A method for determining the contents of five components of Shugan tablets based on double-label multiple-assay method

Abstract

The invention relates to the technical field of quality detection, and discloses a method for determining the contents of five components of Shugan tablets based on a double-label multiple-measurement method. The method comprises the following steps: S1: extracting a test solution; S2: preparing a reference solution; S3: preparing each negative control solution; S4: determining chromatographic conditions; S5: using a double-label linear correction method to construct a retention time prediction model, taking the standard retention times of paeoniflorin and hesperidin as abscissas and the actual retention times of paeoniflorin and hesperidin on a chromatographic column as ordinates, substituting the standard retention times of agallochtetrol, naringin, and neohesperidin into an equation to calculate predicted retention times; and S6: using the double-label multiple-measurement method to determine the contents of paeoniflorin, agallochtetrol, naringin, hesperidin, and neohesperidin in a Shugan tablet sample. The method is capable of simultaneously determining the contents of the five components, namely, paeoniflorin, agallochtetrol, naringin, hesperidin, and neohesperidin, in the Shugan tablets through the double-label multiple-measurement method, with high accuracy and low cost.

Description

Technical Field

[0001] The present invention relates to the technical field of quality detection, and in particular to a method for determining the contents of five components of Shugan Tablets based on a double-label multiple-measurement method. Background Art

[0002] Shugan Tablets are a compound preparation consisting of 12 traditional Chinese medicines: agarwood, white peony root, dried tangerine peel, fructus aurantii, vinegared corydalis, and amomum villosum. This preparation has multiple ingredients and a complex preparation process. However, the current standard (standard number: WS3-B-2047-95) only covers properties and routine inspection items, making it difficult to effectively control product quality. Modern research indicates that agarwood in this preparation primarily contains 2-(2-phenylethyl) chromone derivatives such as agarwood tetraol; white peony root primarily contains monoterpenes and their glycosides, such as paeoniflorin; dried tangerine peel primarily contains flavonoids, such as hesperidin; and fructus aurantii contains flavonoids, such as naringin and neohesperidin. Existing studies have only established a single component determination method for paeoniflorin in Shugan Tablets, which fails to reflect the overall quality of the preparation. Therefore, a multi-component determination method for Shugan Tablets is urgently needed to improve the current quality standard, control its overall quality, and ensure its efficacy and safety.

[0003] However, multi-component content determination methods require a large number of reference substances, significantly increasing testing costs. Quantitative determination analysis multi-component by a single-marker (QAMS) or surrogate reference substance methods can reduce the number of reference substances used and have been successfully applied to the quality evaluation of various Chinese herbal medicine slices and formula preparations. However, these methods can sometimes result in large errors in chromatographic peak positioning and relative calibration factors. Sun Lei proposed the "two reference substances for determination of multiple components" method, which uses two reference substances and a liner calibration with two reference substances (LCTRS) for qualitative analysis of multi-component chromatographic peaks and a relative calibration factor for quantitative analysis of multiple components. This method significantly improves the accuracy of chromatographic peak identification and quantification, improves the applicability of chromatographic columns, and reduces reference substance costs. It has been successfully applied to the multi-component content determination of various Chinese herbal medicine slices and preparations.

[0004] Therefore, a method is needed to simultaneously determine the contents of five components in Shugan Tablets, namely, paeoniflorin, aganetetrol, naringin, hesperidin and neohesperidin, using a dual-label multiple-measurement method. That is, using paeoniflorin and hesperidin as reference substances, LCTRS is used to construct a retention time prediction model, and the chromatographic peaks of the remaining components are located. Agarnetetrol uses paeoniflorin as a reference substance, and naringin and neohesperidin use hesperidin as a reference substance, and the relative correction factors are calculated respectively. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides a method for determining the content of five components of Shugan Tablets based on the double-label multiple-measurement method. It has the advantage of using two reference substances to accurately qualitatively and quantitatively determine the five components at the same time, comprehensively reflecting the quality status of Shugan Tablets, and solving the one-sided quality control problem of single indicator determination.

[0006] To achieve the above-mentioned purpose of accurately qualitatively and quantitatively analyzing the five components of Shugan Tablets using two reference substances and comprehensively reflecting the quality of Shugan Tablets, the present invention provides the following technical solution: a method for determining the contents of the five components of Shugan Tablets based on a dual-label multiple-measurement method, comprising the following steps:

[0007] S1: Extraction of test solution: Grind Shugan tablets into powder and prepare the solution by methanol sonication or methanol reflux method;

[0008] S2: Prepare reference solution: weigh a certain mass fraction of agave tetraol, paeoniflorin, naringin, hesperidin, and neohesperidin reference substances, add a certain volume fraction of methanol to prepare a mixed reference solution;

[0009] S3: Prepare negative control solutions according to the test solution preparation method;

[0010] S4: Determine chromatographic conditions: Use high performance liquid chromatography (HPLC) with a C18 column, 0.05% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, and gradient elution (0-45 min, 15% to 21% B); set appropriate flow rate and column temperature; determine the detection wavelength; and specify the injection volume of the reference and test samples.

[0011] S5: Qualitative analysis: Using paeoniflorin and hesperidin as reference substances, a retention time prediction model was constructed using the double-standard linear calibration method;

[0012] S6: Quantitative analysis: The contents of paeoniflorin, aganetetrol, naringin, hesperidin and neohesperidin in Shugan Tablets were determined by double-label multiple-assay method and compared with the results determined by external standard method. In the test sample, aganetetrol was referenced to paeoniflorin, while naringin and neohesperidin were referenced to hesperidin. According to the formula: C i = A i ×C S / ( f i / s × A S ) were used to calculate the contents of agalloch otetraol, naringin, and neohesperidin in the test samples, where C i is the content of the component to be tested in the test sample, A i is the peak area of ​​the component to be tested 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.

[0013] Preferably, the flow rate in step S4 is 0.9-1.1 mL·min -1 , column temperature is 25~40℃; detection wavelength is 230~260nm; injection volume of reference substance and test substance is 2~20μL; the C 18 Column 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 pack 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 18One or more chromatographic columns, wherein the specifications of the chromatographic columns are 4.6 mm×250 mm, 5 μm.

[0014] Preferably, the step of extracting the test solution in S1 is: grind Shugan tablets, weigh 0.8 g, place it in a stoppered conical flask, add 25 to 50 mL of methanol with a volume fraction of 50% to 70%, weigh it, and then perform ultrasonic treatment for 30 to 90 minutes at 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 50% to 70%, shake it well and filter it, and take the filtrate to obtain the test solution.

[0015] Preferably, the step of preparing the reference solution in S2 is: weighing a certain mass fraction of agave tetraol, paeoniflorin, naringin, hesperidin, and neohesperidin reference substances, adding 70% volume fraction of methanol to prepare a mixed solution containing 5-50 μg of agave tetraol, 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.

[0016] Preferably, in the qualitative analysis, chromatograms of Shugan Tablet samples and reference substances are collected on different reversed-phase chromatographic columns, actual retention times of the samples are recorded, and the mean of the characteristic peak retention times is used as the standard retention time to construct a retention time prediction model.

[0017] Preferably, the characteristic peaks include chromatographic peaks of paeoniflorin, agalloch alazone, naringin, hesperidin, and neohesperidin.

[0018] Preferably, in the quantitative analysis, the relative correction factor is calculated using a multi-point calibration method, where multiple groups of mixed reference solutions are taken and diluted step by step into a series of mixed reference solutions of different concentrations, sampled and measured, and the relative correction factors of the compounds at different concentrations are calculated.

[0019] Preferably, the method further comprises a methodological investigation step, wherein the methodological investigation includes a specificity test, a linear relationship investigation, a precision test, a repeatability test, a stability test and a sample recovery test.

[0020] Preferably, 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.

[0021] Preferably, the method for constructing the retention time prediction model in S5 is to use 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, to calculate the retention time linear equation, and then substitute the standard retention time of agave tetraol 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.

[0022] Compared with the prior art, the present invention provides a method for determining the contents of five components of Shugan Tablets based on a dual-label multiple-measurement method, which has the following beneficial effects:

[0023] 1. This method for determining the content of Shugan tablet ingredients based on the double-label multiple-measurement method improves qualitative accuracy by locating chromatographic peaks using the double-label linear correction method; quantitative analysis is performed using the relative correction factor method, and a group reference strategy is adopted to reduce quantitative deviations caused by structural differences and improve quantitative accuracy.

[0024] 2. This method for determining the content of Shugan tablets based on double-label multiple-measurement method is applicable to a variety of C 18 Column, significantly improving the applicability of chromatographic columns.

[0025] 3. Compared with the traditional multi-index content determination method, this method of determining the content of Shugan tablet ingredients based on the double-label multi-measurement method reduces the number of reference substances used and reduces the cost of inspection and testing.

[0026] 4. This method for determining the content of Shugan tablet components based on the double-label multiple-measurement method uses ultrasonic extraction, which is simple to operate and does not require complex equipment and tedious steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure is an HPLC chromatogram of the five components of the present invention. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] A method for determining the contents of five components of Shugan Tablets based on a double-label multiple-assay method comprises the following steps:

[0030] S1: Extract the test solution: Grind Shugan tablets into powder, weigh 0.8 g, place in a stoppered conical flask, add 25 mL of 70% by volume methanol, weigh, and then sonicate or reflux for 60 minutes. Cool and weigh again, make up the missing weight with 70% by volume methanol, shake well, filter, and take the filtrate to obtain the test solution;

[0031] In the present invention, the instruments used are Agilent 1260 Infinity II high performance liquid chromatograph, Shimadzu LC-20ADXR high performance liquid chromatograph, Thermo U3000 RSLC high performance liquid chromatograph, XPE-205 analytical balance; Minispin 12000 rpm centrifuge; Milli-Q Advantage A ultrapure water machine; Elmasonic P ultrasonic cleaner;

[0032] Reference substances including agallotetraol (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 purchased from the China Food and Drug Administration for content determination. Acetonitrile was chromatographically pure, and all other ingredients were analytically pure. Water was prepared using a Milli-Q system.

[0033] Comparison of extraction methods: 0.8 g of Shugan tablets sample was taken, 25 mL of 70% methanol was added, and ultrasound (60 minutes) and reflux (60 minutes) were compared. The measured sample results are shown in Table 1.

[0034] Table 1 Comparison of extraction methods (mg / g)

[0035]

[0036] According to Table 1, there was no significant difference in the contents of the five components between ultrasonic and reflux extraction. Ultrasonic extraction was chosen in this study because it was simpler.

[0037] Comparison of extraction solvents: The extraction efficiencies of ethanol, methanol, 20% methanol by volume, 50% methanol by volume, and 70% methanol by volume were compared. The measured sample results are shown in Table 2.

[0038] Table 2 Comparison of extraction solvents (mg / g)

[0039]

[0040] According to Table 2, when the extraction solvent is methanol with a volume fraction of 50% and methanol with a volume fraction of 70%, the extraction efficiency of the five components is better than that of other solvents. Because the impurities extracted by methanol with a volume fraction of 70% are less than those extracted by methanol with a volume fraction of 50%, methanol with a volume fraction of 70% is used as the extraction solvent in this scheme.

[0041] Comparison of extraction time: The changes in the contents of the five components after ultrasonic extraction for 30 min, 60 min, and 90 min were compared. The sample measurement results are shown in Table 3.

[0042] Table 3 Comparison of extraction time (mg / g)

[0043]

[0044] According to Table 3, there is no significant difference in the content determination results of the five components when the extraction time is 30 min, 60 min, and 90 min, respectively. To ensure complete extraction, the extraction time is selected as 60 min in this scheme.

[0045] Comparison of extraction volumes: The effects of adding 10 mL, 25 mL, and 50 mL of 70% methanol to the sample on the content determination results were compared. The results are shown in Table 4.

[0046] Table 4 Comparison of different extraction volumes (mg / g)

[0047]

[0048] According to Table 4, there is little difference in extraction efficiency between the sampling volumes of 25 mL and 50 mL. Considering the moderate size of the peak area, the extraction volume of 25 mL was finally selected in this scheme.

[0049] Based on the above experimental results, the final extraction method for the test sample was determined as follows: grind Shugan tablets into powder and weigh 0.8 g, place it in a stoppered conical flask, add 25 mL of 70% methanol by volume, weigh the weight, and perform ultrasonic treatment at a power of 250 W and a frequency of 40 kHz for 60 minutes. After cooling, weigh the weight again, make up the missing weight with 70% methanol by volume, shake well and filter, take the filtrate to obtain the test sample solution.

[0050] S2: Preparation of reference solution: Accurately weigh 15.84 mg of agarwood tetraol, 18.61 mg of paeoniflorin, 20.42 mg of naringin, 16.50 mg of hesperidin, and 19.70 mg of neohesperidin. Place them in 20 mL, 20 mL, 50 mL, 50 mL, and 50 mL volumetric flasks, respectively. Add 70% by volume methanol and sonicate until completely dissolved. Then, dilute to the mark with 70% by volume methanol and shake well to obtain the stock solution of each reference substance. Accurately take 1 mL, 5 mL, 10 mL, 3 mL, and 10 mL of the reference substance stock solutions of agalloch eagle tetraol, paeoniflorin, naringin, hesperidin, and neohesperidin, respectively, and place them in the same 50 mL volumetric flask. Add 70% by volume methanol to the mark, and shake well to prepare a mixed solution containing 15.62 μg of agalloch eagle tetraol, 89.98 μg of paeoniflorin, 78.49 μg of naringin, 64.15 μg of hesperidin, and 78.33 μg of neohesperidin per 1 mL to obtain the reference substance solution.

[0051] S3: prepare each negative control solution according to the test solution preparation method; specifically, take the prescription amount of the remaining medicinal materials except agarwood, and prepare a negative control solution lacking agarwood according to the test solution preparation method; the same method, take the prescription amount of the remaining medicinal materials except white peony root, tangerine peel, and fructus aurantii, and prepare negative control solutions lacking white peony root, tangerine peel, and fructus aurantii respectively according to the test solution preparation method; then take the prescription amount of the remaining medicinal materials except tangerine peel and fructus aurantii, strictly follow the test solution preparation method, and undergo the same pretreatment process to prepare a negative control solution lacking tangerine peel and fructus aurantii at the same time, which will not be described in detail here.

[0052] S4: Determine the chromatographic conditions: Use high performance liquid chromatography, the chromatographic column is C 18 The column was eluted with 0.05% phosphoric acid as mobile phase A and acetonitrile as mobile phase B. The gradient elution was: 0-45 min, 15% to 21% B; the flow rate was 1.0 mL min -1 , column temperature was 35°C; detection wavelength: agarwood tetraol was 252 nm, and the other four components were 230 nm; the injection volume of the reference and test samples was 10 μL;

[0053] The theoretical plate number calculated based on the paeoniflorin peak should be no less than 7000. The HPLC chromatograms of the five components of paeoniflorin, agallotetraol, naringin, hesperidin, and neohesperidin are as follows: Figure 1 As shown, 1-paeoniflorin; 2-agarwood tetraol; 3-naringin; 4-hesperidin; 5-neohesperidin; A-mixed reference solution; B sample solution; C-negative control lacking white peony root; D-negative control lacking agarwood; E-negative control lacking fructus aurantii and tangerine peel; F-negative control lacking fructus aurantii; G-negative control lacking tangerine peel;

[0054] S5: Qualitative analysis: Using paeoniflorin and hesperidin as reference substances, a retention time prediction model was constructed using the double-standard linear calibration method;

[0055] S6: Quantitative analysis: The contents of paeoniflorin, aganetetrol, naringin, hesperidin and neohesperidin in Shugan Tablets were determined by double-label multiple-assay method and compared with the results determined by external standard method. In the test sample, aganetetrol was referenced to paeoniflorin, while naringin and neohesperidin were referenced to hesperidin. According to the formula: C i = A i × C S / ( f i / s × A S ) were used to calculate the contents of agalloch otetraol, naringin, and neohesperidin in the test samples, where C i is the content of the component to be tested in the test sample, A i is the peak area of ​​the component to be tested 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. f i / s is 1.9449, naringin f i / s 1.0596, neohesperidin f i / s It is 1.0359.

[0056] In one embodiment of the present invention, C 18 Column 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 chromatographic columns, the specifications of the chromatographic columns are all 4.6 mm×250 mm, 5 μm.

[0057] In one embodiment of the present invention, the gradient elution program is 0-45 min, and the proportion of mobile phase B increases linearly from 15% to 21%.

[0058] In one embodiment of the present invention, in qualitative analysis, chromatograms of Shugan Tablet samples and reference substances were collected on 16 different reverse-phase chromatographic columns, the actual retention times of the samples were recorded, and the mean of the characteristic peak retention times was used as the standard retention time to construct a retention time prediction model.

[0059] In one embodiment of the present invention, the characteristic peaks include chromatographic peaks of paeoniflorin, agalloch alazonetetraol, naringin, hesperidin, and neohesperidin.

[0060] In one embodiment of the present invention, in quantitative analysis, the relative correction factor is calculated using a multi-point calibration method, where multiple groups of mixed reference solutions are taken, diluted step by step into a series of mixed reference solutions of different concentrations, sampled and measured, and the relative correction factors of the compounds at different concentrations are calculated.

[0061] In one embodiment of the present invention, a methodological investigation step is also included, and the methodological investigation includes a specificity test, a linear relationship investigation, a precision test, a repeatability test, a stability test, and a sample recovery test; each experimental step and the corresponding data table are as follows:

[0062] 1. Methodological investigation

[0063] In this study, 11 batches of Shugan Tablet samples were collected from three manufacturers. Samples S1 and S2 were from Company A (batch numbers 22120569 and 21123573, respectively), S3 was from Company B (batch number 2406001), and S4 to S11 were from Company C (batch numbers 230501, 230502, 240601, 240602, 231101, 231102, 230701, and 230702, respectively). Sample S9 was used for the methodological review.

[0064] 1.1 Specificity test: Mixed reference solution, test solution, negative samples lacking agarwood, white peony root, dried tangerine peel, and fructus aurantii, as well as negative samples lacking both dried tangerine peel and fructus aurantii, were collected. The chromatographic column was Col12: Thermo Acclaim 120 C 18 (Chromatographic column specifications: 4.6 mm × 250 mm, 5 μm); 0.05% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution: 0-45 min, 15%-21% B; flow rate: 1.0 mL min -1 , column temperature: 35 ℃; detection wavelength: 252 nm for agarwood tetraol and 230 nm for the other four components; injection volume: 10 μL each for reference and test sample. Chromatographic conditions were used for determination. The chromatogram is shown in the figure. Figure 1 The results showed that agarwood, white peony root, and fructus aurantii had no interference due to their lack of flavor and had good specificity. However, in the negative control sample lacking dried tangerine peel, a very small chromatographic peak appeared at the hesperidin reference peak, while in the negative control sample lacking both dried tangerine peel and fructus aurantii, no chromatographic peaks appeared at the hesperidin, neohesperidin, and naringin reference peaks. This indicates that fructus aurantii contains a very small amount of hesperidin, while the main source of hesperidin in the preparation is dried tangerine peel. Therefore, the hesperidin content in the preparation is used to evaluate the quality of dried tangerine peel.

[0065] 1.2 Linear relationship investigation Mixed reference solutions of different concentrations were prepared using the Col12 column: ThermoAcclaim 120 C 18 (Chromatographic column specifications: 4.6 mm × 250 mm, 5 μm); 0.05% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution: 0-45 min, 15%-21% B; flow rate: 1.0 mL min -1 Column temperature: 35°C; detection wavelength: 252 nm for agarwood tetraol and 230 nm for the other four components; injection volume: 10 μL each for the reference and test samples. Chromatographic measurements were performed under these conditions, and the peak area of ​​each component was recorded. Regression equations for the five components were calculated by regressing the chromatographic peak area (Y) against the injection volume (X). The results are shown in Table 5.

[0066] Table 5 Linear regression results of the five components in Shugan tablets

[0067]

[0068] 1.3 Precision Test: Grind sample S9 and weigh 0.8 g. Place in a stoppered conical flask and add 25 mL of 70% methanol. Weigh the weight and then sonicate at 250 W and 40 kHz for 60 min. Cool and weigh again. Make up the missing weight with 70% methanol. Shake well and filter. Take the filtrate to prepare the test solution. The chromatographic column is 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, acetonitrile was used as mobile phase B, and gradient elution was performed: 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 agallotetraol and 230 nm for the other four components. The injection volume for both the reference and test samples was 10 μL, and the injections were repeated six times. The peak areas of the five components were measured and the RSDs were calculated. The results showed that the RSDs for the peak areas of agallotetraol, paeoniflorin, naringin, hesperidin, and neohesperidin were 0.17%, 0.22%, 0.10%, 0.19%, and 0.15%, respectively (Table 6), indicating good instrument precision.

[0069] Table 6 Precision test of Shugan tablets

[0070]

[0071] 1.4 Repeatability Test Take 6 portions of sample S9, grind them into powder, weigh 0.8 g, place them in a stoppered conical flask, add 25 mL of 70% by volume methanol, weigh the weight, and then sonicate at a power of 250 W and a frequency of 40 kHz for 60 minutes. After cooling, weigh the weight again, make up the missing weight with 70% by volume methanol, shake well, filter, and take the filtrate. Repeat this process 6 times to prepare 6 sets of test solutions. The chromatographic column is 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, acetonitrile was used as mobile phase B, and gradient elution was performed: 0-45 min, 15%-21% B; the flow rate was 1.0 mL min -1 The column temperature was 35°C; the detection wavelength was 252 nm for agarwood tetraol and 230 nm for the other four components; the injection volume of the reference and test samples was 10 μL. The content of each sample was determined under the chromatographic conditions. The results are shown in Table 7. The average contents of agarwood tetraol, paeoniflorin, naringin, hesperidin, and neohesperidin were 0.5655 mg·g, respectively. -1 、3.118 mg·g-1 , 2.702 mg·g -1 、0.6205 mg·g -1 and 2.658 mg·g -1 , RSDs were 0.30%, 1.83%, 2.09%, 1.32% and 0.15%, respectively, indicating that the method had good repeatability.

[0072] Table 7 Repeatability test results of Shugan Tablets (mg / g)

[0073]

[0074] 1.5 Stability Test: 0.8 g of sample S9 was ground into a fine powder and placed in a stoppered conical flask. 25 mL of 70% by volume methanol was added and the weight was determined. Ultrasonic treatment was performed at a power of 250 W and a frequency of 40 kHz for 60 min. The sample was cooled and weighed again. The missing weight was supplemented with 70% by volume methanol. The sample was shaken and filtered. The filtrate was used to prepare the test solution. After 0, 4, 9, 15, 41, and 61 h, 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, acetonitrile was used as mobile phase B, and gradient elution was performed: 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 agallotetraol and 230 nm for the other four components; the injection volume for both the reference and test samples was 10 μL. The peak areas were recorded under the chromatographic conditions, and the RSD values ​​were calculated. The results are shown in Table 8. The results showed that the RSD values ​​of the peak areas for agallotetraol, paeoniflorin, naringin, hesperidin, and neohesperidin were 1.52%, 0.97%, 0.98%, 0.60%, and 0.50%, respectively, indicating that the test solution was basically stable within 61 hours.

[0075] Table 8 Stability test results

[0076]

[0077] 1.6 Sample recovery test Accurately weigh 6 portions of Shugan Tablets sample S9 with a known content, 0.4 g each, and accurately add an appropriate amount of 5 reference substances to each portion. Place in a stoppered conical flask, add 25 mL of 70% methanol by volume, weigh the weight, and then ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 60 minutes. After cooling, weigh again, make up the missing weight with 70% methanol by volume, shake well, filter, and take the filtrate. Repeat 6 times to prepare 6 groups of test solution. The chromatographic column is 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. Gradient elution was performed: 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 agallotetraol and 230 nm for the other four components; the injection volume for both the reference and test samples was 10 μL. The recoveries and RSDs were calculated, and the results are shown in Table 9. The average recoveries of agallotetraol, paeoniflorin, naringin, hesperidin, and neohesperidin were 100.62%, 103.16%, 98.59%, 101.83%, and 97.72%, respectively, with RSDs of 0.68%, 1.06%, 0.60%, 1.81%, and 1.09%, respectively, indicating that the method was highly accurate.

[0078] Table 9 Recovery test results of Shugan Tablets. n = 6

[0079]

[0080]

[0081] 2. Qualitative research on dual-standard and multiple-measurement

[0082] The principle of the double-standard linear correction method is that there is a linear relationship between the retention times of the components to be measured on different reversed-phase chromatographic columns. Using two standard substances as references, the measured retention times are substituted into the determined linear equation to predict the retention time of the components to be measured and determine the peak position of the chromatographic peak of the components to be measured.

[0083] 2.1 Selection of double labels and calculation of standard retention time 18 Chromatograms of Shugan Tablets samples and reference substances were collected on the column. The chromatographic peaks were located by the retention time of the reference substances and the actual retention time of the samples was recorded. The results showed that Col 17 (ZORBAX Eclipse Plus C 18 , specifications are 4. 6 mm × 250 mm, 5μm), Col 18 (Inspire C 18, specifications are 4. 6 mm × 250 mm, 5 μm) and Col 19 (Dikma Diamonsil C 18(2) , size 4. 6 mm × 250 mm, 5 μm) chromatogram showed poor separation of hesperidin, while Col 20 (Waters Symmetry C 18 Because the chromatograms of the four columns (4.6 mm × 250 mm, 5 μm) showed interference from the agallotetraol impurity, these four columns were not used in method development. The mean retention time of the characteristic peaks on the remaining 16 columns was used as the standard retention time (SRT), which was 11.64 min for paeoniflorin, 15.58 min for agallotetraol, 29.07 min for naringin, 32.13 min for hesperidin, and 36.62 min for neohesperidin. The linear equations and correlation coefficients for each column were calculated using the SRT of the five components as the abscissa and the actual retention time as the ordinate. The results are shown in Table 10. The correlation coefficients for the retention times on all 16 columns were greater than 0.999, indicating a good linear relationship.

[0084] Table 10 Linear equations and correlation coefficients of retention time on different chromatographic columns

[0085]

[0086] 2.2 Determination and Optimization of Double-Labeled Compounds. The prediction accuracy of different compounds used as double-label compounds was calculated, and the results are shown in Table 11. The results show that the combination of Peaks 1 to 4 not only minimized the retention time regression deviation, but also achieved 100% retention time prediction accuracy and column consistency. Considering that reference materials for Peaks 1 (peoniflorin) and 4 (hesperidin) are readily available and inexpensive, paeoniflorin and hesperidin were selected as double-labeled compounds.

[0087] Table 11 Prediction accuracy of different double-labeled compounds

[0088]

[0089] 2.3 Determination of compounds by double-standard linear calibration method The new chromatographic column Hypersil ODS2-C 18 The elution was performed using a 4.6 mm × 250 mm, 5 μm column. 0.05% phosphoric acid was used as mobile phase A and acetonitrile was used as mobile phase B. Gradient elution was performed: 0–45 min, 15%–21% B; the flow rate was 1.0 mL min -1The method was validated using a 10 μL injection volume for both the control and test samples, with the column temperature set at 35°C. The detection wavelengths were 252 nm for agallotetraol and 230 nm for the other four components. The retention time linear equation was calculated 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, respectively) as the ordinate. Substituting the SRTs of agallotetraol, naringin, and neohesperidin (15.58, 29.07, and 32.13 min, respectively) into the equation yielded predicted retention times of 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 the double-label linear calibration method for locating target components in unknown chromatographic columns.

[0090] 2.4 Comparison of the Dual-Label Linear Calibration Method and the Relative Retention Time Method. Paeoniflorin and hesperidin were selected as dual-labeled compounds for dual-label linear calibration. The relative retention time method used naringin, which has a retention time intermediate to that of the other compounds, as a reference. The prediction results of the two methods are shown in Table 12. The results show that the maximum deviation of the predicted values ​​using the relative retention time method was greater than 1.0 min, while the maximum deviation using the dual-label linear calibration method was less than 0.6 min. This indicates that the dual-label linear calibration method has higher prediction accuracy than the relative retention time method and is applicable to a wider range of columns.

[0091] Table 12 Absolute deviation of retention time prediction values ​​of the two methods (min)

[0092]

[0093] 3. Quantitative study of double-label multiple-measurement method

[0094] 3.1 Calculation of Relative Correction Factors An appropriate amount of each reference substance was accurately weighed and prepared into three groups. Each group was then added with 70% methanol by volume to prepare a mixed reference solution containing 71.70 μg of agave tetraol, 132.75 μg of paeoniflorin, 198.73 μg of naringin, 54.28 μg of hesperidin, and 191.53 μg of neohesperidin per 1 mL. Appropriate amounts of the three mixed reference solutions were serially diluted to form a series of mixed reference solutions of varying concentrations. 10 μL of each solution was injected into the liquid chromatograph, and the peak area of ​​each component was recorded. A multi-point calibration method was used to calculate the relative correction factors of the compounds at different concentrations: the relative correction factors of naringin and neohesperidin were calculated using hesperidin as the reference, and the relative correction factor of agave tetraol was calculated using paeoniflorin as the reference. The results are shown in Table 13.

[0095] Table 13 Relative correction factors

[0096]

[0097] 3.2 Correction Factor Reproducibility Study The experiment investigated different brands of high performance liquid chromatographs and chromatographic columns, including Shimadzu LC-20ADXR, ThermoU3000RSLC, and Agilent 1260. The chromatographic columns were Col 3: Agilent ZORBAX SB C 18 ;Col12: Thermo Acclaim 120 C 18 ;Col13: Welch Ultimate plusC 18 (The specifications of the chromatographic columns were all 4.6 mm × 250 mm, 5 μm) 3 chromatographic columns were used, with 0.05% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, and gradient elution was performed: 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 agarwood tetraol and 230 nm for the other four components. The injection volume for both the reference and test samples was 10 μL. The relative calibration factors for each component in the dual-label, multiple-determination method were calculated using a multi-point calibration method and compared with the QAMS method using hesperidin as the reference. The results are shown in Table 14. The results showed that the RSDs of the relative calibration factors for the five components in the dual-label, multiple-determination method were all less than 3.0% across different instruments and columns. However, the RSD for the relative calibration factor of paeoniflorin in the QAMS method was greater than 5.0%. This indicates that different brands of liquid chromatographs and columns did not significantly affect the relative calibration factors in the dual-label, multiple-determination method, but had a significant impact on the relative calibration factors in the QAMS method. Based on this, this study proposed a "chemical property grouping-multiple reference material combination" strategy: target compounds were divided into two groups based on structural characteristics: flavonoids (naringin and neohesperidin) and terpenes (peoniflorin and agave tetraol). Recalculation of RCFs was performed using hesperidin (flavonoid group) and paeoniflorin (terpenoid group), respectively, as in-group reference materials. After optimization, the RSD values ​​for the RCFs of the three components were all less than 3.0%, indicating that the grouping reference material strategy can reduce quantitative bias caused by structural differences. This suggests that in the development of dual-label multiple-assay and QAMS methods for complex systems, when target components exhibit significant chemical property differences, grouping reference materials based on the principle of "structural similarity" can be used to increase the number of reference materials to improve the accuracy of quantification of compounds from different classes.

[0098] Table 14 Relative correction factors for different instruments and columns in the single-measurement multiple-evaluation method and the dual-label multiple-measurement method

[0099]

[0100] 3.3 Durability investigation of correction factors

[0101] 3.3.1 Effect of different flow rates

[0102] Accurately weigh 15.84 mg of agarwood tetraol, 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 20 mL, 20 mL, 50 mL, 50 mL, and 50 mL volumetric flasks, respectively. Add 70% by volume methanol and sonicate until completely dissolved. Then, dilute to the mark with 70% by volume methanol and shake well to obtain the stock solutions of each reference substance. 1 mL, 5 mL, 10 mL, 3 mL, and 10 mL of the reference stock solutions of agallochetol, paeoniflorin, naringin, hesperidin, and neohesperidin were accurately taken and placed in the same 50 mL volumetric flask. The volume was made up to the mark with 70% methanol by volume, and the mixture was shaken to prepare a mixed solution containing 15.62 μg of agallochetol, 89.98 μg of paeoniflorin, 78.49 μg of naringin, 64.15 μg of hesperidin, and 78.33 μg of neohesperidin per mL. The reference solution was obtained. The results of the HPLC-MS / MS experiments were carried out on a Thermo Fisher U3000 liquid chromatograph at different flow rates (0.9, 1.0, and 1.1 mL min-1) on a Col 12 column. -1 ) for each component f i / s The impact of 3 ingredients f i / s The RSDs of the two groups were all less than 1.0%, indicating that different flow rates had a significant effect on the composition. f i / s There was no significant effect. The results are shown in Table 15.

[0103] Table 15 Comparison of correction factors for different flow rates

[0104]

[0105] 3.3.2 Effect of different column temperatures

[0106] Preparation of reference solution: Accurately weigh 15.84 mg of agarwood tetraol, 18.61 mg of paeoniflorin, 20.42 mg of naringin, 16.50 mg of hesperidin, and 19.70 mg of neohesperidin into 20 mL, 20 mL, 50 mL, 50 mL, and 50 mL volumetric flasks, respectively. Add 70% by volume methanol and sonicate until completely dissolved. Then, dilute to the mark with 70% by volume methanol and shake well to obtain the stock solution of each reference substance. 1 mL, 5 mL, 10 mL, 3 mL and 10 mL of the reference stock solutions of agalloch eagle tetraol, paeoniflorin, naringin, hesperidin and neohesperidin were accurately taken respectively and placed in the same 50 mL volumetric flask, and the volume was made up to the mark with 70% methanol by volume. After shaking, a mixed solution containing 15.62 μg of agalloch eagle tetraol, 89.98 μg of paeoniflorin, 78.49 μg of naringin, 64.15 μg of hesperidin and 78.33 μg of neohesperidin per 1 mL was prepared to obtain the reference solution. The effects of different column temperatures (25°C, 35°C and 40°C) on the properties of each component were investigated on a Thermo Fisher U3000 liquid chromatograph on a Col 12 column. f i / s The impact of 3 ingredients f i / s The RSDs of the two groups were all less than 3.0%, indicating that different column temperatures had a significant effect on the composition. f i / s There was no significant effect. The results are shown in Table 16.

[0107] Table 16 Comparison of correction factors for different column temperatures

[0108]

[0109] 3.4 Comparison of results between the dual-label multiple measurement method and the external standard method

[0110] The established dual-label multiple-assay method was used to determine the contents of paeoniflorin, aganetrol, naringin, hesperidin, and neohesperidin in 11 batches of Shugan Tablet samples, and the results were compared with those determined by the external standard method. The results are shown in Table 17. Agarnetrol was measured with paeoniflorin as the reference, and naringin and neohesperidin were measured with hesperidin as the reference, according to the formula: C i =A i ×C S / (f i / s ×A S ) Calculate the contents of agalloch tetrol, naringin and neohesperidin in the test sample respectively; in the formula C i is the content of the component to be tested in the test sample; A i is the peak area of ​​the component to be tested in the test sample;A S is the peak area of ​​the reference substance in the test product; 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; f i / s is 1.9449, naringin f i / s 1.0596, neohesperidin f i / s It is 1.0359.

[0111] The content determination values ​​of external standard method and double standard multiple determination method were t The results of the test showed that the differences of agalloch esters, naringin and neohesperidin p The values ​​were 0.733, 0.970, and 0.993, respectively, with no significant difference between them. In other words, there was no significant difference between the double-label multiple-assay method and the external standard method. This shows that the double-label multiple-assay method can be applied to the analysis and determination of the contents of the five components in Shugan Tablets.

[0112] Table 17 Determination results of double-label multiple-measurement method and external standard method (mg·tablet) -1 )

[0113]

[0114] In one embodiment of the present invention, the specificity test is performed by measuring the chromatograms of the mixed reference solution, the test solution, and each negative control solution to examine whether the chromatographic peaks of each component are interfered.

[0115] In one embodiment of the present invention, the method for constructing the retention time prediction model in S5 is to use 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 to calculate the retention time linear equation, and then substitute the standard retention time of agave tetraol 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.

[0116] Working principle: Preparation of test solution: Grind Shugan tablets into powder, weigh 0.8 g, place in a stoppered conical flask, add 25 mL of 70% methanol by volume, weigh and ultrasonicate for 60 minutes, cool and weigh again, make up the missing weight with 70% methanol by volume, shake well and filter, take the filtrate to obtain the test solution;

[0117] Preparation of reference solution: Prepare reference solution: Accurately weigh 15.84 mg of agarwood tetraol, 18.61 mg of paeoniflorin, 20.42 mg of naringin, 16.50 mg of hesperidin, and 19.70 mg of neohesperidin, and place them in 20 mL, 20 mL, 50 mL, 50 mL, and 50 mL volumetric flasks, respectively. Add 70% by volume methanol and sonicate until completely dissolved. Then, dilute to the mark with 70% by volume methanol and shake well to obtain the stock solutions of each reference substance. Accurately take 1 mL, 5 mL, 10 mL, 3 mL, and 10 mL of the reference substance stock solutions of agalloch eagle tetraol, paeoniflorin, naringin, hesperidin, and neohesperidin, respectively, and place them in the same 50 mL volumetric flask. Add 70% by volume methanol to the mark, and shake well to prepare a mixed solution containing 15.62 μg of agalloch eagle tetraol, 89.98 μg of paeoniflorin, 78.49 μg of naringin, 64.15 μg of hesperidin, and 78.33 μg of neohesperidin per 1 mL to obtain the reference substance solution.

[0118] Preparation of negative control solution: prepare each negative control solution according to the test solution preparation method; specifically, take the prescribed amount of the remaining medicinal materials except agarwood, and prepare a negative control solution lacking agarwood according to the test solution preparation method; in the same way, take the prescribed amount of the remaining medicinal materials except white peony root, tangerine peel, and fructus aurantii, and prepare negative control solutions lacking white peony root, tangerine peel, and fructus aurantii respectively according to the test solution preparation method; then take the prescribed amount of the remaining medicinal materials except tangerine peel and fructus aurantii, strictly follow the test solution preparation method, and undergo the same pretreatment process to prepare a negative control solution lacking both tangerine peel and fructus aurantii;

[0119] Chromatographic conditions: The selected chromatographic column is 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 pack 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 (all with specifications of 4.6 mm × 250 mm, 5 μm) were used. 0.05% phosphoric acid was used as mobile phase A, and acetonitrile was used as mobile phase B. Gradient elution was performed: 0–45 min, 15%–21% B; the flow rate was 1.0 mL min -1 , column temperature was 35°C; detection wavelength: agarwood tetraol was 252 nm, and the other four components were 230 nm; the injection volume of the reference and test samples was 10 μL;

[0120] Qualitative analysis: Using paeoniflorin and hesperidin as reference substances, a retention time prediction model was constructed using the double-standard linear calibration method. The retention time linear equation was calculated using the standard retention time of paeoniflorin (11.65 min) and the standard retention time of hesperidin (32.13 min) as the horizontal axis, and the actual retention time of paeoniflorin and hesperidin on the chromatographic column as the vertical axis. The standard retention time of agallotetraol (15.58 min), naringin (29.07 min), and neohesperidin (32.13 min) were then substituted into the equation to obtain the predicted retention times.

[0121] Quantitative analysis: Take multiple groups of mixed reference solution, dilute them step by step into a series of mixed reference solution with different concentrations, inject 10μL, inject into liquid chromatograph, and record the peak area of ​​each component. Use multi-point calibration method to calculate the relative correction factor of the compound at different concentrations: using hesperidin as reference, calculate the relative correction factor of naringin and neohesperidin, and using paeoniflorin as reference to calculate the relative correction factor of agave tetraol. According to the formula C i =A i ×C S / (f i / s ×A S ) Calculate the content of each component in the test sample.

[0122] In summary, the method for determining the content of Shugan tablets based on double-label multiple-measurement method improves the accuracy of qualitative analysis by positioning the chromatographic peaks using double-label linear correction method; quantitative analysis is performed using relative correction factor method, and the group reference strategy is adopted to reduce the quantitative deviation caused by structural differences and improve the accuracy of quantitative analysis; through the investigation of various chromatographic columns, it is applicable to various C 18 The column significantly improves the applicability of the chromatographic column; compared with the traditional multi-index content determination method, it reduces the number of reference substances used and reduces the inspection and testing costs; the extraction method adopts ultrasonic extraction, which is easy to operate and does not require complex equipment and tedious steps.

[0123] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0124] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining the contents of five components of Shugan Tablets based on a dual-label multiple-assay method, characterized in that: The following steps are involved: S1: Extraction of test solution: Grind Shugan tablets into powder and prepare the solution by methanol sonication or methanol reflux method; S2: Prepare reference solution: weigh a certain mass fraction of agave tetraol, paeoniflorin, naringin, hesperidin and neohesperidin reference substances, add a certain volume fraction of methanol to prepare a mixed reference solution; S3: Prepare negative control solutions according to the test solution preparation method; S4: Determine chromatographic conditions: Use high performance liquid chromatography (HPLC) with a C18 column, 0.05% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, and a gradient elution of 15% to 21% B over a period of 0-45 min. Set an appropriate flow rate and column temperature. Determine the detection wavelength. Determine the injection volume of the reference and test samples. S5: Qualitative analysis: Using paeoniflorin and hesperidin as reference substances, a retention time prediction model was constructed using the double-standard linear calibration method; S6: Quantitative analysis: The contents of paeoniflorin, aganetetrol, naringin, hesperidin and neohesperidin in Shugan Tablets were determined by double-label multiple-assay method and compared with the results determined by external standard method. The aganetetrol in the test sample was referenced to paeoniflorin, while naringin and neohesperidin were referenced to hesperidin. According to the formula: C i = A i × C S / ( f i / s × A S ) were used to calculate the contents of agalloch otetraol, naringin, and neohesperidin in the test samples, where C i is the content of the component to be tested in the test sample, A i is the peak area of ​​the component to be tested 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. The method for determining the contents of the five components of Shugan Tablets based on a dual-label multiple-detection method according to claim 1, characterized in that: The flow rate in S4 is 0.9-1.1 mL·min -1 , column temperature is 25~40℃; detection wavelength is 230~260nm; injection volume of reference substance and test substance is 2~20μL; the C 18 Column selected from Col1: Agilent 5 HC-C 18(2) ;Col2:Agilent5 TC-C 18(2) ;Col 3: Agilent ZORBAX SB C 18 ;Col4:TechMate 120A C 18 ST;Col5:Capcellpak 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 One or more chromatographic columns, wherein the specifications of the chromatographic columns are 4.6 mm×250 mm, 5 μm.

3. The method for determining the contents of the five components of Shugan Tablets based on a dual-label multiple-detection method according to claim 1, characterized in that: The step of extracting the test solution in S1 is as follows: grind Shugan tablets into powder, weigh 0.8 g, place in a stoppered conical flask, add 25-50 mL of methanol with a volume fraction of 50%-70%, weigh, and then perform ultrasonic treatment for 30-90 minutes at a power of 250 W and a frequency of 40 kHz. After cooling, weigh again, make up the missing weight with methanol with a volume fraction of 50%-70%, shake well, filter, and take the filtrate to obtain the test solution.

4. The method for determining the contents of five components of Shugan Tablets based on a dual-label multiple-detection method according to claim 1, characterized in that: The step of preparing the reference solution in S2 is as follows: weighing a certain mass fraction of agallotetraol, paeoniflorin, naringin, hesperidin, and neohesperidin reference substances, adding 70% volume fraction of methanol to prepare a mixed solution containing 5-50 μg of agallotetraol, 10-100 μg of paeoniflorin, 50-200 μg of naringin, 10-70 μg of hesperidin, and 20-200 μg of neohesperidin per 1 mL, thereby obtaining the reference solution.

5. The method for determining the contents of five components of Shugan Tablets based on a dual-label multiple-detection method according to claim 1, characterized in that: In the qualitative analysis, chromatograms of Shugan Tablet samples and reference substances were collected on different reversed-phase chromatographic columns, the actual retention times of the samples were recorded, and the mean of the characteristic peak retention times was used as the standard retention time to construct a retention time prediction model.

6. The method for determining the contents of five components of Shugan Tablets based on a dual-label multiple-detection method according to claim 5, characterized in that: The characteristic peaks include the chromatographic peaks of paeoniflorin, agalloch alazone tetraol, naringin, hesperidin and neohesperidin.

7. The method for determining the contents of the five components of Shugan Tablets based on a dual-label multiple-detection method according to claim 1, characterized in that: In the quantitative analysis, the relative correction factor is calculated using a multi-point correction method, where multiple groups of mixed reference solutions are diluted step by step into a series of mixed reference solutions with different concentrations, injected and measured, and the relative correction factors of the compounds at different concentrations are calculated.

8. The method for determining the contents of five components of Shugan Tablets based on a dual-label multiple-detection method according to claim 1, characterized in that: It also includes a methodological investigation step, which includes a specificity test, a linear relationship investigation, a precision test, a repeatability test, a stability test and a sample recovery test.

9. The method for determining the contents of five components of Shugan Tablets based on a dual-label multiple-detection method according to claim 8, characterized in that: 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.

10. The method for determining the contents of five components of Shugan Tablets based on a dual-label multiple-detection method according to claim 1, characterized in that: The method for constructing the retention time prediction model in S5 is to use the standard retention time of paeoniflorin 11.65 min and the standard retention time of hesperidin 32.13 min as the horizontal axis and the actual retention time of paeoniflorin and hesperidin on the chromatographic column as the vertical axis to calculate the retention time linear equation, and then substitute the standard retention time of agave tetraol 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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