Determination method of 9 components in Xiao Xianxiong Tang based on quantitative analysis of multi-components by single marker

Abstract

The present application relates to a method for detecting the contents of nine components in Xiaoxianxiong Decoction based on a quantitative analysis of multiple components by a single marker, which can realize the simultaneous determination of multiple components using only one control sample. Compared with other methods, this method saves experimental materials, simplifies operation steps, saves determination time, and has high accuracy. It can determine the contents of multiple index components in Xiaoxianxiong Decoction, thereby ensuring the controllability of the quality of the preparation.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis methods, specifically involving a method for determining the content of nine components in the classic formula Xiao Xianxiong Decoction based on one-test-multiple-evaluation. Background Technology

[0002] The classic formula Xiao Xianxiong Decoction described in this invention is derived from Zhang Zhongjing's *Treatise on Febrile Diseases: Differentiation and Treatment of Taiyang Disease*, and is composed of Pinellia ternata, Coptis chinensis, and Trichosanthes kirilowii. It has the effects of clearing heat and resolving phlegm, opening the chest and dispersing nodules, and is an excellent formula for treating phlegm-heat accumulation in the epigastrium, causing pain upon pressure. Trichosanthes kirilowii is the chief herb, sweet, cold, and moistening, with the effects of clearing heat and resolving phlegm, opening the chest and dispersing nodules. When decocting it in this formula, it should be boiled first to "treat the upper body gradually," clearing the obstruction of the chest and diaphragm, clearing lung heat, moistening lung dryness, and treating cough and asthma caused by phlegm-heat. Pinellia ternata is the assistant herb, which can resolve phlegm and disperse nodules, relieve nausea and vomiting, benefit the throat, reduce swelling and relieve pain. Coptis chinensis is also an assistant herb, which can clear heat and dry dampness, purge fire and detoxify. This formula is suitable for chest obstruction caused by misdiagnosis of external pathogens, internal obstruction of heat, and phlegm-heat accumulation. Clinically, Xiao Xianxiong Decoction is mainly used to treat pneumonia, pleurisy, coronary heart disease, intercostal neuralgia, gastritis, hepatobiliary inflammation, and pancreatitis. Currently, research on Xiao Xianxiong Decoction focuses primarily on its efficacy and mechanism of action, with limited reports on its component analysis. Modified versions of Xiao Xianxiong Decoction are commonly used clinically. However, variations in decoction methods make effective control of components difficult, thus affecting efficacy. Traditional Chinese medicine (TCM) is characterized by its multi-component, multi-target, and multi-pathway nature, with different components synergistically exerting their effects. Determining the content of a single component cannot comprehensively assess its quality. The "one-measure-multiple-evaluation" method, using stable, inexpensive, and readily available reference standards as internal standards, simultaneously measures the content of multiple other components. This solves the problems of expensive, scarce, and difficult-to-manage reference standards for TCM, representing an extension and expansion of other analytical methods. Therefore, it is currently widely used for quality control of TCM and compound prescriptions. This experiment used hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, berberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, and analyzed them according to standards, laying the foundation for the research and quality control of Xiaoxianxiong Decoction. Summary of the Invention

[0003] This invention provides an HPLC fingerprint of Xiao Xianxiong Decoction, characterized in that when the HPLC chromatographic conditions are as follows, the HPLC fingerprint of Xiao Xianxiong Decoction is essentially the same as... Figure 1 Consistent;

[0004] The HPLC chromatographic conditions are as follows:

[0005] The chromatographic column was a Spursil C18, 250 mm × 4.6 mm, 5 μm; the mobile phase was acetonitrile (phase A) and 0.05 mol / L potassium dihydrogen phosphate solution adjusted to pH 4.0 with phosphoric acid (phase B); the gradient elution program was: 0–10 min, 2%–10% A; 10–17 min, 10%–15% A; 17–23 min, 15%–37% A; 23–27 min, 37%–40% A; 27–37 min, 40% A; 37–45 min, 40%–2% A; the detection wavelength was 290 nm, cut off at 300 nm; the flow rate was 0.8 mL / min, cut off to 0.6 mL / min from 27–37 min, and returned to 0.8 mL / min after 37 min; the column temperature was 30 °C; and the injection volume was 10 μL.

[0006] Another embodiment of the present invention provides an HPLC fingerprint of the above-mentioned Xiaoxianxiong Decoction, characterized in that the HPLC fingerprint of the Xiaoxianxiong Decoction is substantially the same as... Figure 1 The results were consistent, with 14 characteristic fingerprint chromatographic peaks, including peak 2 for hypoxanthine, peak 6 for vanillic acid, peak 8 for magnoflorine, peak 9 for demethylene berberine, peak 10 for tetrandrine, peak 11 for berberine hydrochloride, peak 12 for berberine hydrochloride, peak 13 for palmatine hydrochloride, and peak 14 for berberine hydrochloride.

[0007] Another embodiment of the present invention provides the application of the above-mentioned HPLC fingerprint of Xiaoxianxiong Decoction in the quality control and component analysis of Xiaoxianxiong Decoction.

[0008] Another embodiment of the present invention provides a method for component analysis of Xiaoxianxiong Decoction, characterized in that the method includes the following steps:

[0009] (1) Preparation of a mixed solution of 9 reference standards:

[0010] Weigh out hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, purslane, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride to prepare mixed standards with mass concentrations of 526 ug / ml, 39 ug / ml, 36 ug / ml, 15 ug / ml, 38 ug / ml, 103 ug / ml, 117 ug / ml, 95 ug / ml, and 322 ug / ml, respectively. Dilute the above mixed standard solutions to six different mass concentrations, which are 0.8, 0.6, 0.4, 0.2, and 0.1 times the original standard concentrations, and store them at 4℃ for later use.

[0011] (2) Preparation of standard curves for each reference standard in step (1):

[0012] Accurately pipette 10 μL of each of the six mixed reference solutions with different mass concentrations obtained in step (1), filter them through a microporous membrane, and perform HPLC detection. Plot standard curves for hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, berberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride with reference concentration as the abscissa (X) and peak area as the ordinate (Y). The linear regression equations are shown in the table below:

[0013]

[0014]

[0015] (3) Preparation of test solution: After evaporating Xiaoxianxiong decoction to dryness, add methanol solution and mix well. Take the supernatant and filter it through a microporous membrane to obtain the test solution.

[0016] (4) Take the test solution obtained in step (3) and detect it by HPLC. Calculate the content of hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, cypermethrin hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride reference standards in Xiaoxianxiong Decoction based on their concentrations, retention times in the fingerprint spectrum, and the linear equation in step (2).

[0017] The chromatographic conditions for HPLC detection described in steps (2) and (4) are as follows:

[0018] The chromatographic column was a Spursil C18, 250 mm × 4.6 mm, 5 μm; the mobile phase was acetonitrile (phase A) and 0.05 mol / L potassium dihydrogen phosphate solution adjusted to pH 4.0 with phosphoric acid (phase B); the gradient elution program was: 0–10 min, 2%–10% A; 10–17 min, 10%–15% A; 17–23 min, 15%–37% A; 23–27 min, 37%–40% A; 27–37 min, 40% A; 37–45 min, 40%–2% A; the detection wavelength was 290 nm, cut off at 300 nm; the flow rate was 0.8 mL / min, cut off to 0.6 mL / min from 27–37 min, and returned to 0.8 mL / min after 37 min; the column temperature was 30 °C; and the injection volume was 10 μL.

[0019] Compared with the prior art, the advantages of this invention are: (1) This invention makes up for the deficiencies of the prior art by establishing a high-performance liquid chromatography (HPLC) fingerprint of Xiaoxianxiong Decoction, providing an effective method for the quality control of Xiaoxianxiong Decoction; (2) The determination method of this invention can achieve simultaneous determination of multiple components using only one reference standard. Compared with other methods, this method saves experimental consumables, simplifies operation steps, saves determination time, and has high accuracy. It can determine the amount of multiple index components in Xiaoxianxiong Decoction, thereby ensuring the controllability of the quality of the preparation; (3) The HPLC fingerprint method and content determination method of Xiaoxianxiong Decoction established by this invention have good analytical evaluation capabilities and have the advantages of accuracy, simplicity, stability and reliability. Therefore, this method can be used as an effective evaluation method for this prescription. Attached Figure Description

[0020] Figure 1 This is the HPLC chromatogram of the mixed reference standards.

[0021] Figure 2 This is the HPLC chromatogram of the Xiaoxianxiong Decoction test sample. Detailed Implementation

[0022] To facilitate a further understanding of the present invention, the following embodiments are provided for more detailed description. However, these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following.

[0023] method:

[0024] 1. Instruments and reagents

[0025] 1.1 Instruments

[0026] Agilent 1260 high-performance liquid chromatograph (Agilent Technologies); Agilent HPLC system (Agilent Technologies); Spursil C18 column (250 mm × 4.6 mm, 5 μm); Agilent 5TC-C18 column (250 mm × 4.6 mm, 5 μm); analytical balance (Sartorius Scientific Instruments Co., Ltd.); water bath (Beijing Kewei Yongxing Instrument Co., Ltd.); ultrasonic instrument (Ningbo Xinyi Ultrasonic Equipment Co., Ltd.).

[0027] 1.2 Reagents and Materials

[0028] Reference standards: xanthine (batch number: CHB201107, purity HPLC ≥ 98%), vanillic acid (batch number: 110776-201503, purity HPLC ≥ 99.8%), magnoflorine (batch number: CHB201113, purity HPLC ≥ 98%), demethylene berberine (batch number: CHB210114, purity HPLC ≥ 98%), tetrandrine (batch number: CHB201126, purity HPLC ≥ 98%), berberine hydrochloride (batch number: 110733-201609, purity HPLC ≥ 89.5%), berberine hydrochloride (batch number: CHB201113, purity HPLC ≥ 98%), berberine hydrochloride (batch number: CHB210114, purity HPLC ≥ 98%), berberine hydrochloride (batch number: CHB201126, purity HPLC ≥ 98%), berberine hydrochloride (batch number: CHB201113, purity HPLC ≥ 89.5%), berberine hydrochloride (batch number: CHB201113, purity HPLC ≥ 98%), berberine hydrochloride (batch number: CHB201113, purity HPLC ≥ 98%), berberine hydrochloride (batch number: CHB201113, purity HPLC ≥ 98%), berberine hydrochloride (batch number: CHB201113, purity HPLC ≥ 89.5 ...98%), berberine The following reagents were purchased from Chengdu Kloma Biotechnology Co., Ltd. and the China National Institutes for Food and Drug Control, respectively: batch number 112026-201802 (purity HPLC ≥ 94.0%), berberine hydrochloride (batch number 110732-201913 (purity HPLC ≥ 85.7%), and berberine hydrochloride (batch number 110713-202015 (purity HPLC ≥ 85.9%). Water was Wahaha purified water. Potassium dihydrogen phosphate (Tianjin Kemio Chemical Reagent Co., Ltd.), phosphoric acid (Tianjin Kemio Chemical Reagent Co., Ltd.), acetonitrile, and methanol were chromatographic grade (Thermo Fisher Scientific). All other reagents were analytical grade.

[0029] 2. Methods and Results

[0030] 2.1 Chromatographic conditions

[0031] An Agilent 1260 high-performance liquid chromatograph (Agilent Technologies) was used. The column was a Spursil C18 (5 μm, 250 × 4.6 mm). The mobile phase was acetonitrile (A)-0.05 mol / L potassium dihydrogen phosphate (B) (pH adjusted to 4 with phosphoric acid). The gradient elution program was as follows: 0–10 min, 2%–10% A; 10–17 min, 10%–15% A; 17–23 min, 15%–37% A; 23–27 min, 37–40% A; 27–37 min, 40% A; 37–45 min, 40%–2% A. The detection wavelength was 290 nm, then 300 nm. The flow rate was 0.8 mL / min, reduced to 0.6 mL / min from 27–37 min, and returned to 0.8 mL / min after 37 min. The column temperature was 30 °C, and the injection volume was 10 μL.

[0032] 2.2 Preparation of the test solution

[0033] Based on historical records of Xiao Xianxiong Decoction and modern clinical safe dosage, the equivalent modern units of measurement are: Trichosanthes kirilowii 30g, Pinellia ternata 12g, and Coptis chinensis 6g. All three herbs were purchased from Shaanxi Xingshengde Pharmaceutical Co., Ltd., and authenticated as genuine products by Professor Meng Xue of the Shaanxi Provincial Academy of Traditional Chinese Medicine. First, Trichosanthes kirilowii was placed in a decoction vessel, 1200mL of deionized water was added, and it was soaked for 30 minutes. It was then simmered over high heat followed by low heat until reduced to 600mL, and filtered to remove the residue. Next, Coptis chinensis and Pinellia ternata were added to the Trichosanthes kirilowii decoction, and the mixture was simmered over high heat followed by low heat until reduced to 400mL, and filtered to remove the residue. The decoction was stored at 4℃ for subsequent preparation. 3mL of the decoction was evaporated to dryness in an electric thermostatic water bath. The solution was dissolved in 10mL of 20% methanol in a dried evaporating dish, and the supernatant was filtered through a 0.45μm microporous membrane to obtain the test solution. This solution was stored at 4℃ for later use.

[0034] 2.3 Preparation of mixed reference solution

[0035] Weigh out hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, berberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride to prepare mixed standards with mass concentrations of 526 ug / ml, 39 ug / ml, 36 ug / ml, 15 ug / ml, 38 ug / ml, 103 ug / ml, 117 ug / ml, 95 ug / ml, and 322 ug / ml, respectively. Dilute the above mixed standard solutions to six different mass concentrations, which are 0.8, 0.6, 0.4, 0.2, and 0.1 times the original standard concentrations, respectively, and store them at 4℃ for later use.

[0036] 2.4 Methodological Examination

[0037] 2.4.1 Chromatographic conditions and system suitability test

[0038] An Agilent 1260 high-performance liquid chromatograph (Agilent Technologies) was used. The chromatographic column was a Spursil C18 (5 μm 250 × 4.6 mm). The mobile phase was acetonitrile (A) - 0.05 mol / L potassium dihydrogen phosphate (B) (pH adjusted to 4 with phosphoric acid). The gradient elution program was as follows: 0–10 min, 2%–10% A; 10–17 min, 10%–15% A; 17–23 min, 15%–37% A; 23–27 min, 37–40% A; 27–37 min, 40% A; 37–45 min, 40%–2% A. The detection wavelength was 290 nm, then 300 nm. The flow rate was 0.8 mL / min, reduced to 0.6 mL / min from 27–37 min, and then returned to 0.8 mL / min after 37 min. The column temperature was 30 °C, and the injection volume was 10 μL. Mixed reference solutions (…) were taken separately… Figure 1 ) and test solution ( Figure 2Injected into the liquid chromatograph, the results are shown in […]. Figure 1 The resolution of each chromatographic peak was greater than 1.5, and the theoretical plate number was greater than 30,000.

[0039] 2.4.2 Examination of Linear Relationships

[0040] Accurately pipette 10 μL of each of the six mixed reference solutions with different mass concentrations under section "2.3" and determine them according to the chromatographic conditions under section "2.1". Plot standard curves for hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, berberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride with reference concentration as the abscissa (X) and peak area as the ordinate (Y). The linear regression equations were obtained, and the results showed that the mass concentrations of the nine components had good linear relationships within the corresponding ranges. The results are shown in the table below.

[0041] Table 1. Linear regression equations for the nine components of Xiao Xianxiong Decoction.

[0042]

[0043] 2.4.3 Precision Experiment

[0044] Take an appropriate amount of this product and prepare the test solution according to section "2.1". Inject and determine 6 times under the chromatographic conditions specified in section "2.1". The peak areas RSDs of hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, berberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride were 3.37%, 3.11%, and 2.10%, respectively.

[0045] The values ​​of 1.94%, 3.23%, 0.33%, 0.65%, 0.37%, and 0.33% indicate that the instrument has good precision in the experiment.

[0046] 2.4.4 Repeatability Test

[0047] Using the same decoction, six samples were prepared in parallel according to the method described in "2.1", and measured six times. The peak area RSDs of hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, berberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride were 3.13%, 3.06%, 2.48%, 2.18%, 2.27%, 1.03%, 1.38%, 0.20%, and 0.64%, respectively, indicating that this method has good repeatability.

[0048] 2.4.5 Stability Test

[0049] A precise extraction of 10 μl of standard solution was performed, and the sample was injected at 0, 2, 4, 6, 8, and 12 h after preparation, according to the chromatographic conditions specified in "2.1". The RSDs of the peak areas of hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, berberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride were 3.03%, 2.98%, 1.77%, 1.22%, 1.98%, 0.88%, 0.90%, 0.63%, and 0.59%, respectively, indicating that the sample has good stability.

[0050] 2.4.6 Recovery Experiment

[0051] Six aliquots of the Xiaoxianxiong Decoction test sample with known concentrations were precisely pipetted, and 1.5 ml of each aliquot was precisely added to the reference stock solutions of hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, berberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride, respectively. The sample recovery test solutions were prepared according to the method in section "2.1". The samples were injected and analyzed under the chromatographic conditions in section "2.1", and the recovery rates were calculated. The RSD values ​​of the recovered samples were 2.83%, 2.24%, 1.41%, 3.48%, 3.14%, 0.98%, 1.24%, 1.42%, and 0.52%, respectively. The results are shown in the table below, indicating that the method has good accuracy.

[0052] Table 2 Results of the sample recovery experiment (n=6)

[0053]

[0054]

[0055]

[0056] 2.5 Determination of Relative Correction Factor

[0057] Accurately pipette the mixed reference solution from section "2.3" and inject it six times consecutively under the chromatographic conditions described in section "2.1". Measure the peak area. Using berberine as an internal reference, determine the relative correction factors for hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, berberine hydrochloride, berberine hydrochloride, and palmatine hydrochloride, respectively. The calculation formulas are as follows:

[0058] f s / i =f s / f i =(A s ×C i ) / (A i ×C s )

[0059] Where, A sC represents the peak area of ​​berberine hydrochloride. i C represents the concentration of analyte i. s The concentration of berberine hydrochloride, A i Let be the peak area of ​​analyte i. The results are shown in Table 3.

[0060] Table 3. Calculation results of relative correction factors for eight components using berberine hydrochloride as an internal reference.

[0061]

[0062]

[0063] 2.6 Robustness test of relative correction factor

[0064] 2.6.1 Effects of different chromatographic columns and instruments on relative correction factors

[0065] The effects of Spursil C18 (250 mm × 4.6 mm, 5 μm) and Agilent 5TC-C18 (250 mm × 4.6 mm, 5 μm) columns, with berberine hydrochloride as an internal reference, on the relative correction factors of each analyte were investigated using an Agilent 1260 high-performance liquid chromatography (HPLC) system and an Agilent 5TC-C18 system, respectively. The results are shown in Table 4. The results indicate that hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, purslane hydrochloride, berberine hydrochloride, palmatine hydrochloride, and the internal reference berberine hydrochloride showed good reproducibility with different chromatographic columns, with RSDs ranging from 0.01% to 2.10%.

[0066] Table 4. Effects of different instrument columns on relative correction factors

[0067]

[0068] 2.6.2 Effect of Different Column Temperatures on the Relative Correction Factors of Eight Components in Xiaoxianxiong Decoction Using the chromatographic conditions described in section "2.1", with berberine hydrochloride as the internal reference, the effects of column temperatures of 25, 30, and 35℃ on the relative correction factors were investigated. The results are shown in Table 5. Hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, berberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and the internal reference berberine hydrochloride showed good applicability of relative correction factors at different column temperatures.

[0069] Table 5. Effect of different column temperatures on relative correction factors

[0070]

[0071] 2.6.3 Localization of chromatographic peaks of the analyte

[0072] The chromatographic peaks of the analytes were located using the relative retention time method. Given the retention time of the internal standard peak, the peaks could be located based on the relative retention times of the eight analytes. The relative retention time of berberine hydrochloride was selected as the standard for analyte location. The relative retention times were calculated under different high-performance liquid chromatography (HPLC) instruments and columns. Under different HPLC instrument and column conditions, the RSD of the relative retention times of each analyte was less than 5.00%. (See Table 6).

[0073] Table 6. Relative retention times measured by different instruments and chromatographic columns.

[0074]

[0075] 2.7 Comparison of test results between the single-test-multiple-evaluation method and the external standard method

[0076] The contents of hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, purslane hydrochloride, berberine hydrochloride, and palmatine hydrochloride in six batches of different Xiaoxianxiong decoction samples were determined by external standard method (ESM). The results were compared with those calculated by the one-test-multiple-evaluation method to verify the accuracy and reliability of the one-test-multiple-evaluation method for the determination of nine components in Xiaoxianxiong decoction. The results of the two methods are shown in Table 7. Data analysis was performed using GraphPad Prism 8.4.2. The independent samples t-test was used to compare the two groups. P > 0.05, indicating that there was no significant difference in the results of the two methods. The one-test-multiple-evaluation method established in this study has good reliability.

[0077]

Claims

1. A method for component analysis of Xiaoxianxiong Decoction, characterized in that... The analytical method includes the following steps: the components are hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, purslane, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride; the analytical method is HPLC detection, and the chromatographic conditions are as follows: the chromatographic column is a Spursil C18, with dimensions of 250 mm × 4.6 mm and a diameter of 5 μm; the mobile phase is acetonitrile (phase A) and 0.05 mol / L potassium dihydrogen phosphate solution adjusted to pH 4.0 with phosphoric acid (phase B); the gradient elution program is: 0–10 min, 2%–10% A; 10–17 min, 10%–15% A; 17–23 min, 15%–37% A; 23–27 min, 37%–40% A; 27–37 min, 40% A; 37–45 min, 40%–2% A; the detection wavelength is 290 nm–300 nm; and the flow rate is 0.8 nm. The flow rate was set to 0.6 mL / min from 27 to 37 min, and then increased to 0.8 mL / min after 37 min. The column temperature was 30°C, and the injection volume was 10 μL.

2. The analytical method according to claim 1, characterized in that... Includes the following steps: (1) Preparation of a mixed solution of 9 reference standards: Weigh out hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, purslane, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride to prepare mixed standards with mass concentrations of 526 μg / ml, 39 μg / ml, 36 μg / ml, 15 μg / ml, 38 μg / ml, 103 μg / ml, 117 μg / ml, 95 μg / ml, and 322 μg / ml, respectively. Dilute the above mixed standard solutions to six different mass concentrations, which are 0.8, 0.6, 0.4, 0.2, and 0.1 times the original standard concentrations, respectively, and store them at 4℃ for later use. (2) Preparation of standard curves for each reference standard in step (1): Accurately pipette 10 μL of each of the six mixed reference solutions with different mass concentrations obtained in step (1), filter them through a microporous membrane, and perform HPLC detection. Plot standard curves for hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, berberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride with reference concentration as the abscissa (X) and peak area as the ordinate (Y). The linear regression equations are shown in the table below: (3) Preparation of test solution: After evaporating Xiaoxianxiong decoction to dryness, add methanol solution and mix well. Take the supernatant and filter it through a microporous membrane to obtain the test solution. (4) Take the test solution obtained in step (3) and detect it by HPLC. Calculate the content of hypoxanthine, vanillic acid, magnoflorine, demethylene berberine, tetrandrine, cypermethrin hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride reference standards in Xiaoxianxiong Decoction based on their concentrations, retention times in the fingerprint spectrum, and the linear equation in step (2).

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