specific chromatographic conditions of the present invention, the detection accuracy of quantitative determination using the multiple components by single marker method is comparable to that of the external standard method.

[0038] Specifically, for the multiple components by single marker method for determining the contents of multiple index components in Wendan Tablets of the present invention, taking hesperidin as the reference substance, the relative correction factors of p-hydroxycinnamic acid, narirutin, and glycyrrhizic acid with respect to hesperidin are established, thereby realizing the simultaneous determination of the contents of 4 components, namely p-hydroxycinnamic acid, narirutin, hesperidin, and glycyrrhizic acid, in Wendan Tablets.

[0039] The determination method of the present invention can accurately and comprehensively evaluate the quality of Wendan Tablets, is simple, rapid, has good stability and repeatability, and is conducive to improving the quality standard of Wendan Tablets. Especially the multiple components by single marker method not only ensures the quality, effectiveness, stability, and controllability of the preparation, but also reduces the detection cost of purchasing reference substances, achieving excellent technical effects and having broad application prospects.

[0040] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions, or changes can be made.

[0041] The above content of the present invention will be further described in detail below through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0042] Figure 1 It is the chromatogram of the test sample of Wendan Tablets for Example 3, wherein, 1 - p-hydroxycinnamic acid, 2 - narirutin, 3 - hesperidin, 4 - glycyrrhizic acid.

[0043] Figure 2 It is the chromatogram of the mixed standard solution for Experimental Example 1, wherein, 1 - p-hydroxycinnamic acid, 2 - narirutin, 3 - hesperidin, 4 - glycyrrhizic acid.

[0044] Figure 3 It is the linear relationship diagram of p-hydroxycinnamic acid.

[0045] Figure 4 It is the linear relationship diagram of narirutin.

[0046] Figure 5 It is the linear relationship diagram of hesperidin.

[0047] Figure 6It is a linear relationship diagram of glycyrrhizic acid. Detailed implementation mode

[0048] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0049] Agilent Technologies 1260 Infinity Ⅱ high performance liquid chromatography system; BSA124S-CW type electronic analytical balance (Satorius), BSA 323S type electronic analytical balance (Satorius); KQ-250DB type numerically controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., 150W, 40KHz), Zeal Quest ultrapure water instrument; The chromatographic column is Inertsil ODS-3 (4.6×250mm, 5μm); Acetonitrile is chromatographically pure, phosphoric acid is chromatographically pure, water is ultrapure water, and other reagents are all analytically pure;

[0050] p-Hydroxycinnamic acid (PRF9070423, purity ≥ 98%, Chengdu Pufa Technology Co., Ltd.), glycyrrhizic acid (PRF10010203, purity ≥ 98%, Chengdu Pufa Technology Co., Ltd.), naringin (purity ≥ 98%, Shanghai Research Center for Traditional Chinese Medicine Standardization), hesperidin (110721-201818, purity 96.2%, National Institutes for Food and Drug Control); The samples of Wendan tablets are from Zhangzhou Pianzihuang Pharmaceutical Co., Ltd.

[0051] Example 1. Method for qualitative determination of multiple index components in Wendan tablets

[0052] Under the following chromatographic conditions, simultaneously determine whether p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid are contained in Wendan tablets (purchased from Zhangzhou Pianzihuang Pharmaceutical Co., Ltd.).

[0053] Preparation of test solution: Take Wendan tablets, grind them into powder, take about 0.5g of the powder, accurately weigh it, accurately add 50ml of 70% ethanol, weigh it, extract by ultrasonic wave at room temperature (ultrasonic power is 250W, frequency 40kHz) for 45min, make up the weight loss, filter, and take the continuous filtrate to obtain.

[0054] Chromatographic conditions: Chromatographic column: Inertsil ODS-3 (4.6×250mm, 5μm); Elution conditions are shown in Table 1; Detection wavelength: 0 - 25min, 256nm; 25 - 42min, 268nm; 42 - 65min, 252nm; Column temperature: 30°C; Flow rate: 1.0ml / min. The number of theoretical plates calculated by hesperidin should not be less than 5000.

[0055] Table 1 Gradient elution conditions

[0056]

[0057] The chromatogram was detected. Then, according to the retention times of p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid under the above chromatographic conditions, qualitative analysis was carried out to determine whether the Wendan tablets sample contained p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid.

[0058] Example 2. Quantitative determination of the contents of multiple index components in Wendan tablets by the external standard method

[0059] Using p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid as reference substances, under the following chromatographic conditions, the contents of p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid in Wendan tablets (purchased from Zhangzhou Pianzihuang Pharmaceutical Co., Ltd.) were simultaneously determined by the external standard method.

[0060] Preparation of the test solution: Take Wendan tablets, grind them into powder, take about 0.5 g of the powder, accurately weigh it, accurately add 50 ml of 70% ethanol, weigh it, extract by ultrasonic wave at room temperature (ultrasonic power is 250 W, frequency is 40 kHz) for 45 min, make up the lost weight, filter, and take the subsequent filtrate, that is obtained.

[0061] Chromatographic conditions: Chromatographic column: Inertsil ODS-3 (4.6×250 mm, 5 μm); Elution conditions are shown in Table 1; Detection wavelength: 0 - 25 min, 256 nm; 25 - 42 min, 268 nm; 42 - 65 min, 252 nm; Column temperature: 30 °C; Flow rate: 1.0 ml / min. The number of theoretical plates calculated by hesperidin should be not less than 5000.

[0062] The chromatogram was detected. Then, according to the standard curves of p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid (the determination method of the standard curves is as described in "Investigation of linear relationship" in Experimental Example 1), the contents of p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid in the Wendan tablets sample were calculated.

[0063] Example 3. Quantitative determination of the contents of multiple index components in Wendan tablets by the method of quantitative analysis of multi-components by single marker (QAMS)

[0064] Using hesperidin as the reference substance, the contents of four components, namely p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid, in Wendan tablets (purchased from Zhangzhou Pianzihuang Pharmaceutical Co., Ltd.) were simultaneously determined by the QAMS method established in the present invention. The specific steps are as follows:

[0065] (1) Preparation of solutions

[0066] Preparation of the reference substance solution: Take an appropriate amount of hesperidin, accurately weigh it, and make a solution containing 0.1 mg of hesperidin per 1 ml with methanol.

[0067] Preparation of test solution: Take Wendan tablets, grind them into powder. Accurately weigh about 0.5 g of the powder, accurately add 50 ml of 70% ethanol, weigh, ultrasonically extract at room temperature for 45 min, make up the weight loss, filter, and take the subsequent filtrate to obtain the solution.

[0068] (2) Determination method

[0069] Accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine under the following chromatographic conditions. The chromatogram is shown in Figure 1 .

[0070] Chromatographic conditions: Chromatographic column: Inertsil ODS-3 (4.6×250 mm, 5 μm); Elution conditions are shown in Table 1; Detection wavelength: 0 - 25 min, 256 nm; 25 - 42 min, 268 nm; 42 - 65 min, 252 nm; Column temperature: 30 °C; Flow rate: 1.0 ml / min. The number of theoretical plates calculated based on hesperidin should be not less than 5000.

[0071] (3) Calculate the content of the component to be measured

[0072] First, calculate the content C of hesperidin in the test sample of Wendan tablets s .

[0073] Table 2 Relative retention time and correction factor

[0074]

[0075]

[0076] Then, using the peak area of hesperidin in the chromatogram as a reference, determine the peak positions of p-coumaric acid, narirutin, and glycyrrhizic acid with the relative retention values (r i / s ) of the chromatographic peaks of the components to be measured and the hesperidin chromatographic peak shown in Table 2. Then, substitute the relative correction factors (f s / i ) shown in Table 2 into the following formula to calculate the contents C of the components to be measured, p-coumaric acid, narirutin, and glycyrrhizic acid i :

[0077] f s / i = f s / f i = (A s × C i ) / (A i × C s )

[0078] Where A s is the peak area of the control sample (i.e., hesperidin), C i is the content of the component to be measured, A i is the peak area of the component to be measured, Cs is the content of the control sample (i.e., hesperidin).

[0079] The beneficial effects of the present invention are demonstrated by the following experimental examples.

[0080] Experimental Example 1, Methodology Investigation

[0081] 1.1 Investigation of linear relationship

[0082] Accurately weigh the reference substances: p-coumaric acid, naringin, hesperidin, glycyrrhizic acid, and prepare stock solutions of 0.8430 mg / ml, 2.0280 mg / ml, 1.0130 mg / ml, and 2.0480 mg / ml respectively. Accurately pipette different volumes of the stock solutions into 10 ml volumetric flasks to prepare mixed standard solution I of 67.4 μg / ml, 202.8 μg / ml, 405.2 μg / ml, and 204.8 μg / ml. Then, successively dilute to obtain mixed standard solution II (I 5 / 10), III (II 5 / 10), IV (III 3.5 / 5), V (IV 3 / 5), VI (V 3 / 5), and detect using the chromatographic conditions of Example 2.

[0083] Then, with the peak area (y) as the ordinate and the reference substance concentration (x) as the abscissa, plot the standard curve (as Figures 3 - 6 ) to obtain the regression equation (see Table 3). The results show that the R values of each reference substance are all 0.9999, indicating that each compound has a good linear relationship within its concentration range.

[0084] Table 3 Standard curves and linear concentration ranges of 4 components in Wendan Tablets

[0085]

[0086] 1.2 Investigation of precision

[0087] Intraday precision: Take each mixed standard solution III prepared in "1.1 Investigation of linear relationship", and using the chromatographic conditions of Example 1, inject samples continuously 6 times within one day, record the peak areas of p-coumaric acid, naringin, hesperidin, and glycyrrhizic acid, and measure the intraday precision. The results are shown in Table 4. The results indicate that the intraday precision of the instrument is good.

[0088] Interday precision: Take each mixed standard solution III prepared in "1.1 Investigation of linear relationship", and using the chromatographic conditions of Example 1, inject samples continuously for three days, inject samples continuously 3 times each day, record the peak areas of p-coumaric acid, naringin, hesperidin, and glycyrrhizic acid, and measure the interday precision. The results are shown in Table 4. The results indicate that the interday precision of the instrument is good.

[0089] Table 4 Precision determination results of 4 components in Wendan Tablets

[0090]

[0091] 1.3 Repeatability test

[0092] Six test solution samples were prepared in parallel according to the method of Example 1, and then the peak areas of each component were determined under the same chromatographic conditions as in Example 1. The contents of p-hydroxycinnamic acid, narirutin, hesperidin, and glycyrrhizic acid were calculated using the standard curve. The average content and RSD value of each component are shown in Table 5, indicating that the method has good repeatability.

[0093] Table 5 Results of repeatability test for 4 components in Wendan Tablets

[0094] Component Average content (mg / g) RSD / %, n = 6 p - Hydroxycinnamic acid 1.2427 0.44 Narirutin 4.8055 0.75 Hesperidin 12.8202 0.34 Glycyrrhizic acid 5.0488 0.19

[0095] 1.4 Stability test

[0096] The same test solution sample was prepared according to the method of Example 1, and injected at 0, 4, 8, 12, 24, 36, 48, 60, and 72 h respectively under the chromatographic conditions described in Example 1. The peak areas at each time point were recorded and the RSD values were calculated.

[0097] Table 6 shows that the peak area of p-hydroxycinnamic acid decreased at 48 - 72 h, and the RSD within 48 h was 2.96%, indicating good stability of p-hydroxycinnamic acid within 48 h; the RSD of narirutin, hesperidin, and glycyrrhizic acid within 48 h was below 1.71%, indicating good stability of narirutin, hesperidin, and glycyrrhizic acid within 72 h. Therefore, the test solution sample of Wendan Tablets has good stability within 48 h.

[0098] Table 6 Results of stability test for 4 components in Wendan Tablets

[0099] Component RSD / %, n = 6 within 48 h RSD / %, n = 6 within 72 h p - Hydroxycinnamic acid 2.74 4.57 Narirutin 1.23 1.71 Hesperidin 0.13 0.19 Glycyrrhizic acid 0.27 0.32

[0100] 1.5 Recovery test

[0101] 0.25 g of Wendan Tablet sample with known content (batch number: 19032105) was accurately weighed, and reference substances at 50%, 100%, and 150% of the known content levels were added respectively. The test solution samples were prepared according to the method of Example 1 and determined under the chromatographic conditions of Example 2. The calculated results of the average recovery rate and RSD are shown in Table 7, indicating good accuracy of the determination method.

[0102] Table 7 Results of recovery test for 4 components in Wendan Tablets

[0103] Component Average recovery rate (%) RSD (%) (n = 6) p - Hydroxycinnamic acid 101.47 1.28 Narirutin 101.17 1.50 Hesperidin 99.75 1.50 Glycyrrhizic acid 98.47 2.75

[0104] 1.6 Detection limit and quantitation limit

[0105] Take the mixed reference substances (the concentration of p-coumaric acid is 13.1 μg / ml, the concentration of rutacridone is 50.1 μg / ml, the concentration of hesperidin is 130.9 μg / ml, and the concentration of glycyrrhizic acid is 50.1 μg / ml), dilute them by an appropriate multiple, measure according to the chromatographic conditions in Example 2, select the noise range at 14 - 15 min, calculate according to the signal-to-noise ratio (S / N) of 3 to obtain the detection limit; calculate according to the signal-to-noise ratio (S / N) of 10 to obtain the quantitation limit. The detection limits and quantitation limits of p-coumaric acid, rutacridone, hesperidin, and glycyrrhizic acid are shown in Table 8 below.

[0106] Table 8 Detection Limits and Quantitation Limits

[0107] p - Hydroxycinnamic acid Narirutin Hesperidin Glycyrrhizic acid Quantification limit (ng) 3.41 2.00 2.62 2.00 Detection limit (ng) 0.53 0.90 0.79 0.60

[0108] 1.7 Durability Test

[0109] 1.7.1 Investigation at Different Column Temperatures

[0110] Take the same batch of Wendan tablets samples in Example 1, prepare the test solution according to the method described in Example 1, under the chromatographic conditions described in Example 1, change the column temperature, and investigate the durability under the column temperature conditions of 25°C, 30°C, and 35°C. The results are shown in Table 9.

[0111] The results show that under the column temperature conditions of 25°C - 35°C, the resolution, symmetry factor, and theoretical plate number of the components to be measured all meet the requirements, indicating that this determination method is applicable within the range of column temperature from 25 to 35°C.

[0112] Table 9 Investigation Results at Different Column Temperatures

[0113]

[0114]

[0115] 1.7.2 Investigation at Different Flow Rates

[0116] Take the same batch of Wendan tablets samples in Example 1, prepare the test solution according to the method described in Example 1, under the chromatographic conditions described in Example 1, change the flow rate of the mobile phase, and investigate the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min. The results are shown in Table 10. The results show that under the conditions of flow rate from 0.8 ml / min to 1.2 ml / min, the resolution, symmetry factor, and theoretical plate number of the components to be measured all meet the requirements, indicating that this method is applicable within the range of flow rate from 0.8 ml / min to 1.2 ml / min.

[0117] Table 10 Investigation Results at Different Flow Rates

[0118]

[0119] 1.7.3 Investigation of Different Phosphoric Acid Concentrations

[0120] Take the same batch of Wendan tablets samples in Example 1, prepare the test solution according to the method described in Example 1. Under the chromatographic conditions described in Example 1, change the phosphoric acid concentration of the mobile phase, and investigate the durability under the conditions of phosphoric acid concentrations of 0.05%, 0.1%, and 0.2%. The results are shown in Table 11. The results show that within the range of 0.05% - 0.2% of the phosphoric acid concentration in the mobile phase, the resolution, symmetry factor, and theoretical plate number of the components to be measured all meet the requirements, indicating that the phosphoric acid concentration of the mobile phase used in this determination method is applicable within the range of 0.05% - 0.2%.

[0121] Table 11 Results of Investigation of Different Phosphoric Acid Concentrations

[0122]

[0123]

[0124] 1.7.4 Investigation of Different Chromatographic Columns

[0125] Take the same batch of Wendan tablets samples in Example 1, prepare the test solution according to the method described in Example 1. Under the chromatographic conditions described in Example 1, change the chromatographic column, and investigate the determination results of different chromatographic columns Inertsil ODS - 3 (4.6×250mm, 5μm), Diamonsil C 18 (4.6×250mm, 5μm), Thermo Syncronis C 18 (4.6×250mm, 5μm), CAPCELL PAK ADME(4.6×250mm, 5μm), Knauer Analytical Chrom Core C 18 (4.6×250mm, 5μm). The results show that the resolution, symmetry factor, and theoretical plate number under the Inertsil ODS - 3 and Diamonsil C 18 chromatographic columns all meet the requirements.

[0126] Table 12 Results of Investigation of Different Chromatographic Columns

[0127]

[0128] Experimental Example 2, Calculation of Relative Correction Factor

[0129] Using hesperidin as the reference substance, and using a batch of Wendan tablets with known contents of p - hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid as test samples I - VI, detect them respectively according to the chromatographic conditions described in Example 3.

[0130] 2.1 Calculation of Relative Retention Value

[0131] Calculate the retention time difference (Δt Ris ) between each component to be measured and hesperidin as the internal reference substance in Samples I-VI. Δt Ris = t i - t s . Calculate the relative retention value (r i / s ) between each component to be measured and hesperidin as the internal reference substance in Samples I-VI. r i / s = t i / t s . t i is the retention time of the component to be measured, and t s is the retention time of the internal reference substance. The results are shown in Table 13.

[0132] Table 13 Retention Time Difference and Relative Retention Value of Chromatographic Peaks of Components to be Measured

[0133]

[0134] The results show that the relative retention value of p-coumaric acid is 0.57, and the retention time difference is -15.74; the relative retention value of narirutin is 0.85, and the retention time difference is -5.66; the relative retention value of glycyrrhizic acid is 1.58, and the retention time difference is 21.36. The RSDs are all less than 1%.

[0135] 2.2 Calculation of Relative Correction Factor

[0136] Using hesperidin as the internal reference substance, calculate the relative correction factor (f s / i ) of each component to be measured in Samples I-VI. f s / i = f s / f i = (A s × C i ) / (A i × C s ), where A s is the peak area of the internal reference substance, C i is the content of the component to be measured (known), A i is the peak area of the component to be measured, and C s is the content of the internal reference substance (known). The calculation results are shown in Table 14.

[0137] Table 14 Relative Correction Factors of Components to be Measured

[0138] <![CDATA[f 对羟基肉桂酸 / 橙皮苷 > <![CDATA[f 芸香柚皮苷 / 橙皮苷 > <![CDATA[f 橙皮苷 / 橙皮苷 > <![CDATA[f 甘草酸 / 橙皮苷 > 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 Average value 0.93 1.00 1.00 1.13 RSD (%) 0.68％ 0.75％ 0.00％ 0.67％

[0139] The results show that using hesperidin as the internal reference substance, the relative correction factors of p-coumaric acid, narirutin, and glycyrrhizic acid are 0.93, 1.00, and 1.13 respectively, and the RSDs are all less than 1%.

[0140] Experimental Example 3 Investigation on the Durability of QAMS Method

[0141] 3.1 Investigation of different chromatographic columns

[0142] Referring to the calculation method of relative correction factor in Experimental Example 2, using the mixed standard solution prepared in Experimental Example 1 as the test sample, under the same chromatographic conditions, changing the type of chromatographic column, the relative correction factors calculated under different chromatographic columns (Inertsil ODS-3 (4.6×250 mm, 5 μm), CAPCELL PAK ADME (4.6×250 mm, 5 μm), Knauer Analytical Chrom Core C18 (4.6×250 mm, 5 μm), Thermo Syncronis C18 (4.6×250 mm, 5 μm), Diamonsil C18 (4.6×250 mm, 5 μm)) were investigated. The results are shown in Table 15. It can be seen that the RSDs of the relative correction factors are all less than 5%, indicating that the relative correction factors of p-hydroxycinnamic acid, naringin, and glycyrrhizic acid calculated with hesperidin as the internal reference substance in the present invention are applicable to the above different types of chromatographic columns.

[0143] Table 15 Results of investigation of correction factors for different chromatographic columns

[0144] Chromatographic column <![CDATA[f 对羟基肉桂酸 / 橙皮苷 > <![CDATA[f 芸香柚皮苷 / 橙皮苷 > <![CDATA[f 橙皮苷 / 橙皮苷 > <![CDATA[f 甘草酸 / 橙皮苷 > <![CDATA[Diamonsil C 18 (n = 6)]]> 0.97 1.05 1.00 1.15 <![CDATA[Thermo Syncronis C 18 (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 Average value 0.96 1.01 1.00 1.16 RSD (%) 2.32％ 3.04％ 0.00％ 2.22％

[0145] 3.2 Investigation of different instruments

[0146] Referring to the calculation method of relative correction factor in Experimental Example 2, using the mixed standard solution prepared in Experimental Example 1 as the test sample, under the same chromatographic conditions, the relative correction factors calculated under different instruments (Agilent 1260Ⅱ, Agilent 1200, Waters e2695 high performance liquid chromatography instruments) were investigated. The results are shown in Table 17. It can be seen that the RSD of the relative correction factor is less than 5% and there is no obvious difference. It indicates that the relative correction factors of p-hydroxycinnamic acid, naringin, and glycyrrhizic acid calculated with hesperidin as the internal reference substance in the present invention are applicable to the above different brand models of high performance liquid chromatographs.

[0147] Table 16 Results of investigation of correction factors for different instruments

[0148] Instrument <![CDATA[f 对羟基肉桂酸 / 橙皮苷 > <![CDATA[f 芸香柚皮苷 / 橙皮苷 > <![CDATA[f 橙皮苷 / 橙皮苷 > <![CDATA[f 甘草酸 / 橙皮苷 > 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 Average value 0.96 0.99 1 1.18 RSD (%) 3.60％ 1.45％ 0.00％ 4.67％

[0149] Experimental Example 4 Comparison of determination results between the multi-component simultaneous determination method (QAMS method) and the external standard method (ESM method)

[0150] 1. Experimental method

[0151] Detect 15 batches of Wendan Tablet samples according to the methods of Example 2 and Example 3 respectively. Calculate the peak areas of p-hydroxycinnamic acid, naringin rutinoside, hesperidin, and glycyrrhizic acid in the Wendan Tablet samples based on the obtained chromatograms, and calculate the contents of p-hydroxycinnamic acid, naringin rutinoside, hesperidin, and glycyrrhizic acid by QAMS method (as described in Example 3 of the present invention) and ESM method (as described in Example 2 of the present invention) respectively.

[0152] Use formula (1) and formula (2) to compare the differences in the determination results between the multi-component assay by single marker method and the external standard method: percentage difference (PD, %), and cosine value of the included angle Cos(θ).

[0153]

[0154]

[0155] Where a and b are the contents (%) of the analyte calculated by QAMS and ESM respectively, and n is the total number of batches of Wendan Tablets.

[0156] 2. Experimental results

[0157] Table 17 Relative retention values of 15 batches of Wendan Tablet samples (n = 3)

[0158] Batch <![CDATA[r 对羟基肉桂酸 / 橙皮苷 > <![CDATA[r 芸香柚皮苷 / 橙皮苷 > <![CDATA[r 橙皮苷 / 橙皮苷 > <![CDATA[r 甘草酸 / 橙皮苷 > 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 Average value 0.58 0.85 1.00 1.60 RSD (%) 1.58％ 0.66％ 0.00％ 1.25％

[0159] Table 18 Results of the contents of 4 components in Wendan Tablets determined by QAMS method and ESM method (n = 3)

[0160]

[0161]

[0162] "-" indicates that there is impurity interference and the content cannot be calculated. The relative retention value results of each batch of samples by QAMS method are shown in Table 17, and the contents and comparison of 4 components in Wendan Tablets determined by ESM method (measured value) and QAMS method (calculated value) are shown in Table 18.

[0163] (1) The average content of hesperidin calculated by the external standard method is 11.6427 mg / g, the average content of p-hydroxycinnamic acid is 1.1479 mg / g, the average content of naringin rutinoside is 4.7781 mg / g, and the average content of glycyrrhizic acid is 3.9654 mg / g; the average content of p-hydroxycinnamic acid calculated by the multi-component assay by single marker method is 1.1339 mg / g, the average content of naringin rutinoside is 4.9386 mg / g, and the average content of glycyrrhizic acid is 4.0279 mg / g. The results show that there is no significant difference between the determination results of the multi-component assay by single marker method and the external standard method for the 4 components in 15 batches of samples.

[0164] (2) The proportion of the calculated value of p-hydroxycinnamic acid to the measured value was 98.17 - 99.30%, PD < 5%, Cos(θ) = 1.0000; the proportion of the calculated value of naringin to the measured value was 99.97 - 104.98%, PD < 5%, Cos(θ) = 0.9999; the proportion of the calculated value of glycyrrhizic acid to the measured value was 99.16 - 102.76%, PD < 5%, Cos(θ) = 0.9999. The results showed that there was no significant difference in the content results of each component measured by the two methods, and the multi-component determination method established in the present invention was feasible.

[0165] The above results showed that there was no obvious difference between the multi-component determination method and the external standard method for the determination results of 4 components in 15 batches of samples. Compared with the external standard method, the multi-component determination method established in the present invention could accurately determine the contents of 4 components (p-hydroxycinnamic acid, naringin, hesperidin, glycyrrhizic acid) in Wendan Tablets at the same time, and was suitable for the quality control of Wendan Tablets.

[0166] Moreover, compared with the 4 reference samples (p-hydroxycinnamic acid, naringin, hesperidin, glycyrrhizic acid reference samples) required by the external standard method, the multi-component determination method established in the present invention only needed to use one reference sample (hesperidin reference sample), effectively reducing the detection cost of purchasing reference substances.

[0167] In summary, the present invention provides a method for simultaneously determining multiple index components in Wendan Tablets. Under the specific chromatographic conditions of the present invention, this method can not only perform qualitative analysis on multiple components (p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid) in Wendan Tablets, but also perform quantitative determination. Under the specific chromatographic conditions of the present invention, the detection accuracy of quantitative determination using the multi-component determination method is equivalent to that of the external standard method.

[0168] In particular, for the multi-component determination method for determining the contents of multiple index components in Wendan Tablets in the present invention, using hesperidin as the reference substance, the relative correction factors of p-hydroxycinnamic acid, naringin, and glycyrrhizic acid to hesperidin were established, thereby realizing the simultaneous determination of the contents of 4 components (p-hydroxycinnamic acid, naringin, hesperidin, glycyrrhizic acid) in Wendan Tablets. The determination method of the present invention can accurately and comprehensively evaluate the quality of Wendan Tablets, is simple and rapid, has good stability and repeatability, and is beneficial to improving the quality standard of Wendan Tablets. Especially the multi-component determination method not only ensures the quality of the preparation to be effective, stable and controllable, but also reduces the detection cost of purchasing reference substances, achieving excellent technical effects and having broad application prospects.

Claims

1. A method for simultaneously determining multiple index components in Wendan Tablets, characterized in that: The method uses Wendan Tablets as the test sample, performs high-performance liquid chromatography detection, and simultaneously determines p-hydroxycinnamic acid, naringin, hesperidin and glycyrrhizic acid in Wendan Tablets; it includes the following steps: (1) Preparation of the test sample solution: Take Wendan Tablets, ultrasonically extract with an organic solvent, and take the extract to obtain the test sample solution; the organic solvent is 60% - 80% ethanol; (2) Inject into a high-performance liquid chromatograph for detection; The chromatographic conditions are as follows: Chromatographic column: Packed with octadecylsilane-bonded silica gel; the octadecylsilane-bonded silica gel is Inertsil ODS-3 or Diamonsil C18; Mobile phase: Composed of phase A and phase B, phase A is acetonitrile, and phase B is an aqueous phosphoric acid solution; the mass concentration of the aqueous phosphoric acid solution is 0.05% - 0.20%; The chromatographic conditions are gradient elution, and the elution conditions are shown in the following table: In the table, the unit of time 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; The detection wavelengths in the chromatographic conditions are: 0 - 25 min, 256 nm; 25 - 42 min, 268 nm; 42 - 65 min, 252 nm; the column temperature is 25 - 35 °C; the flow rate is 0.8 - 1.2 ml / min.

2. The method according to claim 1, wherein: The method is a method for quantitatively determining p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid; the method is an external standard method or a multi-component quantification method with one reference substance.

3. The method according to claim 2, wherein: The external standard method uses p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid as reference substances to determine the contents of p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid in Wendan Tablets; the preparation method of the reference substance solution is as follows: Weigh p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid respectively, and dissolve them with an organic solvent respectively to obtain.

4. The method according to claim 2, wherein: The multi-component quantification method with one reference substance uses hesperidin as the reference substance to determine the contents of p-hydroxycinnamic acid, naringin, hesperidin, and glycyrrhizic acid in Wendan Tablets.

5. The method according to claim 4, wherein: The method includes the following steps: 1) Preparation of the reference substance solution: Weigh hesperidin and dissolve it with an organic solvent to obtain; 2) Preparation of the test sample solution: Weigh Wendan Tablets, extract with an organic solvent, and take the extract to obtain; 3) Respectively suck the test sample solution and the reference substance solution into a high-performance liquid chromatograph to measure the content of hesperidin in Wendan Tablets; 4) Use the relative correction factors between the components to be measured, p-hydroxycinnamic acid, naringin, and glycyrrhizic acid, and hesperidin, and calculate the contents of the components to be measured in Wendan Tablets through the following formula respectively; f s / i =(A s ×C i ) / (A i ×C s ) Among them, f s / i is the relative correction factor between the component to be measured and hesperidin, and A s is the peak area of hesperidin in the chromatogram of the test solution, C i is the content of the component to be measured in Wendan Tablets, and A i is the peak area of the component to be measured in the chromatogram of the test solution, C s is the content of hesperidin in Wendan Tablets.

6. The method according to claim 5, characterized in that: In step 4), the relative correction factor between p-hydroxycinnamic acid and hesperidin is 0.92 - 1.01, the relative correction factor between naringin and hesperidin is 0.97 - 1.05, and the relative correction factor between glycyrrhizic acid and hesperidin is 1.12 - 1.

26.

7. The method according to claim 6, characterized in that: In step 4), the relative correction factor between p-hydroxycinnamic acid and hesperidin is 0.93, the relative correction factor between naringin and hesperidin is 1.00, and the relative correction factor between glycyrrhizic acid and hesperidin is 1.

13.

8. The method according to any one of claims 1 to 7, characterized in that: The mass concentration of the phosphoric acid aqueous solution is 0.10%; and / or, the column temperature is 30 °C; the flow rate is 1.0 ml / min.

9. The method according to any one of claims 1 to 7, characterized in that: The mass-to-volume ratio of the Wendan tablets to the organic solvent is 5 mg / mL to 20 mg / mL; and / or, the extraction time of the ultrasonic extraction is 30 to 60 min, the extraction temperature is room temperature, and the ultrasonic power is 200 to 300 W.

10. The method according to claim 9, characterized in that: The mass-to-volume ratio of the Wendan tablets to the organic solvent is 10 mg / mL; and / or, the extraction time is 45 min, and the ultrasonic power is 250 W.

11. The method according to claim 10, wherein: In the preparation of the test solution, the organic solvent is 70% ethanol.

12. The method according to claim 5, wherein: In the preparation of the reference solution, the organic solvent is an alcohol solvent; and / or, the concentration of the reference solution is 0.025 to 0.405 mg / mL.

13. The method according to claim 12, wherein: In the preparation of the reference solution, the organic solvent is methanol; and / or, the concentration of the reference solution is 0.1 mg / mL.

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