Method for determining contents of five components of liver-soothing tablet based on double-label multi-measurement method

Abstract

The invention relates to the technical field of quality detection, and discloses a method for determining the content of five components of liver-soothing tablets based on a double-label multi-measurement method, and the method comprises the following steps: S1, extracting a test solution; s2, preparing a reference substance solution; s3, preparing each negative control solution; s4, determining chromatographic conditions; s5, constructing a retention time prediction model by using a double-marking linear correction method, taking the standard retention time of paeoniflorin and hesperidin as a horizontal coordinate, taking the actual retention time of paeoniflorin and hesperidin in the chromatographic column as a vertical coordinate, and substituting the standard retention time of agarotetrol, naringin and neohesperidin into the equation to calculate and obtain predicted retention time respectively; s6, determining the contents of paeoniflorin, agarotetrol, naringin, hesperidin and neohesperidin in the liver soothing tablet sample by adopting a double-label multi-determination method; according to the method, the contents of five components, namely paeoniflorin, agarotetrol, naringin, hesperidin and neohesperidin, in the liver-soothing tablet are simultaneously determined by a double-label multi-measurement method, the accuracy is high, and the cost is low.

Description

Technical Field

[0001] The present invention relates to the technical field of quality inspection, and particularly relates to a method for determining the contents of 5 components of Shugan Tablets based on a double-standard multi-measurement method. Background Technique

[0002] Shugan Tablets is a compound preparation composed of 12 Chinese herbal medicines such as Aquilariae Lignum Resinatum, Paeoniae Radix Alba, Citri Reticulatae Pericarpium, Aurantii Fructus Immaturus, Corydalis Rhizoma Yanhusuo Vinegar Processed, and Amomi Fructus Rotundus. This preparation has many medicinal flavors and a relatively complex preparation process. However, the current standard (standard number: WS3-B-2047-95) only has character and routine inspection items and cannot effectively control the product quality. Modern research shows that the main components of Aquilariae Lignum Resinatum in the formula are 2-(2-phenylethyl)chromone derivatives such as aquilarol; Paeoniae Radix Alba mainly contains monoterpenes and their glycoside components such as paeoniflorin; Citri Reticulatae Pericarpium mainly contains flavonoid components such as hesperidin; Aurantii Fructus Immaturus mainly contains flavonoid components such as naringin and neohesperidin. Existing research only establishes a single-component content determination method for paeoniflorin in Shugan Tablets, and it is difficult to reflect the overall quality status of this preparation. In view of this, there is an urgent need to establish a multi-index content determination method for Shugan Tablets to improve the current quality standard, control its overall quality, and ensure effectiveness and safety.

[0003] However, a multi-index content determination method requires more reference substances, significantly increasing the inspection and detection costs. The quantitative determination analysis multi-component by a single-marker (QAMS) or the alternative reference substance method can reduce the use of reference substances and has been successfully applied to the quality evaluation of various Chinese herbal pieces and formula preparations. However, sometimes the chromatographic peak positioning and relative correction factor quantification errors of these methods are relatively large. Sun Lei proposed the "two reference substances for determination of multiple components" (double-standard multi-measurement), that is, two reference substances are selected, and the double-standard linear calibration method (liner calibration with two reference substances, LCTRS) is used for qualitative analysis of multi-component chromatographic peaks and the relative correction factor method for quantitative analysis of multi-components. This method can significantly improve the accuracy of chromatographic peak qualitative and quantitative analysis and the applicable rate of the chromatographic column and can reduce the cost of reference substances. It has now been successfully applied to the multi-index content determination of various Chinese herbal pieces and preparations.

[0004] Therefore, there is a need for a method that can simultaneously determine the contents of five components, namely paeoniflorin, costunolide, naringin, hesperidin, and neohesperidin, in Shugan Tablets by the dual-standard multi-measurement method. That is, taking paeoniflorin and hesperidin as reference substances, using LCTRS to construct a retention time prediction model to locate the chromatographic peaks of the remaining components. Costunolide takes paeoniflorin as the reference substance, and naringin and neohesperidin take hesperidin as the reference substance, and calculate the relative correction factors respectively. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for determining the contents of five components in Shugan Tablets based on the dual-standard multi-measurement method, which has the advantages of being able to accurately qualitatively and quantitatively analyze five components simultaneously with two reference substances, comprehensively reflecting the quality status of Shugan Tablets, and solving the problem of one-sidedness in quality control by single-index determination.

[0006] To achieve the above purpose of accurately qualitatively and quantitatively analyzing five components simultaneously with two reference substances and comprehensively reflecting the quality status of Shugan Tablets, the present invention provides the following technical solutions: A method for determining the contents of five components in Shugan Tablets based on the dual-standard multi-measurement method, comprising the following steps: S1: Extract the test solution: Grind Shugan Tablets finely and prepare it by ultrasonic treatment or refluxing with methanol. S2: Prepare the reference substance solution: Weigh reference substances of costunolide, paeoniflorin, naringin, hesperidin, and neohesperidin with a certain mass fraction, add methanol with a certain volume fraction to make a mixed reference substance solution. S3: Prepare each negative control solution according to the preparation method of the test solution. S4: Determine the chromatographic conditions: Use high-performance liquid chromatography. The chromatographic conditions include using a C18 column, with 0.05% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution for 0 - 45 min, 15% - 21% B; set appropriate flow rate, column temperature; determine the detection wavelength; clarify the injection volumes of the reference substance and the test sample. S5: Qualitative analysis: Take paeoniflorin and hesperidin as reference substances and use the dual-standard linear correction method to construct a retention time prediction model. S6: Quantitative analysis: Use the dual-standard multi-measurement method to determine the contents of paeoniflorin, costunolide, naringin, hesperidin, and neohesperidin in the Shugan Tablet sample, and compare with the results determined by the external standard method. In the test sample, costunolide takes paeoniflorin as the reference, and naringin and neohesperidin take hesperidin as the reference. According to the formula: C i = A i × C S / ( f i / s × A S) Calculate the contents of agarotetrol, naringin, and neohesperidin in the test sample respectively, where C i is the content of the component to be measured in the test sample, A i is the peak area of the component to be measured in the test sample, A S is the peak area of the reference substance in the test sample, C S is the concentration of the reference substance in the test sample measured by the external standard method, f i / s is the correction factor.

[0007] Preferably, in the step S4, the flow rate is 0.9 - 1.1 mL·min -1 , the column temperature is 25 - 40 °C; the detection wavelength: 230 - 260 nm; the injection volume of the reference substance and the test sample is 2 - 20 μL; the C 18 column is selected from Col1: Agilent 5 HC-C 18(2) ; Col2: Agilent 5 TC-C 18(2) ; Col 3: Agilent ZORBAX SB C 18 ; Col4: TechMate 120AC 18 ST; Col5: Capcell pak C 18 MG; Col6: Phenomenex Luna C 18(2) ; Col7: Titank C 18 ; Col8: Exsil mono C 18 ; Col9: Shim-pack GIST C 18 ; Col10: Zafex Supfex-YX-C 18 ; Col11: YMC-Triart C 18 ; Col12: Thermo Acclaim 120 C 18 ; Col13: Welch Ultimate plus C 18 ; Col14: ZORBAX Eclipse XDB C 18 ; Col15: Kromasil C 18 ; Col16: Shim-pack VP-ODS C 18 One or more of the chromatographic columns, and the specifications of the chromatographic columns are all 4.6 mm × 250 mm, 5 μm.

[0008] Preferably, the step of extracting the test solution in S1 is as follows: Grind the Shugan tablets finely, weigh 0.8 g, place it in a stoppered conical flask, add 25 - 50 mL of methanol with a volume fraction of 50% - 70%, weigh it, perform ultrasonic treatment for 30 - 90 minutes under the conditions of a power of 250 W and a frequency of 40 kHz, weigh it again after cooling, make up the missing weight with methanol with a volume fraction of 50% - 70%, shake well and filter, and take the consecutive filtrate to obtain the test solution.

[0009] Preferably, the step of preparing the reference solution in S2 is as follows: Weigh reference substances of caryoptin, paeoniflorin, naringin, hesperidin, and neohesperidin with a certain mass fraction, add methanol with a volume fraction of 70%, and prepare a mixed solution containing 5 - 50 μg of caryoptin, 10 - 100 μg of paeoniflorin, 50 - 200 μg of naringin, 10 - 70 μg of hesperidin, and 20 - 200 μg of neohesperidin per 1 mL to obtain the reference solution.

[0010] Preferably, in the qualitative analysis, chromatograms of the Shugan tablet samples and reference substances are collected on different reversed-phase chromatographic columns, the actual retention time of the samples is recorded, and the average value of the retention time of the characteristic peaks is used as the standard retention time to construct a retention time prediction model.

[0011] Preferably, the characteristic peaks include the chromatographic peaks of paeoniflorin, caryoptin, naringin, hesperidin, and neohesperidin.

[0012] Preferably, in the quantitative analysis, the calculation of the relative correction factor adopts the multi-point calibration method. Take multiple groups of mixed reference solutions, and gradually dilute them into a series of mixed reference solutions with different concentrations, inject samples for determination, and calculate the relative correction factors of the compounds at different concentrations.

[0013] Preferably, it further includes a methodological investigation step, and the methodological investigation includes specificity test, linear relationship investigation, precision test, repeatability test, stability test, and sample addition recovery test.

[0014] Preferably, in the specificity test, chromatograms of the mixed reference solution, test solution, and each negative control solution are measured to investigate whether the chromatographic peaks of each component are interfered.

[0015] Preferably, the method for constructing the retention time prediction model in S5 is as follows: Using the standard retention time of paeoniflorin, 11.65 min, and the standard retention time of hesperidin, 32.13 min, as the abscissa, and the actual retention time of paeoniflorin and hesperidin on the chromatographic column as the ordinate, calculate the retention time linear equation, and then substitute the standard retention time of caryoptin, 15.58 min, the standard retention time of naringin, 29.07 min, and the standard retention time of neohesperidin, 32.13 min, into the equation to calculate the predicted retention times respectively.

[0016] Compared with the prior art, the present invention provides a method for determining the contents of 5 components in Shugan Tablets based on the double-standard multi-measurement method, having the following beneficial effects: 1. For the method for determining the component contents in Shugan Tablets based on the double-standard multi-measurement method, the chromatographic peaks are located by adopting the double-standard linear correction method, improving the accuracy of qualitative analysis; quantitative analysis is carried out by the relative correction factor method, and a grouped reference strategy is adopted, reducing the quantitative deviation caused by structural differences and improving the accuracy of quantitative analysis.

[0017] 2. For the method for determining the component contents in Shugan Tablets based on the double-standard multi-measurement method, through the investigation of various chromatographic columns, it is applicable to various C 18 columns, significantly improving the application rate of chromatographic columns.

[0018] 3. For the method for determining the component contents in Shugan Tablets based on the double-standard multi-measurement method, compared with the traditional multi-index content determination method, the number of reference substances used is reduced, and the inspection and detection cost is lowered.

[0019] 4. For the method for determining the component contents in Shugan Tablets based on the double-standard multi-measurement method, the extraction method adopts ultrasonic extraction, which is simple to operate and does not require complex equipment and cumbersome steps. Description of the Drawings

[0020] Figure 1 It is the HPLC chromatogram of 5 components of the present invention. Detailed Embodiments

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] A method for determining the contents of 5 components in Shugan Tablets based on the double-standard multi-measurement method includes the following steps: S1: Extract the test solution: Grind Shugan Tablets finely, weigh 0.8 g, place it in a stoppered conical flask, add 25 mL of methanol with a volume fraction of 70%, weigh it, perform ultrasonic or reflux treatment for 60 minutes, cool it, weigh it again, make up the missing weight with methanol with a volume fraction of 70%, shake well and filter, and take the subsequent filtrate to obtain the test solution; In the present invention, the instruments used include an Agilent 1260 Infinity Ⅱ high performance liquid chromatograph, a Shimadzu LC-20ADXR high performance liquid chromatograph, a Thermo U3000 RSLC high performance liquid chromatograph, an XPE-205 analytical balance; a Minispin centrifuge with a speed of 12,000 rpm; a Milli-Q Advantage A ultrapure water machine; an Elmasonic P ultrasonic cleaner; Reference substances including agarotetrol (batch number: 111980-201904, purity 98.6 %), naringin (batch number: 110722-202417, purity 96.1 %), hesperidin (batch number: 110721-202220, purity 97.2 %), neohesperidin (batch number: 111857-201804, purity 99.4 %), and paeoniflorin (batch number: 110736-202145, purity 96.7 %) were all purchased from the National Institutes for Food and Drug Control for use in content determination. Acetonitrile was of chromatographic grade, and the rest were of analytical grade. The test water was prepared by the Milli-Q system.

[0023] Among them, for the comparison of extraction methods: 0.8 g of the Shugan tablets sample was taken and 25 mL of methanol with a volume fraction of 70% was added. Ultrasonic extraction (60 minutes) and reflux extraction (60 minutes) were compared, and the sample results are shown in Table 1.

[0024] Table 1 Comparison results of extraction methods (mg / g)

[0025] According to Table 1, there was no significant difference in the contents of the 5 components extracted by ultrasonic and reflux methods. Since ultrasonic extraction is more convenient, ultrasonic extraction was selected in this scheme.

[0026] For the comparison of extraction solvents: The extraction efficiencies of ethanol, methanol, methanol with a volume fraction of 20%, methanol with a volume fraction of 50%, and methanol with a volume fraction of 70% were compared respectively, and the sample results are shown in Table 2.

[0027] Table 2 Comparison results of extraction solvents (mg / g)

[0028] According to Table 2, when the extraction solvent was methanol with a volume fraction of 50% and methanol with a volume fraction of 70%, the extraction efficiencies of the 5 components were better than those of other solvents. Since the impurities extracted by methanol with a volume fraction of 70% were less than those by methanol with a volume fraction of 50%, methanol with a volume fraction of 70% was used as the extraction solvent in this scheme.

[0029] Comparison of extraction time: The changes in the contents of 5 components after ultrasonic extraction for 30 min, 60 min, and 90 min were compared, and the sample determination results are shown in Table 3.

[0030] Table 3 Comparison results of extraction time (mg / g)

[0031] According to Table 3, there were no significant differences in the determination results of the contents of the 5 components after extraction for 30 min, 60 min, and 90 min respectively. To ensure complete extraction, the extraction time was selected as 60 min in this scheme.

[0032] Comparison of extraction volume: The effects of accurately adding 10 mL, 25 mL, and 50 mL of methanol with a volume fraction of 70% to the samples on the content determination results were compared, and the results are shown in Table 4.

[0033] Table 4 Comparison results of different extraction volumes (mg / g)

[0034] According to Table 4, there was little difference in the extraction efficiency between the sampling volumes of 25 mL and 50 mL. Considering the appropriate peak area size, the extraction volume of 25 mL was finally selected in this scheme.

[0035] Based on the above experimental results, the extraction method of the test sample was finally determined as follows: Grind the Shugan tablets finely, weigh 0.8 g, place it in a stoppered conical flask, add 25 mL of methanol with a volume fraction of 70%, weigh it, perform ultrasonic treatment for 60 minutes under the conditions of a power of 250 W and a frequency of 40 kHz, cool it, weigh it again, make up the missing weight with methanol with a volume fraction of 70%, shake well and filter, take the subsequent filtrate to obtain the test sample solution.

[0036] S2: Preparation of reference solution: Accurately weigh 15.84 mg of asiaticotriol, 18.61 mg of paeoniflorin, 20.42 mg of naringin, 16.50 mg of hesperidin, and 19.70 mg of neohesperidin as reference substances, and place them in volumetric flasks of 20 mL, 20 mL, 50 mL, 50 mL, and 50 mL respectively. Add methanol with a volume fraction of 70% and ultrasonically dissolve until completely dissolved, then dilute to the scale with methanol with a volume fraction of 70%, shake well to obtain the respective reference stock solutions. Accurately take 1 mL, 5 mL, 10 mL, 3 mL, and 10 mL of the asiaticotriol, paeoniflorin, naringin, hesperidin, and neohesperidin reference stock solutions and place them in the same 50 mL volumetric flask, dilute to the scale with methanol with a volume fraction of 70%, shake well to prepare a mixed solution containing 15.62 μg of asiaticotriol, 89.98 μg of paeoniflorin, 78.49 μg of naringin, 64.15 μg of hesperidin, and 78.33 μg of neohesperidin per 1 mL, that is, obtain the reference solution; S3: Prepare each negative control solution according to the preparation method of the test solution. Specifically, take the remaining medicinal materials except Aquilariae Lignum Resinatum in the prescribed amounts, and prepare the negative control solution without Aquilariae Lignum Resinatum according to the preparation method of the test solution. Similarly, take the remaining medicinal materials except Paeoniae Radix Alba, Citri Reticulatae Pericarpium, and Aurantii Fructus Immaturus in the prescribed amounts respectively, and prepare the negative control solutions without Paeoniae Radix Alba, without Citri Reticulatae Pericarpium, and without Aurantii Fructus Immaturus according to the preparation method of the test solution. Then, take the remaining medicinal materials except Citri Reticulatae Pericarpium and Aurantii Fructus Immaturus in the prescribed amounts, and strictly follow the preparation method of the test solution. After the same pretreatment process, prepare the negative control solution without both Citri Reticulatae Pericarpium and Aurantii Fructus Immaturus, which will not be elaborated here.

[0037] S4: Determine the chromatographic conditions: Use high performance liquid chromatography. The chromatographic column is C 18 column. Use 0.05% phosphoric acid as mobile phase A and acetonitrile as mobile phase B for gradient elution: 0 - 45 min, 15% - 21% B; the flow rate is 1.0 mL·min -1 , the column temperature is 35 °C; the detection wavelengths are: 252 nm for agallochaol, and 230 nm for the other four components; the injection volumes of both the reference substance and the test solution are 10 μL; The theoretical plate number calculated based on the paeoniflorin peak should be not less than 7000. The HPLC chromatograms of the five components, namely paeoniflorin, agallochaol, naringin, hesperidin, and neohesperidin, are as shown Figure 1 below, where 1 - paeoniflorin; 2 - agallochaol; 3 - naringin; 4 - hesperidin; 5 - neohesperidin; A - mixed reference substance solution; B - sample solution; C - negative control without Paeoniae Radix Alba; D - negative control without Aquilariae Lignum Resinatum; E - negative control without both Aurantii Fructus Immaturus and Citri Reticulatae Pericarpium; F - negative control without Aurantii Fructus Immaturus; G - negative control without Citri Reticulatae Pericarpium; S5: Qualitative analysis: Use paeoniflorin and hesperidin as reference substances, and construct a retention time prediction model using the double - standard linear correction method; S6: Quantitative analysis: Use the double - standard multi - measurement method to determine the contents of paeoniflorin, agallochaol, naringin, hesperidin, and neohesperidin in the Shugan Tablets sample, and compare with the results determined by the external standard method. In the test solution, agallochaol is referenced to paeoniflorin, and naringin and neohesperidin are referenced to hesperidin. According to the formula: C i = A i × C S / ( f i / s × A S ), calculate the contents of agallochaol, naringin, and neohesperidin in the test solution respectively, where C i is the content of the component to be measured in the test solution, A i is the peak area of the component to be measured in the test solution, AS is the peak area of the reference substance in the test sample, C S is the concentration of the reference substance in the test sample measured by the external standard method, f i / s is the correction factor. For f i / s agarotetrol, it is 1.9449, and for f i / s naringin, it is 1.0596, and for f i / s neohesperidin, it is 1.0359.

[0038] In one embodiment of the present invention, the C 18 column is selected from Col1: Agilent 5 HC-C 18(2) ; Col2: Agilent 5TC-C 18(2) ; Col 3: Agilent ZORBAX SB C 18 ; Col4: TechMate 120A C 18 ST; Col5: Capcell pakC 18 MG; Col6: Phenomenex Luna C 18(2) ; Col7: Titank C 18 ; Col8: Exsil mono C 18 ; Col9: Shim-pack GIST C 18 ; Col10: Zafex Supfex-YX-C 18 ; Col11: YMC-Triart C 18 ; Col12: ThermoAcclaim 120 C 18 ; Col13: Welch Ultimate plus C 18 ; Col14: ZORBAX Eclipse XDB C 18 ; Col15: Kromasil C 18 ; Col16: Shim-pack VP-ODS C 18 one or more of the chromatographic columns, and the specifications of the chromatographic columns are all 4.6 mm × 250 mm, 5 μm.

[0039] In one embodiment of the present invention, the gradient elution program is 0 - 45 min, and the proportion of mobile phase B linearly increases from 15% to 21%.

[0040] In one embodiment of the present invention, in qualitative analysis, chromatograms of the Shugan tablets sample and the reference substance are collected on 16 different reversed-phase chromatographic columns, the actual retention time of the sample is recorded, the average value of the retention time of the characteristic peaks is used as the standard retention time, and a retention time prediction model is constructed.

[0041] In one embodiment of the present invention, the characteristic peaks include the chromatographic peaks of paeoniflorin, costunolide, naringin, hesperidin, and neohesperidin.

[0042] In one embodiment of the present invention, in quantitative analysis, the multi-point calibration method is used to calculate the relative correction factor. Multiple groups of mixed reference substance solutions are taken and successively diluted into a series of mixed reference substance solutions with different concentrations, injected for determination, and the relative correction factor of the compound at different concentrations is calculated.

[0043] In one embodiment of the present invention, it also includes a methodology investigation step. The methodology investigation includes specificity test, linear relationship investigation, precision test, repeatability test, stability test, and spiking recovery test; the specific experimental steps and corresponding data tables are as follows: 1. Methodology Investigation In this scheme, 11 batches of Shugan tablets samples from 3 production enterprises are collected. S1 - S2 are from enterprise A (batch numbers are 22120569 and 21123573), S3 is from enterprise B (batch number is 2406001), and S4 - S11 are from enterprise C (batch numbers are 230501, 230502, 240601, 240602, 231101, 231102, 230701, 230702). Sample S9 is selected for the methodology investigation.

[0044] 1.1 Specificity Test Take the mixed reference substance solution, the test solution, the negative samples lacking costus root, white peony root, dried tangerine peel, and immature bitter orange, and the negative sample lacking both dried tangerine peel and immature bitter orange. The chromatographic column is Col12: Thermo Acclaim 120 C 18 (the specification of the chromatographic column is 4.6 mm × 250 mm, 5 μm); 0.05% phosphoric acid is used as mobile phase A, and acetonitrile is used as mobile phase B for gradient elution: 0 - 45 min, 15% - 21% B; flow rate: 1.0 mL·min -1 , column temperature: 35 °C; detection wavelength: 252 nm for costunolide, and 230 nm for the other four components; injection volume: 10 μL for both the reference substance and the test solution. Determine under the above chromatographic conditions, and the chromatogram is shown in Figure 1The results showed that Aquilariae Lignum Resinatum, Paeoniae Radix Alba, and Aurantii Fructus Immaturus had no interference and good specificity. However, in the negative control sample without Citri Reticulatae Pericarpium, a very small chromatographic peak appeared at the peak of hesperidin reference substance, while no chromatographic peaks appeared at the peaks of hesperidin, neohesperidin, and naringin reference substances in the negative control without both Citri Reticulatae Pericarpium and Aurantii Fructus Immaturus. This indicated that Aurantii Fructus Immaturus contained trace amounts of hesperidin, and the main source of hesperidin in the preparation was Citri Reticulatae Pericarpium. Therefore, the quality of Citri Reticulatae Pericarpium was evaluated based on the content of hesperidin in the preparation.

[0045] 1.2 Investigation of linear relationship Prepare mixed reference substance solutions with different concentrations. The chromatographic column was Col12: ThermoAcclaim 120 C 18 (the specifications of the chromatographic column were 4.6 mm × 250 mm, 5 μm); 0.05% phosphoric acid was used as mobile phase A, and acetonitrile was used as mobile phase B for gradient elution: 0 - 45 min, 15% - 21% B; flow rate: 1.0 mL·min -1 , column temperature: 35 °C; detection wavelength: 252 nm for agallochaol, and 230 nm for the other four components; injection volume: 10 μL each for reference substances and test samples. The chromatographic conditions were used for determination, and the peak areas of each component were recorded. The regression equation of the five components was calculated by regressing the chromatographic peak area (Y) against the injection volume (X). The results are shown in Table 5.

[0046] Table 5 Linear regression results of five components in Shugan Tablets

[0047] 1.3 Precision test Weigh 0.8 g of the ground sample S9, place it in a stoppered conical flask, add 25 mL of methanol with a volume fraction of 70%, weigh it, and perform ultrasonic treatment for 60 minutes under the conditions of a power of 250 W and a frequency of 40 kHz. After cooling, weigh it again, and make up for the missing weight with methanol with a volume fraction of 70%. Shake well and filter. Take the subsequent filtrate to prepare the test sample solution. The chromatographic column was Col12: Thermo Acclaim 120 C 18 (the specifications of the chromatographic column were 4.6 mm × 250 mm, 5 μm), 0.05% phosphoric acid was used as mobile phase A, and acetonitrile was used as mobile phase B for gradient elution: 0 - 45 min, 15% - 21% B; flow rate was 1.0 mL·min -1, the column temperature was 35 °C; detection wavelengths: for kinoresinol, it was 252 nm, and for the other four components, it was 230 nm; under the chromatographic conditions where the injection volumes of both the reference substance and the test sample were 10 μL, the injection was repeated 6 times, and the RSD values were calculated by measuring the peak areas of the 5 components. The results showed that the RSD values of the peak areas of kinoresinol, paeoniflorin, naringin, hesperidin, and neohesperidin were 0.17%, 0.22%, 0.10%, 0.19%, and 0.15% respectively (see Table 6), indicating good instrument precision.

[0048] Table 6 Precision Test of Shugan Tablets

[0049] 1.4 Repeatability Test Take 6 portions of sample S9, grind them finely, weigh 0.8 g, place them in a stoppered conical flask, add 25 mL of methanol with a volume fraction of 70%, weigh it, perform ultrasonic treatment for 60 minutes under the conditions of a power of 250 W and a frequency of 40 kHz, cool and then weigh again, make up the missing weight with methanol with a volume fraction of 70%, shake well and filter, take the subsequent filtrate, and prepare six groups of test sample solutions by repeating 6 times. The chromatographic column was Col12: Thermo Acclaim 120 C 18 (the specifications of the chromatographic column were 4.6 mm × 250 mm, 5 μm), using 0.05% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, and performing gradient elution: 0 - 45 min, 15% - 21% B; the flow rate was 1.0 mL·min -1 , the column temperature was 35 °C; detection wavelengths: for kinoresinol, it was 252 nm, and for the other four components, it was 230 nm; under the chromatographic conditions where the injection volumes of both the reference substance and the test sample were 10 μL, the content of each sample was measured, and the measurement results are shown in Table 7. The results showed that the average contents of kinoresinol, paeoniflorin, naringin, hesperidin, and neohesperidin were 0.5655 mg·g -1 、3.118 mg·g -1 、2.702 mg·g -1 、0.6205 mg·g -1 and 2.658 mg·g -1 , and the RSD values were 0.30%, 1.83%, 2.09%, 1.32%, and 0.15% respectively, indicating good repeatability of this method.

[0050] Table 7 Results of Repeatability Test of Shugan Tablets (mg / g)

[0051] 1.5 Stability test Weigh 0.8 g of the ground sample S9, place it in a stoppered conical flask, add 25 mL of methanol with a volume fraction of 70%, weigh it, and perform ultrasonic treatment for 60 minutes under the conditions of a power of 250 W and a frequency of 40 kHz. After cooling, weigh it again, make up the missing weight with methanol with a volume fraction of 70%, shake well and filter. Take the subsequent filtrate to prepare the test solution. At 0, 4, 9, 15, 41, and 61 h respectively, the chromatographic column is Col12: Thermo Acclaim 120 C 18 (The specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm), using 0.05% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution: 0 - 45 min, 15% - 21% B; the flow rate is 1.0 mL·min -1 , the column temperature is 35°C; the detection wavelengths are 252 nm for agarotetrol and 230 nm for the other four components; record the peak areas under the chromatographic conditions where the injection volumes of both the reference substance and the test solution are 10 μL, calculate the RSD value, and the measurement results are shown in Table 8. The results show that the RSD values of the peak areas of agarotetrol, paeoniflorin, naringin, hesperidin, and neohesperidin are 1.52%, 0.97%, 0.98%, 0.60%, and 0.50% respectively, indicating that the test solution is basically stable within 61 hours.

[0052] Table 8 Results of stability test

[0053] 1.6 Test for recovery of added samples Weigh accurately 6 portions of the known-content Shugan tablets sample S9, 0.4 g for each portion, accurately add appropriate amounts of 5 reference substances respectively, place them in stoppered conical flasks, add 25 mL of methanol with a volume fraction of 70%, weigh them, and perform ultrasonic treatment for 60 minutes under the conditions of a power of 250 W and a frequency of 40 kHz. After cooling, weigh them again, make up the missing weight with methanol with a volume fraction of 70%, shake well and filter. Take the subsequent filtrate, repeat 6 times to prepare 6 groups of test solutions in total. Using the chromatographic column Col12: Thermo Acclaim 120 C 18 (The specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm), using 0.05% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution: 0 - 45 min, 15% - 21% B; the flow rate is 1.0 mL·min -1, the column temperature was 35 °C; detection wavelength: for costunolide, it was 252 nm, and for the other four components, it was 230 nm; under the chromatographic conditions where the injection volumes of both the reference substance and the test sample were 10 μL, the recovery rates and RSD values were calculated, and the results are shown in Table 9. The average recovery rates of costunolide, paeoniflorin, naringin, hesperidin, and neohesperidin were 100.62%, 103.16%, 98.59%, 101.83%, and 97.72% respectively, and the RSDs were 0.68%, 1.06%, 0.60%, 1.81%, and 1.09% respectively, indicating that this method had good accuracy.

[0054] Table 9 Results of the recovery test of Shugan Tablets. n = 6

[0055]

[0056] 2. Qualitative study of dual - standard multi - determination The principle of the dual - standard linear calibration method is that there is a linear relationship in the retention times of the components to be measured on different reversed - phase chromatographic columns. Using 2 reference substances as references, the measured retention time is substituted into the determined linear equation to predict the retention time of the component to be measured, and the peak position of the chromatographic peak of the component to be measured is determined.

[0057] 2.1 Selection of dual - standards and calculation of standard retention times Chromatograms of Shugan Tablets samples and reference substances were collected on 20 different C 18 columns. Chromatographic peak positions were located through the retention times of the reference substances, and the actual retention times of the samples were recorded. The results showed that for Col 17 (ZORBAX Eclipse Plus C 18 , with a specification of 4.6 mm × 250 mm, 5 μm), Col 18 (Inspire C 18 , with a specification of 4.6 mm × 250 mm, 5 μm), and Col 19 (Dikma Diamonsil C 18(2) , with a specification of 4.6 mm × 250 mm, 5 μm), the resolution of hesperidin in the chromatograms was poor, while for Col 20 (Waters Symmetry C 18, with a specification of 4.6 mm×250 mm, 5 μm), there was interference from the agalloquilol impurity in the chromatogram, so these four chromatographic columns were not involved in the method development. The average retention time of the characteristic peaks on the remaining 16 chromatographic columns was used as the standard retention time (standard retention time, abbreviated as SRT), which were paeoniflorin 11.64 min, agalloquilol 15.58 min, naringin 29.07 min, hesperidin 32.13 min, and neohesperidin 36.62 min in sequence. Taking the SRT of the 5 components as the abscissa and the actual retention time as the ordinate, the linear equations and correlation coefficients of each chromatographic column were calculated, and the results are shown in Table 10. The results showed that the correlation coefficients of the retention times of the 16 chromatographic columns were all greater than 0.999, indicating a good linear relationship.

[0058] Table 10 Linear equations and correlation coefficients of retention times on different chromatographic columns

[0059] 2.2 Determination and optimization of double-standard compounds Calculate the prediction accuracy rates of different compounds as double standards, and the results are shown in Table 11. The results showed that when combining peaks 1-4, not only was the regression deviation of the retention time the smallest, but also the prediction accuracy rate of the retention time and the chromatographic column compliance rate were both 100%. Considering that the reference substances of peak 1 (paeoniflorin) and peak 4 (hesperidin) were also relatively easy to obtain and had a relatively low price, paeoniflorin and hesperidin were selected as the double-standard compounds.

[0060] Table 11 Prediction accuracy rates of different double-standard compounds

[0061]

[0062] 2.3 Determination of compounds by the double-standard linear correction method Using a new chromatographic column Hypersil ODS2-C 18 (4.6 mm×250 mm, 5 μm), using 0.05% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, gradient elution was carried out: 0-45 min, 15% - 21% B; the flow rate was 1.0 mL·min -1, the column temperature was 35 °C; detection wavelengths: for kinoresinol, it was 252 nm, and for the other four components, it was 230 nm; method validation was carried out under the chromatographic conditions where the injection volumes of both the reference substance and the test sample were 10 μL. Using the SRTs of paeoniflorin and hesperidin (11.65 and 32.13 min respectively) as the abscissa and the actual retention times (8.09 and 22.87 min) as the ordinate, the linear equation of the retention time was calculated. Substituting the SRTs of kinoresinol, naringin, and neohesperidin (15.58, 29.07, and 32.13 min respectively) into the equation, the predicted retention times were calculated to be 10.935, 20.664, and 26.109 min respectively. Compared with the actual retention times (10.733, 20.180, and 26.410 min), the absolute deviations were 0.20, 0.48, and -0.30 min respectively, indicating the feasibility of this dual-standard linear calibration method for locating target components in an unknown chromatographic column.

[0063] 2.4 Comparison between the dual-standard linear calibration method and the relative retention time method Paeoniflorin and hesperidin were selected as the dual-standard compounds for calibration by the dual-standard linear calibration method. The relative retention time method used naringin at the middle position of the retention time as the reference substance. The prediction results of the two methods are shown in Table 12. The results showed that the maximum deviation of the predicted values of the relative retention time method was greater than 1.0 min, while the maximum deviation of the dual-standard linear calibration method was less than 0.6 min. It can be seen that the dual-standard linear calibration method has higher prediction accuracy and is applicable to a larger number of chromatographic columns than the relative retention time method.

[0064] Table 12 Absolute deviations of the predicted retention times of the two methods (min)

[0065] 3. Quantitative study of the dual-standard multi-determination method 3.1 Calculation of relative correction factors Appropriate amounts of each reference substance were accurately weighed, a total of 3 groups, and each was made into a mixed reference substance solution containing 71.70 μg of kinoresinol, 132.75 μg of paeoniflorin, 198.73 μg of naringin, 54.28 μg of hesperidin, and 191.53 μg of neohesperidin per 1 mL with methanol of 70% volume fraction. Appropriate amounts of the 3 groups of mixed reference substance solutions were respectively serially diluted into a series of mixed reference substance solutions with different concentrations, and 10 μL of each was injected into the liquid chromatograph, and the peak areas of the chromatographic peaks of each component were recorded. The relative correction factors of the compounds at different concentrations were calculated using the multi-point calibration method: using hesperidin as the reference substance, the relative correction factors of naringin and neohesperidin were calculated, and using paeoniflorin as the reference substance, the relative correction factor of kinoresinol was calculated. The results are shown in Table 13.

[0066] Table 13 Relative correction factors

[0067] 3.2 Reproducibility Investigation of Correction Factors In the experiment, high-performance liquid chromatographs and columns of different brands, namely Shimadzu LC-20ADXR, Thermo U3000RSLC, and Agilent 1260, were investigated. According to the column as Col 3: Agilent ZORBAX SB C 18 ; Col12: Thermo Acclaim 120 C 18 ; Col13: Welch Ultimate plus C 18 (the specifications of all chromatographic columns were 4.6 mm×250 mm, 5 μm), a total of 3 chromatographic columns. 0.05% phosphoric acid was used as mobile phase A, and acetonitrile was used as mobile phase B for gradient elution: 0 - 45 min, 15% - 21% B; the flow rate was 1.0 mL·min -1 , the column temperature was 35°C; the detection wavelengths were 252 nm for asiatic acid and 230 nm for the other four components; the injection volumes of the reference substance and the test substance were both 10 μL. The samples were analyzed under the above chromatographic conditions, and the relative correction factors of each component in the double-standard multi-detection method were calculated by the multi-point correction method and compared with the QAMS method using only hesperidin as the reference. The results are shown in Table 14. The results showed that the RSD values of the relative correction factors of the 5 components in the double-standard multi-detection method on different instruments and columns were all less than 3.0%, but the RSD value of the relative correction factor of paeoniflorin in the QAMS method was greater than 5.0%. It indicated that different brands of liquid chromatographs and columns had no significant influence on the relative correction factors in the double-standard multi-detection method, but had a greater influence on the relative correction factors in the QAMS method. Based on this, this study proposed the strategy of "chemical property grouping - multi-reference substance combination": the target compounds were divided into two groups according to their structural characteristics, flavonoids (naringin, neohesperidin) and terpenoids (paeoniflorin, asiatic acid), and hesperidin (flavonoid group) and paeoniflorin (terpenoid group) were used as the reference substances within the group to recalculate the RCF respectively. The optimized data showed that the RSD values of the RCF of 3 components were all less than 3.0%, indicating that the grouping reference strategy could reduce the quantitative deviation caused by structural differences. It was suggested that in the development of the double-standard multi-detection method and the QAMS method for complex systems, when the chemical properties of the target components differed greatly, the reference substances could be set by grouping according to the principle of "structural similarity", and the number of reference substances could be increased to improve the quantitative accuracy of different types of compounds.

[0068] Table 14 Relative Correction Factors of One-Reference Substance Multi-Evaluation Method and Double-Standard Multi-Detection Method on Different Instruments and Columns

[0069] 3.3 Durability Investigation of Correction Factors 3.3.1 Influence of Different Flow Rates Accurately weigh 15.84 mg of agallochawood alcohol, 18.61 mg of paeoniflorin, 20.42 mg of naringin, 16.50 mg of hesperidin, and 19.70 mg of neohesperidin as reference substances. Respectively place them in volumetric flasks of 20 mL, 20 mL, 50 mL, 50 mL, and 50 mL. Add methanol with a volume fraction of 70% and ultrasonicate until completely dissolved, then dilute to the mark with methanol with a volume fraction of 70%, shake well, and thus obtain the stock solutions of each reference substance. Accurately pipette 1 mL, 5 mL, 10 mL, 3 mL, and 10 mL of the stock solutions of agallochawood alcohol, paeoniflorin, naringin, hesperidin, and neohesperidin into the same 50 mL volumetric flask, dilute to the mark with methanol with a volume fraction of 70%, and shake well to prepare a mixed solution containing 15.62 μg of agallochawood alcohol, 89.98 μg of paeoniflorin, 78.49 μg of naringin, 64.15 μg of hesperidin, and 78.33 μg of neohesperidin per 1 mL, thus obtaining the reference substance solution; Use a Thermo Fisher U3000 liquid chromatograph to examine the effects of different flow rates (0.9, 1.0, 1.1 mL·min -1 ) on each component f i / s The RSDs of 3 components f i / s are all less than 1.0%, indicating that different flow rates have f i / s no significant effect on the components, and the results are shown in Table 15.

[0070] Table 15 Comparison of correction factors at different flow rates

[0071] 3.3.2 Influence of different column temperatures Prepare the reference substance solution: Accurately weigh 15.84 mg of agallochawood alcohol, 18.61 mg of paeoniflorin, 20.42 mg of naringin, 16.50 mg of hesperidin, and 19.70 mg of neohesperidin as reference substances. Respectively place them in volumetric flasks of 20 mL, 20 mL, 50 mL, 50 mL, and 50 mL. Add methanol with a volume fraction of 70% and ultrasonicate until completely dissolved, then dilute to the mark with methanol with a volume fraction of 70%, shake well, and thus obtain the stock solutions of each reference substance. Accurately pipette 1 mL, 5 mL, 10 mL, 3 mL, and 10 mL of the stock solutions of agallochawood alcohol, paeoniflorin, naringin, hesperidin, and neohesperidin into the same 50 mL volumetric flask, dilute to the mark with methanol with a volume fraction of 70%, and shake well to prepare a mixed solution containing 15.62 μg of agallochawood alcohol, 89.98 μg of paeoniflorin, 78.49 μg of naringin, 64.15 μg of hesperidin, and 78.33 μg of neohesperidin per 1 mL, thus obtaining the reference substance solution. Use a Thermo Fisher U3000 liquid chromatograph to examine the effects of different column temperatures (25 °C, 35 °C, 40 °C) on each componentf i / s The influence of 3 components f i / s The RSDs of all were less than 3.0%, indicating that different column temperatures had f i / s no significant influence on the components. The results are shown in Table 16.

[0072] Table 16 Comparison of correction factors at different column temperatures

[0073] 3.4 Comparison of results between the double-standard multi-detection method and the external standard method The contents of paeoniflorin, costunolide, naringin, hesperidin and neohesperidin in 11 batches of Shugan tablets were determined by the established double-standard multi-detection method and compared with the results determined by the external standard method. The results are shown in Table 17. Costunolide was referenced to paeoniflorin, and naringin and neohesperidin were referenced to hesperidin. According to the formula: C i =A i ×C S / (f i / s ×A S ) The contents of costunolide, naringin and neohesperidin in the test samples were calculated respectively; in the formula C i is the content of the component to be measured in the test sample; A i is the peak area of the component to be measured in the test sample; A S is the peak area of the reference substance in the test sample; C S is the concentration of the reference substance in the test sample measured by the external standard method; f i / s is the correction factor; the f i / s of costunolide is 1.9449, the f i / s of naringin is 1.0596, and the f i / s of neohesperidin is 1.0359.

[0074] The content determination values of the external standard method and the double-standard multi-detection method were compared by t test. The results showed that the p values of costunolide, naringin and neohesperidin were 0.733, 0.970 and 0.993 respectively, and there were no significant differences, that is, there was no significant difference between the double-standard multi-detection method and the external standard method. Thus, it can be seen that the double-standard multi-detection method can be applied to the analysis and determination of the contents of 5 components in Shugan tablets.

[0075] Table 17 Results of Dual - standard Multiple - determination Method and External - standard Method (mg·tablet -1 )

[0076] In one embodiment of the present invention, the specificity test examines whether the chromatographic peaks of each component are interfered by measuring the chromatograms of the mixed reference solution, the test solution, and each negative control solution.

[0077] In one embodiment of the present invention, the method for constructing the retention time prediction model in S5 is as follows: taking the standard retention time of paeoniflorin, 11.65 min, and the standard retention time of hesperidin, 32.13 min, as the abscissa, and the actual retention time of paeoniflorin and hesperidin on the chromatographic column as the ordinate, calculating the retention time linear equation. Then, substituting the standard retention time of agallochaol, 15.58 min, the standard retention time of naringin, 29.07 min, and the standard retention time of neohesperidin, 32.13 min, into the equation to calculate the predicted retention times respectively.

[0078] Working principle: Preparation of test solution: Grind the Shugan tablets finely, weigh 0.8 g, place it in a stoppered conical flask, add 25 mL of methanol with a volume fraction of 70%, weigh it, then perform ultrasonic treatment for 60 minutes. After cooling, weigh it again, make up the missing weight with methanol with a volume fraction of 70%, shake well and filter. Take the subsequent filtrate to obtain the test solution; Preparation of reference solution: Prepare the reference solution: Weigh accurately 15.84 mg of agallochaol, 18.61 mg of paeoniflorin, 20.42 mg of naringin, 16.50 mg of hesperidin, and 19.70 mg of neohesperidin as reference substances, and place them in volumetric flasks of 20 mL, 20 mL, 50 mL, 50 mL, and 50 mL respectively. Add methanol with a volume fraction of 70% and ultrasonicate until completely dissolved, then make up the volume to the scale with methanol with a volume fraction of 70%, shake well to obtain each reference stock solution. Accurately take 1 mL, 5 mL, 10 mL, 3 mL, and 10 mL of the reference stock solutions of agallochaol, paeoniflorin, naringin, hesperidin, and neohesperidin respectively and place them in the same 50 - mL volumetric flask, make up the volume to the scale with methanol with a volume fraction of 70%, shake well to prepare a mixed solution containing 15.62 μg of agallochaol, 89.98 μg of paeoniflorin, 78.49 μg of naringin, 64.15 μg of hesperidin, and 78.33 μg of neohesperidin per 1 mL, that is, obtain the reference solution; Preparation of negative control solution: Each negative control solution was prepared according to the preparation method of the test solution. Specifically, the remaining medicinal materials except Aquilariae Lignum Resinatum were taken in the prescribed amounts and the negative control solution without Aquilariae Lignum Resinatum was prepared according to the preparation method of the test solution. Similarly, the remaining medicinal materials except Paeoniae Radix Alba, Citri Reticulatae Pericarpium, and Aurantii Fructus Immaturus were taken in the prescribed amounts and the negative control solutions without Paeoniae Radix Alba, without Citri Reticulatae Pericarpium, and without Aurantii Fructus Immaturus were respectively prepared according to the preparation method of the test solution. Then, the remaining medicinal materials except Citri Reticulatae Pericarpium and Aurantii Fructus Immaturus were taken in the prescribed amounts, and following the preparation method of the test solution strictly and through the same pretreatment process, the negative control solution without both Citri Reticulatae Pericarpium and Aurantii Fructus Immaturus was prepared. Chromatographic conditions: The selected chromatographic columns were Col1: Agilent 5 HC-C 18(2) ; Col2: Agilent 5 TC-C 18(2) ; Col 3: Agilent ZORBAX SB C 18 ; Col4: TechMate 120A C 18 ST; Col5: Capcell pak C 18 MG; Col6: Phenomenex Luna C 18(2) ; Col7: Titank C 18 ; Col8: Exsil mono C 18 ; Col9: Shim-pack GIST C 18 ; Col10: Zafex Supfex-YX-C 18 ; Col11: YMC-Triart C 18 ; Col12: ThermoAcclaim 120 C 18 ; Col13: Welch Ultimate plus C 18 ; Col14: ZORBAX Eclipse XDB C 18 ; Col15: Kromasil C 18 ; Col16: Shim-pack VP-ODS C 18 A total of 16 chromatographic columns (the specifications of the chromatographic columns were all 4.6 mm × 250 mm, 5 μm), with 0.05% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, for gradient elution: 0 - 45 min, 15% - 21% B; the flow rate was 1.0 mL·min -1 , the column temperature was 35°C; the detection wavelengths: for (+)-Agarospirol was 252 nm, and for the other four components were 230 nm; the injection volumes of the reference substance and the test solution were both 10 μL; Qualitative analysis: Using paeoniflorin and hesperidin as reference substances, a retention time prediction model was constructed by the double-standard linear calibration method. Taking the standard retention time of paeoniflorin, 11.65 min, and the standard retention time of hesperidin, 32.13 min, as the abscissa, and the actual retention times of paeoniflorin and hesperidin on the chromatographic column as the ordinate, the linear equation of the retention time was calculated. Then, substituting the standard retention time of 15.58 min for costunolide, 29.07 min for naringin, and 32.13 min for neohesperidin into the equation, the predicted retention times were obtained respectively. Quantitative analysis: Multiple groups of mixed reference substance solutions were taken and successively diluted into a series of mixed reference substance solutions with different concentrations. 10 μL of the sample was injected into the liquid chromatograph, and the peak areas of the chromatographic peaks of each component were recorded. The relative correction factors of the compounds at different concentrations were calculated by the multi-point calibration method: Using hesperidin as the reference substance, the relative correction factors of naringin and neohesperidin were calculated; using paeoniflorin as the reference substance, the relative correction factor of costunolide was calculated. According to the formula C i =A i ×C S / (f i / s ×A S ) the contents of each component in the test sample were calculated.

[0079] In summary, for the method for determining the component contents of Shugan Tablets based on the double-standard multi-detection method, by using the double-standard linear calibration method to locate the chromatographic peaks, the accuracy of qualitative analysis was improved; by using the relative correction factor method for quantitative analysis and adopting the grouping reference strategy, the quantitative deviation caused by structural differences was reduced, and the accuracy of quantitative analysis was improved; by investigating various chromatographic columns, it was applicable to a variety of C 18 columns, significantly improving the applicable rate of the chromatographic columns; compared with the traditional multi-index content determination method, the number of reference substances used was reduced, and the test and detection costs were lowered; the extraction method used ultrasonic extraction, which was simple to operate and did not require complex equipment and cumbersome steps.

[0080] It should be noted that, in this document, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the phrase "comprising a..." does not exclude the presence of additional identical elements in the process, method, article or device comprising said element.

[0081] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining the contents of 5 components in Shugan Tablets based on a dual-label multi-detection method, characterized in that: It includes the following steps: S1: Extract the test solution: Grind the Shugan tablets finely and prepare it by ultrasonic treatment with methanol or refluxing with methanol. S2: Prepare the reference solution: Weigh reference substances of caryoptin, paeoniflorin, naringin, hesperidin, and neohesperidin with a certain mass fraction, add methanol with a certain volume fraction to make a mixed reference solution. S3: Prepare each negative control solution according to the preparation method of the test solution. S4: Determine the chromatographic conditions: Use high-performance liquid chromatography. The chromatographic conditions include using a C18 column, taking 0.05% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, performing gradient elution for 0 - 45 min, with 15% - 21% B; set appropriate flow rate and column temperature; determine the detection wavelength; clarify the injection volumes of the reference substance and the test sample. S5: Qualitative analysis: Use paeoniflorin and hesperidin as reference substances and construct a retention time prediction model by the double-standard linear calibration method. S6: Quantitative analysis: The contents of paeoniflorin, costunolide, naringin, hesperidin and neohesperidin in the Shugan Tablets sample were determined by the double-standard multi-measurement method and compared with the results determined by the external standard method. In the test sample, costunolide was referenced to paeoniflorin, and naringin and neohesperidin were referenced to hesperidin. According to the formula: C i = A i × C S / ( f i / s × A S ), calculate the contents of costunolide, naringin and neohesperidin in the test sample respectively, where C i is the content of the component to be measured in the test sample, A i is the peak area of the component to be measured in the test sample, A S is the peak area of the reference substance in the test sample, C S is the concentration of the reference substance in the test sample measured by the external standard method, f i / s is the correction factor.

2. A method for determining the contents of 5 components in Shugan tablets based on the double-label multi-measurement method according to claim 1, characterized in that: In the step S4, the flow rate is 0.9 to 1.1 mL·min -1 , the column temperature is 25 to 40 °C; the detection wavelength is 230 to 260 nm; the injection volumes of the reference substance and the test substance are 2 to 20 μL; the C 18 column is selected from Col1: Agilent 5 HC-C 18(2) ; Col2: Agilent 5 TC-C 18(2) ; Col 3: Agilent ZORBAX SB C 18 ; Col4: TechMate 120A C 18 ST; Col5: Capcell pak C 18 MG; Col6: Phenomenex Luna C 18(2) ; Col7: Titank C 18 ; Col8: Exsil monoC 18 ; Col9: Shim-pack GIST C 18 ; Col10: Zafex Supfex-YX-C 18 ; Col11: YMC-Triart C 18 ; Col12: Thermo Acclaim 120 C 18 ; Col13: Welch Ultimate plus C 18 ; Col14: ZORBAXEclipse XDB C 18 ; Col15: Kromasil C 18 ; Col16: Shim-pack VP-ODS C 18 one or more of the chromatographic columns, and the specifications of the chromatographic columns are all 4.6 mm×250 mm, 5 μm.

3. A method for determining the contents of 5 components in Shugan tablets based on a double-label multi-measurement method according to claim 1, characterized in that: The step of extracting the test solution in S1 is as follows: Grind the Shugan tablets finely, weigh 0.8 g, place it in a stoppered conical flask, add 25 - 50 mL of methanol with a volume fraction of 50% - 70%, weigh it, perform ultrasonic treatment for 30 - 90 minutes under the conditions of a power of 250 W and a frequency of 40 kHz, weigh it again after cooling, make up the missing weight with methanol with a volume fraction of 50% - 70%, shake well and filter, and take the subsequent filtrate to obtain the test solution.

4. A method for determining the contents of 5 components in Shugan tablets based on a dual-label multi-measurement method according to claim 1, characterized in that: The step of preparing the reference solution in S2 is as follows: Weigh reference substances of caryoptin, paeoniflorin, naringin, hesperidin, and neohesperidin with a certain mass fraction, add methanol with a volume fraction of 70% to make a mixed solution containing 5 - 50 μg of caryoptin, 10 - 100 μg of paeoniflorin, 50 - 200 μg of naringin, 10 - 70 μg of hesperidin, and 20 - 200 μg of neohesperidin per 1 mL, and thus obtain the reference solution.

5. A method for determining the contents of 5 components in Shugan tablets based on the double-label multi-measurement method according to claim 1, characterized in that: In the said qualitative analysis, by collecting the chromatograms of the Shugan tablet samples and reference substances on different reversed-phase chromatographic columns, recording the actual retention times of the samples, and taking the mean value of the retention times of the characteristic peaks as the standard retention time, a retention time prediction model is constructed.

6. A method for determining the contents of 5 components in Shugan tablets based on the double-label multi-measurement method according to claim 5, characterized in that: The said characteristic peaks include the chromatographic peaks of paeoniflorin, caryoptin, naringin, hesperidin, and neohesperidin.

7. A method for determining the contents of 5 components in Shugan Tablets based on the double-label multi-measurement method according to claim 1, characterized in that: In the said quantitative analysis, the relative correction factor is calculated by the multi-point calibration method. Take multiple groups of mixed reference solutions, respectively dilute them step by step into a series of mixed reference solutions with different concentrations, inject samples for determination, and calculate the relative correction factors of the compounds at different concentrations.

8. A method for determining the contents of 5 components in Shugan tablets based on the double-label multi-measurement method according to claim 1, characterized in that: It also includes the steps of methodology investigation. The said methodology investigation includes specificity test, linear relationship investigation, precision test, repeatability test, stability test, and recovery test of sample addition.

9. A method for determining the contents of 5 components of Shugan tablets based on a dual-label multi-measurement method according to claim 8, characterized in that: The said specificity test examines whether the chromatographic peaks of each component are interfered by measuring the chromatograms of the mixed reference solution, the test solution, and each negative control solution.

10. A method for determining the contents of 5 components in Shugan Tablets based on the double-label multi-measurement method according to claim 1, characterized in that: The method for constructing the retention time prediction model in S5 is as follows: taking the standard retention time of paeoniflorin, 11.65 min, and the standard retention time of hesperidin, 32.13 min, as the abscissa, and the actual retention times of paeoniflorin and hesperidin on the chromatographic column as the ordinate, calculating the retention time linear equation, and then substituting the standard retention time of costunolide, 15.58 min, the standard retention time of naringin, 29.07 min, and the standard retention time of neohesperidin, 32.13 min, into the equation to calculate the predicted retention times respectively.

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