A method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu granules

Abstract

The present invention relates to the technical field of drug production and detection, and specifically discloses a method for determining the content of paeoniflorin in Huangqi Guizhi Wuwutang granules, comprising the following steps: analyzing paeoniflorin by high performance liquid chromatography, and sequentially performing sample pretreatment, chromatographic analysis and content determination. The pretreatment uses an HP-20 resin column, which is sequentially eluted with different concentrations of ethanol, and 50% and 70% ethanol eluates are collected and concentrated. A C18 chromatographic column is selected, acetonitrile-0.1% phosphoric acid aqueous solution is used as the mobile phase, a diode array detector is detected at a wavelength of 230nm, and a multi-wavelength deconvolution algorithm is applied for quantitative analysis. The determination method of the present invention has strong specificity, and stability, repeatability, intermediate precision, accuracy, etc. all meet the requirements, and the durability of the chromatographic conditions is good.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules. Background Art

[0002] Huangqi Guizhi Wuwu Decoction comes from Zhang Zhongjing's "Golden Chamber" in the Han Dynasty. The original prescription is: 3 liang of Huangqi, 3 liang of Shaoyao, 3 liang of Guizhi, 6 liang of ginger, and 12 jujubes. The above five flavors are boiled with 6 liters of water to obtain 2 liters, and 7 he of warm water is taken three times a day. The effect of this prescription is to invigorate qi and warm the meridians, harmonize blood and relieve arthralgia. It is mainly used to treat blood arthralgia syndrome, with symptoms such as numbness of the skin, slight aversion to wind and cold, pale tongue with white fur, and slightly astringent and tight pulse. Huangqi in the prescription is sweet and warm and invigorates qi as the main medicine. Guizhi dispels wind and cold and warms the meridians and relieves arthralgia, while white peony nourishes blood and nourishes the skin to relieve blood arthralgia. Both are assistant medicines. Ginger is pungent and warm, dispersing wind evil; jujube is sweet and warm, invigorating qi and nourishing blood, and both are used as adjuvants. Paeoniflorin is one of the active ingredients in Huangqi Guizhi Wuwu Granules, which has pharmacological effects such as anti-allergy and sedation. By measuring its content, the efficacy of the drug can be indirectly evaluated. Ensure that the content of paeoniflorin in the drug reaches an effective therapeutic concentration, thereby ensuring the therapeutic effect of the drug. The process route of this party is to sample aromatic water and directly include cyclodextrin. The inclusion compound is not blended before the traditional drying preparation, but is directly blended into the concentrated solution for drying after the inclusion is completed, which reduces the process links, improves efficiency, and retains volatile oily active ingredients better. The existing method for determining the content of paeoniflorin may not be able to accurately distinguish paeoniflorin from other compounds with similar structures, resulting in interference with the measurement results. During the measurement process, due to the influence of various factors, the content of paeoniflorin may change, resulting in unstable measurement results. Therefore, the present invention proposes a method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction Granules, which can effectively solve the above-mentioned technical problems. Summary of the invention

[0003] In response to the technical problems mentioned in the background technology, the present invention proposes a method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules.

[0004] The present invention adopts the following technical solution:

[0005] A method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules comprises the following steps:

[0006] S1. Sample pretreatment: Take an HP-20 macroporous adsorption resin column, first add 3 column volumes of 95% ethanol, control the flow rate at 1.0 mL / min, and then add 3 column volumes of water for activation; accurately weigh 0.18 g-0.22 g of Huangqi Guizhi Wuwu Decoction granules, place in a 100 mL stoppered conical flask, accurately add 50 mL of solvent, stopper, weigh, ultrasonically treat for 30-90 min, cool to 25°C-35°C, weigh again, make up the lost weight with water, shake well, and obtain the loading solution;

[0007] The loading solution was loaded onto the column at a flow rate of 1.0 mL / min, and the following elutions were performed in sequence: Step 1: Accurately add 3 column volumes of water for elution, and discard the effluent; Step 2: Accurately add 2 column volumes of 30% ethanol for elution, collect the eluate, and mark it as fraction 1; Step 3: Accurately add 2 column volumes of 50% ethanol for elution, collect the eluate, and mark it as fraction 2; Step 4: Accurately add 2 column volumes of 70% ethanol for elution, collect the eluate, and mark it as fraction 3;

[0008] Take fraction 2 and fraction 3, combine them, place them in an evaporator to recover the solvent under reduced pressure, control the temperature at 45℃±2℃, transfer the recovered concentrated solution to a 25mL volumetric flask, dilute to the mark with 50% ethanol, shake well, and obtain the pretreatment solution;

[0009] S2. Selection of chromatographic conditions:

[0010] Chromatographic operating conditions and parameters: Chromatographic column: C18 chromatographic column, 4.6*250mm, 5um, mobile phase A: acetonitrile; mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution, flow rate 1.0mL / min; injection volume 10μL; column temperature 30℃, injection chamber temperature 25℃, diode array detector, scanning range: 200-400nm, detection wavelength 230nm, liquid chromatography with a volume ratio of mobile phase A to mobile phase B of 14%:86%;

[0011] S3. Preparation of reference solution: Take the paeoniflorin reference substance, weigh it accurately, and add methanol to make a solution containing 80 μg per 1 mL;

[0012] S4. Preparation of the test solution: Take 10 mL of the pretreatment solution, place it in a 25 mL volumetric flask, add methanol to the mark, shake well, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0013] S5. Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, collect multi-wavelength signals, and separate and quantitatively analyze the chromatographic peaks using multi-wavelength signal deconvolution algorithm.

[0014] Preferably, the following method is used to separate and quantitatively analyze the chromatographic peaks by a multi-wavelength signal deconvolution algorithm: a wavelength scan is performed on the chromatogram at a sampling interval of 0.001 min by a diode array detector, a signal data matrix in the range of 200-400 nm is collected, and signal data of main wavelengths of 230 nm, 280 nm, and 290 nm are separated and extracted from the data matrix to obtain a three-dimensional original data sequence; a 9-point sliding average calculation is performed on the signal data of the main wavelength of 230 nm in the three-dimensional original data sequence, and the signal intensities of the 4 points before and after each data point are weighted averaged to obtain a main peak baseline characteristic value sequence; a three-dimensional peak shape characteristic model is established by correlating the main peak baseline characteristic value sequence with the auxiliary wavelength data of 280 nm and 290 nm, and the co-elution region signal is deconvoluted and separated to obtain the peak area data of each component; according to the peak area data of each component, the content of each component is calculated according to the external standard method, with the reference solution as the external standard, to obtain the content determination result.

[0015] Preferably, the method performs an association operation on the main peak baseline characteristic value sequence and the auxiliary wavelength data of 280nm and 290nm to establish a three-dimensional peak shape characteristic model, deconvolutes and separates the co-elution region signal, and obtains the peak area data of each component, including: aligning the main peak baseline characteristic value sequence with the auxiliary wavelength data of 280nm and 290nm on the time axis, establishing a sampling point pairing relationship at intervals of 0.001min, and obtaining a three-dimensional signal pairing sequence; calculating the response ratio of the three wavelengths at each sampling point according to the three-dimensional signal pairing sequence, and establishing a signal intensity ratio matrix for each data point in the co-elution region. , and obtain a wavelength characteristic distribution diagram; according to the change law of the signal intensity ratio in the wavelength characteristic distribution diagram, the co-elution area is segmented into 0.05min time windows, and the signal distribution characteristics in each window are calculated to obtain the target component response interval; according to the signal distribution in the target component response interval, a three-dimensional peak shape mathematical model is established, and the contours of the overlapping chromatographic peaks are fitted to obtain the peak shape fitting parameters of each component; the signal of the co-elution area is mathematically deconvoluted by the peak shape fitting parameters, and the overlapping peaks are quantitatively integrated according to the maximum absorption wavelength of each component to obtain the peak area data of each component.

[0016] Preferably, in the step of selecting chromatographic conditions: octadecylsilane bonded silica gel is used as filler; acetonitrile-0.1% phosphoric acid solution volume ratio 14:86 is used as mobile phase; detection wavelength is 230nm. The theoretical plate number calculated based on the paeoniflorin peak should be no less than 5000.

[0017] Preferably, in the sample pretreatment step, the solvent is one of ethanol, methanol, acetonitrile and water.

[0018] Preferably, in the sample pretreatment step, the mass concentration of ethanol is 49.5%-50.5%.

[0019] Preferably, in the sample pretreatment step, ultrasonic treatment is replaced by heating reflux.

[0020] Preferably, in the sample pretreatment step, the ultrasonic treatment time is 28.5 minutes to 31.5 minutes.

[0021] Preferably, the Huangqi Guizhi Wuwu Granules are prepared in the following manner:

[0022] Take astragalus, white peony root, cinnamon twig, ginger and jujube and boil them together with water, collect the aromatic water, combine the filtrate, and concentrate under reduced pressure to prepare the Huangqi Guizhi Wuwu Decoction extract;

[0023] Take an appropriate amount of β-CD (β-cyclodextrin), add all the aromatic water collected by extraction, stir evenly, carry out inclusion in a colloid mill, and collect the aromatic water inclusion liquid;

[0024] Take qualified extract and aromatic water inclusion liquid, place them in a mixing tank, stir them evenly in a water bath, control the relative density to be 1.07-1.10 (60°C), filter through a 200-mesh sieve while hot, and spray dry.

[0025] Preferably, the mass ratio of the astragalus, white peony root, cinnamon twig, jujube and ginger is 2.07:2.07:2.07:1.80:4.14, and the astragalus, white peony root, cinnamon twig, jujube and ginger are all raw medicinal slices.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Granules has strong specificity, and the stability, repeatability, intermediate precision, accuracy, etc. all meet the requirements, and the durability of the chromatographic conditions is good, so the liquid chromatography conditions provided by the present invention can be used for the content determination of paeoniflorin in the classic Huangqi Guizhi Wuwu Decoction. For different objects, the mobile phase used for gradient elution is different, the proportion is different, and the gradient elution time control is different, so that the content of paeoniflorin in the granules can be accurately obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the chromatogram of the specificity test.

[0029] Figure 2 This is the peak purity chart of paeoniflorin reference substance.

[0030] Figure 3 This is the peak purity diagram of the test sample paeoniflorin.

[0031] Figure 4 This is the standard curve of paeoniflorin.

[0032] Figure 5 Chromatograms for the durability-different flow rate test.

[0033] Figure 6 This is the chromatogram of the durability test at different column temperatures.

[0034] Figure 7 Chromatograms for the robustness test with different mobile phase ratios.

[0035] Figure 8 For robustness - different column chromatograms.

[0036] Fig. 9 For durability - different acidity chromatograms. DETAILED DESCRIPTION

[0037] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. 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 creative work are within the scope of protection of the present invention.

[0038] Instruments and reagents

[0039] Liquid chromatograph: Thermo Ultimate 3000 DAD; Agilent 1100 Series DAD.

[0040] Chromatographic column: Cosmosil 5C18-MS-II 4.6*250mm 5um.

[0041] Thermo Hypersil GOLD 4.6*250mm 5um.

[0042] Dikma Diamonsil C18(2) 4.6*250mm 5um.

[0043] Electronic analytical balance: Mettler-Toledo Instruments (Shanghai) Co., Ltd. XS105DU (one hundred thousandth)

[0044] Mettler-Toledo Instruments (Shanghai) Co., Ltd. TLE104 / 02 (1 in 10,000).

[0045] Mettler-Toledo Instruments (Shanghai) Co., Ltd. TLE303E / 02 (one thousandth).

[0046] Ultrasonic cleaner: KQ600DA from Kunshan Ultrasonic Instrument Co., Ltd.

[0047] Acetonitrile was chromatographically pure, CNW; phosphoric acid was analytically pure, Guangzhou Chemical Reagent Factory; ethanol was analytically pure, Guangzhou Public School Reagent Factory; water was Watsons drinking water, Guangzhou Watsons Food and Beverage Co., Ltd.

[0048] Paeoniflorin reference substance (batch number: 110736-202145, purity: 94.6%) was purchased from China Food and Drug Inspection Institutes.

[0049] The present invention provides a method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules, comprising the following steps:

[0050] S1. Sample pretreatment: Take an HP-20 macroporous adsorption resin column, first add 3 column volumes of 95% ethanol, control the flow rate at 1.0 mL / min, and then add 3 column volumes of water for activation; accurately weigh 0.18 g-0.22 g of Huangqi Guizhi Wuwu Decoction granules, place in a 100 mL stoppered conical flask, accurately add 50 mL of solvent, stopper, weigh, ultrasonically treat for 30-90 min, cool to 25°C-35°C, weigh again, make up the lost weight with water, shake well, and obtain the loading solution;

[0051] The loading solution was loaded onto the column at a flow rate of 1.0 mL / min, and the following elutions were performed in sequence: Step 1: Accurately add 3 column volumes of water for elution, and discard the effluent; Step 2: Accurately add 2 column volumes of 30% ethanol for elution, collect the eluate, and mark it as fraction 1; Step 3: Accurately add 2 column volumes of 50% ethanol for elution, collect the eluate, and mark it as fraction 2; Step 4: Accurately add 2 column volumes of 70% ethanol for elution, collect the eluate, and mark it as fraction 3;

[0052] Take fraction 2 and fraction 3, combine them, place them in an evaporator to recover the solvent under reduced pressure, control the temperature at 45℃±2℃, transfer the recovered concentrated solution to a 25mL volumetric flask, dilute to the mark with 50% ethanol, shake well, and obtain the pretreatment solution;

[0053] S2. Selection of chromatographic conditions:

[0054] Chromatographic operating conditions and parameters: Chromatographic column: C18 chromatographic column, 4.6*250mm, 5um, mobile phase A: acetonitrile; mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution, flow rate 1.0mL / min; injection volume 10μL; column temperature 30℃, injection chamber temperature 25℃, diode array detector, scanning range: 200-400nm, detection wavelength 230nm, liquid chromatography with a volume ratio of mobile phase A to mobile phase B of 14%:86%.

[0055] Octadecylsilane bonded silica gel was used as filler; acetonitrile-0.1% phosphoric acid solution volume ratio 14:86 was used as mobile phase; the detection wavelength was 230nm. The theoretical plate number calculated based on the paeoniflorin peak should be no less than 5000;

[0056] S3. Preparation of reference solution: Take the paeoniflorin reference substance, weigh it accurately, and add methanol to make a solution containing 80 μg per 1 mL;

[0057] S4. Preparation of the test solution: Take 10 mL of the pretreatment solution, place it in a 25 mL volumetric flask, add methanol to the mark, shake well, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0058] S5. Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, collect multi-wavelength signals, and separate and quantitatively analyze the chromatographic peaks using multi-wavelength signal deconvolution algorithm.

[0059] In this embodiment, the high performance liquid chromatography method refers to General Rules 0512 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia.

[0060] In this embodiment, the following method is used to separate and quantitatively analyze the chromatographic peaks by a multi-wavelength signal deconvolution algorithm: a wavelength scan is performed on the chromatogram at a sampling interval of 0.001 min by a diode array detector, a signal data matrix in the range of 200-400 nm is collected, and signal data of main wavelengths of 230 nm, 280 nm, and 290 nm are separated and extracted from the data matrix to obtain a three-dimensional original data sequence; a 9-point sliding average calculation is performed on the signal data of the main wavelength of 230 nm in the three-dimensional original data sequence, and the signal intensities of 4 points before and after each data point are weighted averaged to obtain a main peak baseline characteristic value sequence; a three-dimensional peak shape characteristic model is established by correlating the main peak baseline characteristic value sequence with the auxiliary wavelength data of 280 nm and 290 nm, and the co-elution region signal is deconvoluted and separated to obtain the peak area data of each component; according to the peak area data of each component, the content of each component is calculated according to the external standard method, with the reference solution as the external standard, to obtain the content determination result.

[0061] In this embodiment, the main peak baseline characteristic value sequence is associated with the auxiliary wavelength data of 280nm and 290nm to establish a three-dimensional peak shape characteristic model, and the co-elution region signal is deconvoluted and separated to obtain the peak area data of each component, including: aligning the main peak baseline characteristic value sequence with the auxiliary wavelength data of 280nm and 290nm on the time axis, establishing a sampling point pairing relationship at intervals of 0.001min, and obtaining a three-dimensional signal pairing sequence; calculating the response ratio of the three wavelengths at each sampling point according to the three-dimensional signal pairing sequence, and establishing a signal intensity ratio matrix for each data point in the co-elution region. array to obtain a wavelength characteristic distribution diagram; according to the change law of the signal intensity ratio in the wavelength characteristic distribution diagram, the co-elution area is segmented into 0.05min time windows, and the signal distribution characteristics in each window are calculated to obtain the target component response interval; according to the signal distribution in the target component response interval, a three-dimensional peak shape mathematical model is established, and the contours of the overlapping chromatographic peaks are fitted to obtain the peak shape fitting parameters of each component; the signal of the co-elution area is mathematically deconvoluted by the peak shape fitting parameters, and the overlapping peaks are quantitatively integrated according to the maximum absorption wavelength of each component to obtain the peak area data of each component.

[0062] In this embodiment, in the sample pretreatment step, the solvent is one of ethanol, methanol, acetonitrile and water.

[0063] In a preferred embodiment of the present invention, in the sample pretreatment step, the mass concentration of ethanol is 49.5%-50.5%, preferably 18%.

[0064] In a preferred embodiment of the present invention, in the sample pretreatment step, 0.18 g to 0.22 g, preferably 0.20 g, of Astragalus and Cinnamon Twig Five Ingredients Granules are taken.

[0065] In this embodiment, in the sample pretreatment step, ultrasonic treatment is replaced by heating reflux.

[0066] In a preferred embodiment of the present invention, in the sample pretreatment step, the ultrasonic treatment time is 28.5 minutes to 31.5 minutes, preferably 30 minutes.

[0067] In a preferred embodiment of the present invention, in step S4, 10 mL of the pretreatment solution is taken and placed in a 25 mL volumetric flask, methanol is added to the mark, shaken well, filtered through a 0.45 μm filter membrane, and the filtrate is taken to obtain;

[0068] In this embodiment, the Huangqi Guizhi Wuwu Granules were prepared in the following manner:

[0069] Take astragalus, white peony root, cinnamon twig, ginger and jujube and boil them together with water, collect the aromatic water, combine the filtrate, and concentrate under reduced pressure to prepare the Huangqi Guizhi Wuwu Decoction extract;

[0070] Take an appropriate amount of β-CD and add it to all the aromatic water collected by extraction, stir evenly, carry out inclusion in a colloid mill, and collect the aromatic water inclusion liquid; the specific inclusion parameters can be given in the embodiment

[0071] Take qualified extract and aromatic water inclusion liquid, place them in a mixing tank, stir them evenly in a water bath, control the relative density to be 1.07-1.10 (60°C), filter through a 200-mesh sieve while hot, and spray dry.

[0072] In a preferred embodiment of the present invention, the mass ratio of the astragalus, white peony root, cinnamon twig, jujube and ginger is 2.07:2.07:2.07:1.80:4.14, and the astragalus, white peony root, cinnamon twig, jujube and ginger are all raw medicinal slices.

[0073] In a preferred embodiment of the present invention, the specific method for preparing the Huangqi Guizhi Wuwu Decoction extract is as follows: taking Huangqi, white peony root, cinnamon twig, jujube, and ginger in a mass ratio of 2.07:2.07:2.07:1.80:4.14, adding five times the amount of water, boiling and extracting for 50 minutes, collecting the filtrate, filtering it while hot through a 200-mesh gauze, and collecting all the aromatic water. The filtrate is concentrated under reduced pressure at 50-60°C to obtain an extract with a relative density of 1.07-1.10 (60°C).

[0074] In a preferred embodiment of the present invention, the added amount of β-CD should be 6% of the total amount of the decoction pieces.

[0075] In a preferred embodiment of the present invention, the inclusion time should be 20 minutes.

[0076] In a preferred embodiment of the present invention, the qualified extract and aromatic water inclusion liquid are placed in a batching tank, and the water bath temperature should be 60-80°C.

[0077] In a preferred embodiment of the present invention, the qualified extract and the aromatic water inclusion liquid are placed in a mixing tank and stirred in a 60-80° C. water bath for at least 30 minutes.

[0078] In a preferred embodiment of the present invention, the air inlet temperature is controlled at 160° C., the peristaltic pump flow rate is 6-8 rpm / min, and the rotation speed is 2400 rpm during spray drying.

[0079] Embodiment 1: The present invention provides a method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules, comprising the following steps:

[0080] S1. Sample pretreatment: Take an HP-20 macroporous adsorption resin column, first add 3 column volumes of 95% ethanol, control the flow rate at 1.0 mL / min, and then add 3 column volumes of water for activation; accurately weigh 0.18 g-0.22 g of Huangqi Guizhi Wuwu Decoction granules, place in a 100 mL stoppered conical flask, accurately add 50 mL of solvent, stopper, weigh, ultrasonically treat for 30-90 min, cool to 25°C-35°C, weigh again, make up the lost weight with water, shake well, and obtain the loading solution;

[0081] The loading solution was loaded onto the column at a flow rate of 1.0 mL / min, and the following elutions were performed in sequence: Step 1: Accurately add 3 column volumes of water for elution, and discard the effluent; Step 2: Accurately add 2 column volumes of 30% ethanol for elution, collect the eluate, and mark it as fraction 1; Step 3: Accurately add 2 column volumes of 50% ethanol for elution, collect the eluate, and mark it as fraction 2; Step 4: Accurately add 2 column volumes of 70% ethanol for elution, collect the eluate, and mark it as fraction 3;

[0082] Take fraction 2 and fraction 3, combine them, place them in an evaporator to recover the solvent under reduced pressure, control the temperature at 45℃±2℃, transfer the recovered concentrated solution to a 25mL volumetric flask, dilute to the mark with 50% ethanol, shake well, and obtain the pretreatment solution;

[0083] S2. Selection of chromatographic conditions: octadecylsilane bonded silica gel as filler; acetonitrile-0.1% phosphoric acid solution (14:86) as mobile phase; detection wavelength at 230nm. The theoretical plate number calculated based on the paeoniflorin peak should be no less than 5000;

[0084] S3. Preparation of reference solution: Take the paeoniflorin reference substance, weigh it accurately, and add methanol to make a solution containing 80 μg per 1 mL;

[0085] S4. Preparation of the test solution: Take 10 mL of the pretreatment solution, place it in a 25 mL volumetric flask, add methanol to the mark, shake well, filter through a 0.45 μm filter membrane, and take the filtrate to obtain;

[0086] S5. Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0087] In this embodiment, in the sample pretreatment step, the mass concentration of ethanol is 49.5%-50.5%.

[0088] Example 2: The difference between Example 2 and Example 1 is that in the sample pretreatment step of this example, the solvent is methanol, and the rest are the same.

[0089] Example 3: The difference between Example 3 and Example 1 is that in the sample pretreatment step of this example, the solvent is acetonitrile, and the rest are the same.

[0090] Example 4: The difference between Example 4 and Example 1 is that in the sample pretreatment step of this example, the solvent is water, and the rest are the same.

[0091] The measurement results of Examples 1-4 are shown in Table 1:

[0092] Table 1

[0093]

[0094] Example 5: The difference between Example 4 and Example 1 is that in the sample pretreatment step of this example, 0.1 g of Astragalus and Cinnamon Twig Five Ingredients Granules is taken, and the rest are the same.

[0095] The results of the measurements of Example 1 and Example 4 are shown in Table 2:

[0096] Table 2

[0097]

[0098] Example 6: The difference between Example 5 and Example 1 is that in this example, in the sample pretreatment step, ultrasonic treatment for 28.5 minutes to 31.5 minutes (preferably 30 minutes) is replaced by heating under reflux for 28.5 minutes to 31.5 minutes (preferably 30 minutes), and the rest are the same.

[0099] The results of the measurements of Example 1 and Example 5 are shown in Table 3:

[0100] Table 3

[0101]

[0102] Example 7: The difference between Example 5 and Example 1 is that in the sample pretreatment step of this example, the ultrasonic treatment is performed for 60 minutes, and the rest is the same.

[0103] Example 8: The difference between Example 7 and Example 1 is that in the sample pretreatment step of this example, ultrasonic treatment is performed for 90 minutes, and the rest are the same.

[0104] The measurement results of Example 1 and Examples 6-7 are shown in Table 4:

[0105] Table 4

[0106]

[0107] In summary: Example 1 of the present invention is a preferred embodiment, and the following test is performed on it according to the determination method.

[0108] Specificity test:

[0109] Paeoniflorin is an index component of the classic Huangqi Guizhi Wuwu Decoction. According to the treatment method of the test sample, prepare the negative sample solution lacking white peony root and inject the blank solvent, and record the chromatogram. Figure 1 . Figure 1 In the above table, S1: ethanol, S2: white peony negative, S3: paeoniflorin, S4: material standard.

[0110] Result analysis: There is no chromatographic peak at the retention time corresponding to paeoniflorin in the negative sample lacking white peony root and the blank solvent chromatogram, and there is no interference, indicating that the method of the present invention is specific for determining the content of paeoniflorin.

[0111] Peak purity check:

[0112] Take the above test solution, inject it into the liquid chromatograph, use the above chromatographic conditions to perform full wavelength detection with the DAD detector, and calculate the peak purity. Figure 2-3 .

[0113] Result analysis: No impurity peaks were detected in the paeoniflorin peak in the sample chromatogram, indicating that under this chromatographic condition, the purity of the paeoniflorin chromatographic peak met the requirements.

[0114] Investigation of linear relationship:

[0115] Accurately weigh 10.79 mg of paeoniflorin reference substance, place it in a 25 mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain a reference substance solution mother solution with a concentration of 408.2936 μg / mL. Accurately measure 1 mL respectively, place it in 2, 5, 10, and 20 mL volumetric flasks, add methanol to dilute to the scale, shake well, and obtain reference substance solutions with concentrations of 204.1468, 81.6587, 40.8294, and 20.4147 ug / mL, respectively. Accurately pipette 10 μL of each of the above reference substance solutions, inject it into the liquid chromatograph, and determine it according to the liquid chromatography method (General Rules 0512 of Part Four of the 2020 Edition of the Chinese Pharmacopoeia). Draw a standard curve with concentration (μg / mL) as the horizontal axis and peak area as the vertical axis. See Figure 4 , Table 5.

[0116] Table 5-Linear relationship table of paeoniflorin

[0117]

[0118] Regression equation: y=0.1561x+0.1068

[0119] Correlation coefficient: r=1.0000

[0120] Result analysis: The concentration of paeoniflorin had good linearity in the range of 20.41-408.29 μg / mL.

[0121] Precision test:

[0122] a Instrument precision test

[0123] Take the paeoniflorin reference solution and inject it continuously 6 times according to the determined determination method, as shown in Table 6 for details.

[0124] Table 6 - Precision test results (n=6)

[0125]

[0126] Result analysis: After 6 consecutive injections, the RSD of the peak area of ​​peony inoside reference substance was less than 1.5%, indicating that the instrument had good precision.

[0127] b Repeatability test:

[0128] The experimenter (A) takes about 0.2g (6 portions) of this product (batch number J230504), weighs it accurately, and operates according to the determined test solution preparation and determination method. See Table 6 for details.

[0129] Table 6 - Repeatability test results (n=6)

[0130]

[0131] Result analysis: After 6 consecutive injections, the RSD of the paeoniflorin content in the benchmark sample was less than 1.5%, indicating that the repeatability of this method was good.

[0132] cIntermediate precision test

[0133] Select different measurement time, different HPLC, different experimenters (B), take this product (batch number: J230504), take an appropriate amount, take about 0.2g, weigh accurately, and operate according to the determined test solution preparation and measurement items. The results are shown in Tables 7-9.

[0134] Table 7- Results of intermediate precision test of peony root glucoside (different personnel)

[0135]

[0136] Result analysis: The average content of paeoniflorin in the samples determined by different experimenters was 19.22 mg / g, with an RSD of less than 1.5%. The intermediate precision of this method was good.

[0137] Table 8- Results of intermediate precision test of peony root glucoside (different instruments)

[0138]

[0139] Result analysis: The average content of paeoniflorin in samples determined by different instruments was 19.37 mg / g, and the RSD was less than 1.5%. The intermediate precision of this method was good.

[0140] Table 9- Results of intermediate precision test of peony root glucoside (different dates)

[0141]

[0142] Result analysis: The average content of paeoniflorin in samples measured on different dates was 19.16 mg / g, with an RSD of less than 1.5%. The intermediate precision of this method was good.

[0143] In summary, under the conditions of precision (instrument precision, repeatability, and intermediate precision), the RSD of the paeoniflorin content in the samples was less than 1.5%, indicating that the method had good precision.

[0144] Stability test:

[0145] Take this product (batch number: J230504), mix well, take about 0.2g, weigh accurately, and operate according to the preparation and determination method of the test solution. After 0, 2, 4, 10, 14, and 24 hours of preparation, sample and measure respectively. See Table 10 for details.

[0146] Table 10 - Stability test results

[0147]

[0148] Result analysis: The RSD of the peak area of ​​peony inoside was less than 1.5%, and the test solution had good stability within 24 hours.

[0149] Accuracy test:

[0150] Using the sample recovery method, accurately weigh 20.27 mg of paeoniflorin reference substance (batch number: 110736-202145, purity: 94.6%) and place it in a 500 mL volumetric flask. Add an appropriate amount of ethanol to dissolve and dilute to the scale. Shake well to obtain a reference substance solution with a concentration of 38.3508 μg / mL.

[0151] Take about 0.1g of this product (batch number: J230504), accurately weigh 6 portions, put them in a 50mL stoppered conical bottle, accurately add 50mL (6 portions) of the above-mentioned peony inoside reference solution, seal and weigh, ultrasonically treat (power 600W, frequency 40kHz) for 28.5 minutes to 31.5 minutes, cool to 25℃ to 35℃, and then use ethanol to make up the lost weight, shake well, filter, accurately take 10μL of the filtrate, inject it into the liquid chromatograph, and determine it. See Table 11 for details.

[0152] The content of paeoniflorin in the corresponding physical object (dry paste powder) of Huangqi Guizhi Wuwu Decoction (batch number: J230504) is 19.19 mg / g.

[0153] Table 11 - Accuracy test results (n=6)

[0154]

[0155] Result analysis: The average recovery rate of paeoniflorin was 97.1% and the RSD was less than 1.5%, indicating that the method had good accuracy.

[0156] Durability test:

[0157] The durability of this chromatographic condition was investigated with different flow rates, column temperatures, mobile phase ratios, columns, and acid concentrations. Figure 5-9 , Table 12.

[0158] Table 12 - Durability test results

[0159]

[0160] Figure 5 Medium, S1: 0.8mL / min, S2: 1.0mL / min, S3: 1.2mL / min.

[0161] Figure 6 Medium, S1: 25℃, S2: 30℃, S3: 35℃.

[0162] Figure 7 In, S1: 12:88, S2: 14:86, S3: 16:84.

[0163] Figure 8 Medium, S1: Thermo Hypersil GOLD 4.6*250mm 5um, S2: Cosmosil 5C18-MS-II4.6*250mm 5um, S3: Dikma Diamonsil C18(2) 4.6*250mm 5um.

[0164] Fig. 9 In the assay, S1: acetonitrile-0.05% phosphoric acid; S2: acetonitrile-0.1% phosphoric acid; S3: acetonitrile-0.15% phosphoric acid.

[0165] Result analysis: The results of the method of the present invention under different flow rates, different column temperatures, different mobile phase ratios, different acidities, and different chromatographic column conditions are basically consistent, indicating that the method has good durability.

[0166] In summary, the method for determining the content of paeoniflorin in the classic Huangqi Guizhi Wuwu Decoction has strong specificity, and its stability, repeatability, intermediate precision, accuracy, etc. all meet the requirements, and the durability of the chromatographic conditions is good. Therefore, this liquid chromatography condition can be used for the determination of the content of paeoniflorin in the classic Huangqi Guizhi Wuwu Decoction.

[0167] The above research results show that the method for determining the content of paeoniflorin in the classic prescription of Shaoyao Gancao Decoction has strong specificity, and its stability, repeatability, intermediate precision, accuracy, etc. all meet the requirements, and the durability of the chromatographic conditions is good. Therefore, this liquid chromatography condition can be used for the determination of the content of paeoniflorin in the classic prescription of Shaoyao Gancao Decoction.

[0168] Example 9: Separation and quantitative analysis of chromatographic peaks by multi-wavelength signal deconvolution algorithm is achieved in the following manner:

[0169] (1) The wavelength of the chromatogram is scanned by a diode array detector at a sampling interval of 0.001 min to collect a signal data matrix in the range of 200-400 nm. The signal data of the main wavelengths of 230 nm, 280 nm, and 290 nm are separated and extracted from the data matrix to obtain a three-dimensional raw data sequence;

[0170] (2) A 9-point sliding average calculation is performed based on the signal data with a main wavelength of 230 nm in the three-dimensional original data sequence, and the signal intensity of the four points before and after each data point is weighted averaged to obtain the main peak baseline characteristic value sequence;

[0171] (3) A three-dimensional peak shape characteristic model was established by correlating the main peak baseline characteristic value sequence with the auxiliary wavelength data of 280 nm and 290 nm, and the co-elution region signal was deconvoluted and separated to obtain the peak area data of each component;

[0172] (4) According to the peak area data of each component, the content of each component is calculated according to the external standard method, using the reference solution as the external standard to obtain the content determination result.

[0173] Specifically, the signal is collected by a diode array detector, which is a photoelectric conversion device composed of a plurality of photodiodes arranged and capable of simultaneously collecting spectral signals of multiple wavelengths. When scanning, the sampling interval is set to 0.001 minutes, and this high-frequency sampling can ensure the accurate description of the chromatographic peak shape. In the ultraviolet spectrum range of 200-400nm, peony glycosides and related compounds have characteristic absorption. By collecting the full spectrum of signals in this range, a data matrix containing three dimensions of wavelength, time and absorbance is formed. From the data matrix, the signal data of the three characteristic wavelengths of 230nm, 280nm and 290nm are extracted. 230nm is the maximum absorption wavelength of peony glycosides, and the selection of this wavelength as the main wavelength for quantitative analysis has a high sensitivity; and 280nm and 290nm as auxiliary wavelengths can provide spectral characteristic information of the target compound, which is helpful for the accurate identification and quantification of the target peak in complex samples. The signal data of these three wavelengths constitute a three-dimensional raw data sequence, laying the foundation for subsequent data processing.

[0174] In order to improve the data quality, the signal data of the main wavelength of 230nm was processed by a 9-point sliding average algorithm. The sliding average algorithm is a commonly used data smoothing method that reduces the impact of random noise by weighted averaging continuous data points in the time series. In the specific operation, for each data point, the 4 points before and after it are taken for weighted calculation, and the weight coefficient is in the form of symmetrical distribution, so that the data point closer to the center point has a greater weight. The main peak baseline characteristic value sequence obtained after this treatment has a better peak shape profile, which is conducive to subsequent quantitative analysis. After obtaining the main peak baseline characteristic value sequence, it is associated with the data of the two auxiliary wavelengths of 280nm and 290nm. This step first requires ensuring the precise alignment of the three wavelength data on the time axis, and establishing a pairing relationship between the sampling points at intervals of 0.001 minutes. For each data point in the co-elution area, the response ratio of the three wavelengths is calculated to construct a signal intensity ratio matrix. This matrix reflects the relative response characteristics of different compounds at each wavelength, providing an important basis for peak deconvolution. Subsequently, the co-elution area was segmented into 0.05-minute time windows to analyze the distribution characteristics of the signal in each window. The response range of the target component was determined by observing the change pattern of the signal intensity ratio. Based on this information, a three-dimensional peak shape feature model was established, which comprehensively considered the information of the three dimensions of time, wavelength and signal intensity, and could more accurately describe the contour characteristics of overlapping peaks.

[0175] In the actual sample analysis process, when multiple components are detected to co-elute on the chromatogram, the established three-dimensional peak shape characteristic model is used to perform mathematical deconvolution calculations on the overlapping chromatographic peaks. During the deconvolution process, the overlapping peaks are decomposed into the chromatographic peaks of each individual component according to the response characteristics of each component at different wavelengths, and quantitative integration is performed according to the maximum absorption wavelength of each component to obtain the peak area data of each component. Finally, the external standard method is used for quantitative analysis. The prepared peony glycoside reference solution is used as the external standard. The standard curve is established according to the concentration and peak area of ​​the reference. The actual content of peony glycoside in the sample is calculated by comparing the peak area of ​​the target in the sample solution with the standard curve. In actual operation, when the peony glycoside in Huangqi Guizhi Wuwu Decoction Granules is analyzed by high performance liquid chromatography, the collected raw data is subjected to multi-wavelength signal deconvolution processing, which significantly improves the separation and quantitative accuracy. By performing multi-wavelength data acquisition on the co-elution area, a data matrix containing three characteristic wavelengths is established, and the data is subjected to sliding average processing to eliminate baseline noise, and then the overlapping peaks are deconvoluted and separated using the three-dimensional peak shape characteristic model. During the deconvolution process, the target compound paeoniflorin was accurately identified and quantified by analyzing the response ratio characteristics at different wavelengths. The entire data processing process made full use of multidimensional spectral information to achieve accurate determination of the content of paeoniflorin in complex Chinese medicine preparations. Through this method, even when the sample components are complex and the chromatographic peaks overlap, the accurate quantification of paeoniflorin can be achieved, providing a reliable analytical method for the quality control of Chinese medicine preparations. For example: the spectral data in the range of 200-400nm was collected by a diode array detector. A complex overlapping peak region was found on the chromatogram, and a three-dimensional data matrix was constructed by extracting data at three characteristic wavelengths of 230nm, 280nm and 290nm. The data at the main wavelength of 230nm was processed by a 9-point sliding average, which significantly reduced the baseline noise. Subsequently, the retention time range of the target peak was identified by analyzing the change law of the response ratio of the three wavelength data. Using the established three-dimensional peak shape feature model, the overlapping peaks were successfully parsed into individual components, and the content of paeoniflorin in the sample was accurately quantified by comparing the response characteristics with those of the paeoniflorin reference substance.

[0176] Example 10: A three-dimensional peak shape characteristic model is established by correlating the main peak baseline characteristic value sequence with the auxiliary wavelength data of 280 nm and 290 nm, and the co-elution region signal is deconvoluted and separated to obtain the peak area data of each component, including:

[0177] (1) The time axis of the main peak baseline characteristic value sequence was aligned with the auxiliary wavelength data of 280 nm and 290 nm, and a sampling point pairing relationship was established at intervals of 0.001 min to obtain a three-dimensional signal pairing sequence;

[0178] (2) Calculate the response ratio of the three wavelengths at each sampling point based on the three-dimensional signal pairing sequence, establish a signal intensity ratio matrix for each data point in the co-flow area, and obtain a wavelength characteristic distribution map;

[0179] (3) Based on the change law of the signal intensity ratio in the wavelength characteristic distribution diagram, the co-elution area is segmented into 0.05 min time windows, the signal distribution characteristics in each window are calculated, and the target component response interval is obtained;

[0180] (4) Based on the signal distribution of the target component response range, a three-dimensional peak shape mathematical model is established, and the profiles of the overlapping chromatographic peaks are fitted to obtain the peak shape fitting parameters of each component;

[0181] (5) The signal in the co-elution region is mathematically deconvoluted using the peak shape fitting parameters, and the overlapping peaks are quantitatively integrated according to the maximum absorption wavelength of each component to obtain the peak area data of each component.

[0182] Specifically, after collecting the baseline characteristic value sequence of the main wavelength 230nm and the data of the auxiliary wavelengths 280nm and 290nm through the diode array detector, the time axis alignment of the data is first required. Time axis alignment refers to the precise matching of the data points collected at different wavelengths in the time dimension, using 0.001 minutes as the sampling interval to ensure the precise correspondence between the data points. In the process of time axis alignment, a strict one-to-one correspondence is established for the three wavelength data at each time point to form a three-dimensional signal pairing sequence. Based on the established three-dimensional signal pairing sequence, the response ratio of the three wavelengths is calculated for each sampling point. The response ratio refers to the proportional relationship between the detection signal intensity at different wavelengths at the same time point. Two sets of ratio data are obtained by calculation: the ratio of 280nm to 230nm, and the ratio of 290nm to 230nm. These ratio data constitute a signal intensity ratio matrix, which reflects the spectral characteristics of different compounds at each wavelength. By plotting the change curves of these ratios over time, a wavelength characteristic distribution diagram is obtained, which intuitively shows the spectral characteristic differences of each component in the co-elution area. The wavelength characteristic distribution diagram provides important compound identification information. Based on the change law of the signal intensity ratio, the co-elution area is segmented into 0.05-minute time windows. The selection of the time window takes into account the broadening effect of the chromatographic peak and the data acquisition frequency. The 0.05-minute window width can ensure sufficient data points for feature extraction without causing excessive smoothing. In each time window, the signal distribution characteristics of the three wavelengths are calculated, including the mean, standard deviation and ratio change trend of the signal intensity. By analyzing these characteristic parameters, the response range of the target component, that is, the precise retention time range of paeoniflorin and its related compounds on the chromatogram, is determined.

[0183] After obtaining the response interval of the target component, a three-dimensional peak shape mathematical model is established according to the signal distribution characteristics in each interval. The model uses time, wavelength and signal intensity as three dimensions to describe the profile characteristics of overlapping chromatographic peaks through mathematical functions. The model is established using the nonlinear least squares method to perform curve fitting on the measured data points to obtain the peak shape fitting parameters of each component, including characteristic parameters such as peak position, peak height, and peak width.

[0184] Finally, the obtained peak shape fitting parameters were used to perform mathematical deconvolution calculations on the signals in the co-elution area. During the deconvolution process, the overlapping chromatographic peaks were decomposed into independent single-component peaks based on the spectral characteristics of each component at different wavelengths. The peak area of ​​each component peak separated was integrated at its maximum absorption wavelength. Among them, paeoniflorin was quantitatively integrated at 230nm, and the content of paeoniflorin in the sample was finally obtained by comparing the peak area with that of the reference substance.

[0185] For example, chromatogram data from 0 to 30 minutes are collected. When the time axis is aligned, the data points of the three wavelengths are paired at intervals of 0.001 minutes, forming a three-dimensional paired sequence containing 30,000 data points. When calculating the response ratio, it was found that a complex overlapping peak region appeared near the retention time of 12.5 minutes. The signal intensity ratio in this region was observed to change significantly through the wavelength characteristic distribution diagram, indicating that there are multiple co-eluting components. This region was segmented and analyzed according to the time window of 0.05 minutes, and a total of 20 time windows of data were obtained. In each time window, the signal distribution characteristics of the three wavelengths were analyzed, and the response interval of paeoniflorin was accurately determined to be 12.3-12.7 minutes in combination with the spectral characteristics of the paeoniflorin standard. Subsequently, a three-dimensional peak shape model was established, and the peak shape parameters of each component were obtained by fitting calculation, among which the paeoniflorin peak had good symmetry and the width was consistent with the characteristics of the theoretical chromatographic peak. Finally, through deconvolution calculation, the overlapping peaks were successfully separated into independent component peaks, and the paeoniflorin peak was accurately quantitatively integrated at a wavelength of 230nm.

[0186] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0187] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules, characterized in that: The following steps are involved: S1. Sample pretreatment: Take an HP-20 macroporous adsorption resin column, first add 3 column volumes of 95% ethanol, control the flow rate at 1.0 mL / min, and then add 3 column volumes of water for activation; accurately weigh 0.18 g-0.22 g of Huangqi Guizhi Wuwu Decoction granules, place in a 100 mL stoppered conical flask, accurately add 50 mL of solvent, stopper, weigh, ultrasonically treat for 30-90 min, cool to 25°C-35°C, weigh again, make up the lost weight with water, shake well, and obtain the loading solution; The loading solution was loaded onto the column at a flow rate of 1.0 mL / min and eluted in the following order: Step 1: Accurately add 3 column volumes of water for elution and discard the effluent; Step 2: Accurately add 2 times column volume of 30% ethanol for elution, collect the eluate and mark it as fraction 1; Step 3: Accurately add 2 times column volume of 50% ethanol for elution, collect the eluate and mark it as fraction 2; Step 4: Accurately add 2 times column volume of 70% ethanol for elution, collect the eluate and mark it as fraction 3; Take fraction 2 and fraction 3, combine them, place them in an evaporator to recover the solvent under reduced pressure, control the temperature at 45℃±2℃, transfer the recovered concentrated solution to a 25mL volumetric flask, dilute to the mark with 50% ethanol, shake well, and obtain the pretreatment solution; S2. Selection of chromatographic conditions: Chromatographic operating conditions and parameters: Chromatographic column: C18 chromatographic column, 4.6*250mm, 5um, mobile phase A: acetonitrile; mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution, flow rate 1.0mL / min; injection volume 10μL; column temperature 30℃, injection chamber temperature 25℃, diode array detector, scanning range: 200-400nm, detection wavelength 230nm, liquid chromatography with a volume ratio of mobile phase A to mobile phase B of 14%:86%; S3. Preparation of reference solution: Take the paeoniflorin reference substance, weigh it accurately, and add methanol to make a solution containing 80 μg per 1 mL; S4. Preparation of the test solution: Take 10 mL of the pretreatment solution, place it in a 25 mL volumetric flask, add methanol to the mark, shake well, filter through a 0.45 μm filter membrane, and take the filtrate to obtain; S5. Determination method: 10 μL of reference solution and test solution are respectively accurately aspirated, injected into liquid chromatograph, multi-wavelength signals are collected, and chromatographic peaks are separated and quantitatively analyzed by multi-wavelength signal deconvolution algorithm. The following method is used to separate and quantitatively analyze chromatographic peaks by multi-wavelength signal deconvolution algorithm: the wavelength of the chromatogram is scanned by diode array detector at a sampling interval of 0.001 min, and a signal data matrix within the range of 200-400 nm is collected. Signal data of main wavelengths 230 nm, 280 nm, and 290 nm are separated and extracted from the data matrix to obtain three-dimensional original data sequence; performing a 9-point sliding average calculation on the signal data of the main wavelength 230nm in the three-dimensional original data sequence, performing a weighted average on the signal intensities of the 4 points before and after each data point, and obtaining a main peak baseline characteristic value sequence; performing an association operation on the main peak baseline characteristic value sequence and the auxiliary wavelength data of 280nm and 290nm, establishing a three-dimensional peak shape characteristic model, performing deconvolution separation on the co-elution region signal, and obtaining the peak area data of each component; calculating the content of each component according to the peak area data of each component according to the external standard method, using the reference solution as the external standard, and obtaining the content determination result; The method of performing an association operation between the main peak baseline characteristic value sequence and the auxiliary wavelength data of 280nm and 290nm, establishing a three-dimensional peak shape characteristic model, and performing deconvolution separation on the co-elution region signal to obtain the peak area data of each component includes: performing time axis alignment between the main peak baseline characteristic value sequence and the auxiliary wavelength data of 280nm and 290nm, establishing a sampling point pairing relationship at intervals of 0.001min, and obtaining a three-dimensional signal pairing sequence; calculating the response ratio of the three wavelengths at each sampling point according to the three-dimensional signal pairing sequence, and establishing a signal intensity ratio matrix for each data point in the co-elution region to obtain a wavelength characteristic distribution diagram; According to the variation law of the signal intensity ratio in the wavelength characteristic distribution diagram, the co-elution area is segmented into 0.05min time windows, and the signal distribution characteristics in each window are calculated to obtain the target component response interval; according to the signal distribution in the target component response interval, a three-dimensional peak shape mathematical model is established, and the contours of the overlapping chromatographic peaks are fitted to obtain the peak shape fitting parameters of each component; the signal in the co-elution area is mathematically deconvoluted by the peak shape fitting parameters, and the overlapping peaks are quantitatively integrated according to the maximum absorption wavelength of each component to obtain the peak area data of each component.

2. The method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules according to claim 1, characterized in that: In the step of selecting chromatographic conditions: octadecylsilane bonded silica gel is used as filler; acetonitrile-0.1% phosphoric acid solution is used as mobile phase; the detection wavelength is 230nm, and the theoretical plate number calculated based on the paeoniflorin peak should be no less than 5000.

3. The method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules according to claim 1, characterized in that: In the sample pretreatment step, the solvent is one of ethanol, methanol, acetonitrile and water.

4. The method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules according to claim 3, characterized in that: In the sample pretreatment step, the mass concentration of ethanol is 49.5%-50.5%.

5. The method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules according to claim 1, characterized in that: In the sample preparation step, ultrasonic treatment was replaced by heating reflux.

6. The method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules according to claim 1, characterized in that: In the sample preparation step, the ultrasonic treatment time was 28.5 min-31.5 min.

7. The method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules according to claim 1, characterized in that: The Huangqi Guizhi Wuwu Granules were prepared in the following manner: Take astragalus, white peony root, cinnamon twig, ginger and jujube and boil them together with water, collect the aromatic water, combine the filtrate, and concentrate under reduced pressure to prepare the Huangqi Guizhi Wuwu Decoction extract; Take an appropriate amount of β-CD, add all the aromatic water collected by extraction, stir evenly, carry out inclusion in a colloid mill, and collect the aromatic water inclusion liquid; Take qualified extract and aromatic water inclusion liquid, put them in a mixing tank, stir them evenly in a water bath, control the relative density to be 1.07-1.10, filter them through a 200-mesh sieve while hot, and spray dry them.

8. The method for determining the content of paeoniflorin in Huangqi Guizhi Wuwu Decoction granules according to claim 7, characterized in that: The mass ratio of the astragalus, white peony root, cinnamon twig, jujube and ginger is 2.07:2.07:2.07:1.80:4.14, and the astragalus, white peony root, cinnamon twig, jujube and ginger are all raw medicinal pieces.

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