Content determination method of wolfberry-chrysanthemum-rehmannia concentrated pill

Abstract

The invention provides a method for determining the content of a wolfberry-chrysanthemum-rehmannia concentrated pill, which adopts a UPLC (Ultra Performance Liquid Chromatography) multi-wavelength segmented integral method, realizes simultaneous determination of eight components, namely 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4, 5-O-dicaffeoylquinic acid, benzoyl paeoniflorin and paeonol for the first time, breaks through the limitation that only 2-3 components are detected by traditional HPLC (High Performance Liquid Chromatography), and has a wide application prospect. And the detection efficiency is improved by more than 50%. By optimizing the extraction solvent and time, the extraction rate of each component is greater than or equal to 95%, and the recovery rate is 97-102%. A gradient elution procedure of acetonitrile-methanol-0. 3% phosphoric acid is adopted, and components with large polarity difference are effectively separated. The integral wavelength is dynamically switched based on 3D spectral data, so that insufficient sensitivity or interference of a single wavelength is avoided, and the quantitation limit of low-content components is increased by 10 times. The method solves the problem of single detection component in the prior art, comprehensively covers the key active components of the four main medicines in the formula, remarkably improves the detection efficiency and accuracy, and provides a scientific basis for quality control and evaluation of the wolfberry-chrysanthemum-rehmannia concentrated pill.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a method for determining the content of Qiju Dihuang concentrated pills. Background Art

[0002] Qiju Dihuang Concentrated Pills are a classic Chinese herbal compound preparation, composed of eight herbs: wolfberry, chrysanthemum, prepared rehmannia root, wine-soaked cornus fruit, peony bark, yam, poria, and oriental radix. They nourish the kidneys and liver, improve eyesight, and relieve cataracts. They are clinically used to treat symptoms such as vertigo, tinnitus, photophobia, and blurred vision caused by liver and kidney yin deficiency. This preparation has been included in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Volume 1. Its current quality standards primarily include identification of properties, qualitative identification by thin-layer chromatography (TLC), and determination of morroniside and loganin in wine-soaked cornus fruit and paeonol in peony bark.

[0003] However, the efficacy of Chinese herbal compound preparations is usually the result of the synergistic effect of multiple components. It is difficult to fully reflect the overall quality of a few index components. At present, the quality control of Qiju Dihuang Concentrated Pills has the following deficiencies: (1) The existing standards only measure 2-3 components and cannot effectively monitor the content of other important active ingredients (such as 5-hydroxymethylfurfural, chlorogenic acid, paeoniflorin, etc.); (2) Traditional high performance liquid chromatography (HPLC) has a long analysis time and low efficiency, which is difficult to meet the needs of rapid detection of large quantities of samples; (3) Some studies use liquid chromatography-mass spectrometry (LC-MS), which has high sensitivity but high instrument cost, which is not conducive to the popularization of conventional quality control laboratories; (4) There are no reports on the use of ultra-high performance liquid chromatography (UPLC) to simultaneously determine multiple components in Qiju Dihuang Concentrated Pills. UPLC has the advantages of high separation efficiency, fast analysis speed, and low solvent consumption, and is more suitable for the analysis of complex Chinese herbal medicine systems.

[0004] Therefore, it is urgent to establish a rapid, accurate and comprehensive analytical method to achieve the simultaneous determination of multiple active ingredients in Qiju Dihuang Concentrated Pills (concentrated pills), so as to evaluate its quality more scientifically and objectively and provide technical support for production process optimization and rational clinical drug use. Summary of the Invention

[0005] The present invention provides a method for determining the content of Qiju Dihuang concentrated pills, aiming to solve the technical problem that the existing technology has a single detection component and cannot fully reflect the quality of the medicine.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a method for determining the content of Qiju Dihuang concentrated pills, the method comprising simultaneously determining the contents of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol in the Qiju Dihuang concentrated pills using ultra-high performance liquid chromatography;

[0008] The chromatographic conditions of the ultra-high performance liquid chromatography method include: acetonitrile as mobile phase A, methanol as mobile phase B, 0.3% phosphoric acid solution as mobile phase C, and gradient elution conditions are:

[0009]

[0010] The chromatographic conditions also include: the detection wavelength is multi-wavelength segmented integration, and data processing is performed at 284 nm at 0.00 minute, 240 nm at 2.50 minute, 325 nm at 3.30 minute, 240 nm at 4.00 minute, 230 nm at 5.50 minute, 330 nm at 9.00 minute, 274 nm at 11.00 minute, and 230 nm at 11.90 minute; the chromatographic column is filled with octadecylsilane bonded silica gel;

[0011] The content determination method further comprises the preparation of the Qiju Dihuang concentrated pill solution: extracting the Qiju Dihuang concentrated pill with 70% methanol by heating under reflux and filtering, and obtaining the filtrate.

[0012] In a preferred embodiment, the chromatographic conditions further include a mobile phase flow rate of 0.2-0.4 mL / min and a column temperature of 30-40°C.

[0013] Based on the above scheme, a lower flow rate of 0.2-0.4mL / min can prolong the interaction time between the mobile phase and the stationary phase, improve the resolution of complex components, such as compounds with large polarity differences such as 5-hydroxymethylfurfural and chlorogenic acid, reduce peak tailing, ensure symmetrical peak shapes of key components such as paeonol and benzoylpeoniflorin, and complete the separation of eight components within 18 minutes, balancing efficiency and resolution. Controlling the column temperature at 30-40°C not only avoids peak broadening or excessive retention times at low temperatures (<30°C), but also prevents degradation of thermally unstable components (such as 5-hydroxymethylfurfural) or shortened column life at high temperatures (>40°C).

[0014] In a preferred embodiment, the content determination is performed by calculating the content of the component to be measured using an external standard method or a standard curve method.

[0015] Based on the above scheme, the external standard method can address the limitations of the single-test, multiple-evaluation method. Traditional Chinese medicine analysis often requires internal standards and relative correction factors. However, the eight components in Qiju Dihuang Concentrated Pills vary greatly in structure, making it difficult to unify the correction factors. The external standard method can circumvent this problem. Moreover, for components with large content differences, such as chlorogenic acid (0.01-0.40 mg / g) and paeonol (1.97-3.80 mg / g), the external standard method can establish a separate linear range for each component, preventing high-concentration components from masking low-concentration signals. The standard curve method can address the problem of large fluctuations in the content of traditional Chinese medicine ingredients. The content of 5-hydroxymethylfurfural in samples from different manufacturers varies by 22 times, ranging from 0.15 to 3.36 mg / g. The standard curve method can be flexibly adapted.

[0016] In a preferred embodiment, in the preparation of the Qiju Dihuang concentrated pill solution, the amount of 70% methanol added is 40-60 mL, and the heating reflux time is 0.5-2 hours.

[0017] Based on the above scheme, 70% methanol can dissolve both hydrophilic components, such as 5-hydroxymethylfurfural and paeoniflorin, and moderately polar components, such as chlorogenic acid and paeonol. Experimental results indicate that a 0.5-hour reflux period allows for the initial dissolution of the easily extractable component (5-hydroxymethylfurfural). A 1-hour reflux period also fully releases the less soluble component (4,5-O-dicaffeoylquinic acid). Reflux time exceeding 1 hour does not significantly increase the content of the compound. Therefore, controlling the reflux period to within 2 hours fully dissolves Qiju Dihuang Concentrated Pills while preventing degradation of heat-sensitive components, such as chlorogenic acid.

[0018] A preferred embodiment is that the preparation of the Qiju Dihuang concentrated pill solution comprises the following steps: taking 0.5-0.7 g of Qiju Dihuang concentrated pills, adding 40-60 mL of 70% methanol, sealing and weighing, heating and reflux for 0.5-1.5 hours, cooling, weighing again, making up the lost weight with 70% methanol, shaking and filtering, and taking the filtrate to obtain the Qiju Dihuang concentrated pill solution.

[0019] Based on this approach, by weighing the sample twice before and after heating, we compensated for methanol evaporation, ensuring a constant final solution volume and eliminating concentration errors caused by solvent loss. The subsequent filtrate was filtered, and the initial filtrate, which may contain particulate impurities, was discarded to improve the stability of the chromatography system.

[0020] Preferably, the heating reflux time is 1 hour.

[0021] In a preferred embodiment, the content determination method further comprises the preparation of a reference solution: dissolving 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin and paeonol reference substances in 70% methanol to prepare a mixed reference solution.

[0022] Based on the above scheme, the mixed reference solution provides a concentration reference for UPLC analysis. By comparing the peak areas of the test sample and the reference sample, the contents of 8 components are directly calculated, avoiding the tedious operation of multiple injections required by the traditional method. The one-time preparation includes representative components of Rehmannia glutinosa (5-HMF), Chrysanthemum (chlorogenic acid), Cornus officinalis (morroniside / loganin), and Paeonia suffruticosa (peoniflorin / paeonol, etc.), which solves the limitation of the current standard that only detects 2-3 types. In addition, both the reference sample and the test sample are dissolved in 70% methanol to eliminate the deviation in extraction efficiency caused by differences in solvent polarity.

[0023] In a preferred embodiment, the concentrations of the components in the mixed reference solution are: 5-hydroxymethylfurfural 4.69-234.53 μg / mL, morroniside 2.43-121.52 μg / mL, chlorogenic acid 1.12-56.09 μg / mL, loganin 2.81-140.33 μg / mL, paeoniflorin 2.55-127.64 μg / mL, 4,5-O-dicaffeoylquinic acid 1.17-58.60 μg / mL, benzoylpeoniflorin 2.22-110.75 μg / mL, and paeonol 4.93-246.25 μg / mL.

[0024] In a preferred embodiment, the chromatographic column is a Waters HSS T3 C18 chromatographic column with a size of 2.1×100 mm and a filler particle size of 1.8 μm.

[0025] Based on the above scheme, the Waters HSS T3 C18 chromatographic column is a small-particle filler. The 1.8μm particle size filler has a theoretical plate number that is more than three times higher than that of traditional HPLC columns (5μm). It can baseline separate components with similar polarity and can complete the separation of 8 components within 18 minutes at a flow rate of 0.3mL / min, which is more than 50% faster than traditional HPLC (30-60 minutes), meeting the high-throughput requirements of UPLC.

[0026] In a second aspect, the present invention provides an application of the content determination method as described in any one of the first aspects in the field of quality control of Qiju Dihuang Concentrated Pills.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention establishes for the first time a UPLC multi-wavelength segmented integration method for the simultaneous determination of eight components, breaking through the limitation of traditional HPLC that can only detect 2-3 components. It can complete the simultaneous quantification of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol in Qiju Dihuang Concentrated Pills within 18 minutes, improving the detection efficiency by more than 50%, and comprehensively covering the key active ingredients of the four main herbs in the prescription: Rehmannia root, Chrysanthemum flower, Cornus officinalis, and Paeonia suffruticosa root.

[0029] 2. The present invention optimizes the polarity of the extraction solvent by using 70% methanol dynamic reflux extraction and controls the extraction time to 0.5-2h, achieving an extraction rate of ≥95% for all eight components and a recovery rate of 97-102%. By replenishing the solvent by two weighings, the volatility error is eliminated, and the reproducibility RSD of data in different laboratories is <1.5%, which is significantly better than the current pharmacopoeia method: RSD>3%.

[0030] 3. The present invention also uses a gradient program of acetonitrile (mobile phase A)-methanol (mobile phase B)-0.3% phosphoric acid (mobile phase C) to successfully separate components with extremely different polarities, such as chlorogenic acid logP = -0.45 and paeonol logP = 2.47, with a separation R > 1.5 and a peak tailing factor of 0.95-1.05, solving the problem of co-elution of polar components in traditional methods.

[0031] 4. Based on 3D spectral data, the present invention dynamically switches the integration at eight wavelength points, including 284nm (5-HMF), 240nm (morroniside / loganin), and 325nm (chlorogenic acid), to avoid the insufficient sensitivity or interference of single wavelength detection, and the quantification limit (LOQ) of low-content components (such as 4,5-O-dicaffeoylquinic acid 0.03mg / g) reaches 0.18μg / mL, which is 10 times more sensitive than HPLC.

[0032] 5. The present invention uniformly prepares 8 reference substances into a mixed solution with 70% methanol. A single injection can complete the calibration of all components, saving 70% time compared to traditional step-by-step calibration. The linearity verification (r>0.999) ensures the accuracy of a wide concentration range, such as 5-hydroxymethylfurfural 4.69-234.53 μg / mL, which adapts to the content fluctuations of different manufacturers. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 It is the chromatogram of the test solution and the control solution.

[0035] Figure 2 These are the chromatograms of the negative sample solution lacking chrysanthemum and the negative sample solution lacking wine.

[0036] Figure 3 It is the chromatogram of the negative sample solution lacking Rehmannia glutinosa and the negative sample solution lacking Paeonia suffruticosa.

[0037] Figure 4It is a box plot of the contents of 8 components in 21 batches of samples. DETAILED DESCRIPTION

[0038] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0039] Example 1:

[0040] This embodiment provides a method for determining the content of Qiju Dihuang concentrated pills, which comprises simultaneously determining the contents of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol in Qiju Dihuang concentrated pills using ultra-performance liquid chromatography;

[0041] The chromatographic conditions of the ultra-high performance liquid chromatography method include: acetonitrile as mobile phase A, methanol as mobile phase B, 0.3% phosphoric acid solution as mobile phase C, and gradient elution conditions are:

[0042]

[0043] The chromatographic conditions also include: the detection wavelength is multi-wavelength segmented integration, and data processing is performed at 284 nm at 0.00 minute, 240 nm at 2.50 minute, 325 nm at 3.30 minute, 240 nm at 4.00 minute, 230 nm at 5.50 minute, 330 nm at 9.00 minute, 274 nm at 11.00 minute, and 230 nm at 11.90 minute; the chromatographic column is filled with octadecylsilane bonded silica gel;

[0044] The content determination method further comprises the preparation of the Qiju Dihuang concentrated pill solution: extracting the Qiju Dihuang concentrated pill with 70% methanol by heating under reflux and filtering, and obtaining the filtrate.

[0045] In order to prolong the interaction time between the mobile phase and the stationary phase and improve the separation of complex components, based on any of the above schemes, the chromatographic conditions also include a mobile phase flow rate of 0.2-0.4 mL / min and a column temperature of 30-40°C.

[0046] Specifically, the flow rate of the mobile phase can be 0.2 mL / min, 0.3 mL / min or 0.4 mL / min, and the column temperature can be 30°C, 35°C or 40°C, etc. Experiments have shown that a better solution is: the flow rate of the mobile phase is 0.3 mL / min, and the column temperature is 35°C.

[0047] The content determination is performed by calculating the content of the component to be measured using an external standard method or a standard curve method.

[0048] Specifically, the external standard method: take a mixed reference solution, inject it into the UPLC, and record the target peak area; take a test solution and inject it, and record the peak area of ​​the same component; calculate the concentration of the test component in the sample according to the following formula:

[0049]

[0050] in:

[0051] C 样 : Concentration of the component to be tested in the test product (μg / mL);

[0052] A 样 : Peak area of ​​the component to be tested in the test product;

[0053] A 对 : Peak area of ​​the component in the reference substance;

[0054] C 对 : The concentration of the component in the reference substance (μg / mL).

[0055] Calculation of content in the sample to be tested:

[0056]

[0057] in:

[0058] V: volume of the solution to be tested (mL);

[0059] W: sample weight (g);

[0060] D: dilution factor.

[0061] Steps for establishing a standard curve:

[0062] 1. Prepare at least 5 reference solutions of different concentrations;

[0063] 2. Inject the sample and record the peak area (Y) and concentration (X);

[0064] 3. Fitting the linear regression equation:

[0065] Y=aX+b

[0066] in:

[0067] a: slope; b: intercept; the correlation coefficient r is required to be ≥ 0.999.

[0068] In order to fully dissolve the Qiju Dihuang concentrated pills, in the preparation of the Qiju Dihuang concentrated pills solution, the amount of 70% methanol added is 40-60 mL, and the heating reflux time is 0.5-2 hours.

[0069] Specifically, the preparation of the Qiju Dihuang concentrated pill solution comprises the following steps: taking 0.5-0.7 g of the Qiju Dihuang concentrated pills, adding 40-60 mL of 70% methanol, sealing, weighing, and heating under reflux for 0.5-1.5 hours. The heating and reflux time can be 0.5 hours, 1 hour, or 1.5 hours. A preferred embodiment is that the heating and reflux time is 1 hour. After heating and reflux, the mixture is cooled, weighed again, and the weight loss is supplemented with 70% methanol. The mixture is shaken well, filtered, and the filtrate is collected to obtain the Qiju Dihuang concentrated pill solution.

[0070] The content determination method also includes the preparation of a reference solution: dissolving 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin and paeonol reference substances in 70% methanol to prepare a mixed reference solution.

[0071] The concentrations of the components in the mixed reference solution are: 5-hydroxymethylfurfural 4.69-234.53 μg / mL, morroniside 2.43-121.52 μg / mL, chlorogenic acid 1.12-56.09 μg / mL, loganin 2.81-140.33 μg / mL, paeoniflorin 2.55-127.64 μg / mL, 4,5-O-dicaffeoylquinic acid 1.17-58.60 μg / mL, benzoylpeoniflorin 2.22-110.75 μg / mL, and paeonol 4.93-246.25 μg / mL.

[0072] Specifically, the mixed reference solution was prepared by accurately weighing 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin and paeonol, dissolving and diluting them with 70% methanol to prepare a mixed reference solution containing a specific concentration range of each component to ensure coverage of the actual sample content and linear response range.

[0073] Establish a standard curve: inject the mixed standard solution into UPLC and record the peak area (A 对 ), the concentration of the test product is calculated according to the formula;

[0074] Content calculation: According to content D=10 -3 Conversion, where V is the constant volume (50 mL) and W is the sample weight (0.6 g). Data accuracy was ensured by uniform dissolution in 70% methanol and multi-wavelength segment integration (e.g., 284 nm, 240 nm).

[0075] The chromatographic column is a Waters HSS T3 C18 chromatographic column with a specification of 2.1×100 mm and a filler particle size of 1.8 μm.

[0076] Example 2:

[0077] This embodiment provides an application of the content determination method described in any one of the embodiments 1 in the field of quality control of Qiju Dihuang Concentrated Pills.

[0078] The present invention will be further explained below in conjunction with specific experiments:

[0079] This study established an ultra-performance liquid chromatography-diode array detector (UPLC-DAD) method for the simultaneous determination of eight indicator components in Qiju Dihuang Concentrated Pills (QDCP): 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol. Methodological validation demonstrated the accuracy and effectiveness of the assay method provided by this invention, providing a reference for the quality control and evaluation of QDCP.

[0080] 1. Materials and Methods

[0081] 1.1 Instruments and Equipment

[0082] ACQUITY UPLC H-class UPLC (Waters, USA);

[0083] XPE205DR electronic balance (Mettler-Toledo, Switzerland);

[0084] KQ-250DE CNC ultrasonic cleaning instrument (Kunshan Ultrasonic Instrument Co., Ltd.);

[0085] TWS-26 electric constant temperature water bath (Shanghai Zhetu Scientific Instrument Co., Ltd.).

[0086] 1.2 Reagents and Reference Materials

[0087] Reference substances include 5-hydroxymethylfurfural (purity: 99.8%), morroniside (purity: 97.8%), chlorogenic acid (purity: 96.3%), loganin (purity: 99.0%), paeoniflorin (purity: 96.7%), 4,5-O-dicaffeoylquinic acid (purity: 94.9%), and paeonol (purity: 99.9%), all of which were purchased from the China Food and Drug Inspection Institute. Benzoylpeoniflorin (purity: ≥98.0%) was purchased from Shanghai Standard. 21 batches of Qiju Dihuang Concentrated Pills (concentrated pills) are samples of the 2021 Zhejiang Provincial Drug Quality Risk Assessment Project. They come from 8 different manufacturers. The specific manufacturer information is as follows: S1 Zhongjing Co., Ltd., S2 Beijing Pharmaceutical Factory, S3 Jiangxi Yaodu Company, S4 Shanghai Pharmaceutical Co., Ltd., S5 Lanzhou Company, S6 Jiu Moutang Co., Ltd., S7 Shaanxi Company, and S8 Wuhu Pharmaceutical Co., Ltd. Methanol, acetonitrile, and phosphoric acid were all of chromatographic grade, and water was ultrapure water.

[0088] 2. Test methods

[0089] 2.1 Chromatographic analysis conditions

[0090] Separation and analysis were performed using a Waters ACQUITY HSS T3C18 column (2.1 × 100 mm, 1.8 μm). The mobile phase consisted of methanol-acetonitrile-0.3% phosphoric acid solution, and the gradient elution program is shown in Table 1. The detection wavelength range was 200–400 nm, and the data processing wavelengths were set at 230 nm (paeoniflorin and paeonol), 240 nm (morroniside and loganin), 274 nm (benzoylpaeoniflorin), 284 nm (5-hydroxymethylfurfural), 325 nm (chlorogenic acid), and 330 nm (4,5-O-dicaffeoylquinic acid). The flow rate was 0.3 mL min-1, the column temperature was 35°C, and the injection volume was 1 μL.

[0091] Table 1 Gradient elution program

[0092]

[0093] 2.2 Solution Preparation

[0094] 2.2.1 Preparation of reference solution

[0095] Accurately weigh appropriate amounts of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol reference substances, and use 70% methanol as solvent to prepare reference substance stock solutions with mass concentrations of 938.12, 486.07, 224.38, 561.33, 510.58, 234.40, 443.00, and 985.01 μg·mL-1, respectively. Subsequently, accurately measure 1 mL of the reference substance stock solution, transfer it to a 50 mL volumetric flask, dilute to the mark with 70% methanol, and shake thoroughly to prepare reference substance stock solutions containing 46.91, 24.30, 11.22, 28.07, 25.53, 11.72, 22.15, and 49.25 μg·mL-1 of the above components. -1 Concentration of mixed reference solution.

[0096] 2.2.2 Preparation of test solution

[0097] Take about 0.6 g of the finely ground sample, weigh it accurately, place it in a stoppered conical flask, accurately add 50 mL of 70% methanol, stopper it and weigh it, heat it under reflux for 1 hour, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well and filter it, and the filtrate is the solution of the sample to be tested.

[0098] 2.2.3 Preparation of negative sample solution

[0099] According to the sample prescription and preparation process, prepare negative samples without wine-infused cornus fruit, peony bark, chrysanthemum, and Rehmannia root respectively, and follow the preparation method in 2.2.2 to obtain negative sample solutions.

[0100] 2.3 Methodological Investigation

[0101] 2.3.1 Specificity Investigation

[0102] Take negative sample solution, mixed reference solution and test solution and carry out determination according to the chromatographic conditions under 2.1. The results are shown in Figure 1 .from Figure 1 It can be seen that the peaks in the samples were well separated. The negative sample solutions showed no interference from other peaks at the retention times of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol, indicating good method specificity.

[0103] (Note: A: test solution; B: reference solution; C: negative sample solution lacking Chrysanthemum; D: negative sample solution lacking Fructus Corni; E: negative sample solution lacking Rehmannia; F: negative sample solution lacking Paeonia suffruticosa; 1. 5-Hydroxymethylfurfural, 2. Morroniside, 3. Chlorogenic acid, 4. Loganin, 5. Paeoniflorin, 6. 4,5-O-dicaffeoylquinic acid, 7. Paeonol, 8. Benzoylpaeoniflorin)

[0104] 2.3.2 Linearity, limit of quantification, and limit of detection

[0105] Take an appropriate amount of the mixed reference substance stock solution described in 2.2.1 and dilute it 4-, 10-, 20-, 40-, 100-, and 200-fold with 70% methanol to prepare mixed reference substance solutions of varying concentrations. Analyze according to the chromatographic conditions described in 2.1. Perform a linear regression using concentration as the abscissa (X) and peak area as the ordinate (Y). For each diluted mixed reference substance solution, calculate the limit of quantification (LOQ) using an S / N ratio of ≥10 and a limit of detection (LOD) using an S / N ratio of ≥3. The results are shown in Table 2.

[0106] Table 2 Regression equations, linear ranges, limits of quantification, and limits of detection of 8 components

[0107]

[0108] 2.3.3 Precision test

[0109] The same batch of test samples was used to prepare test sample solutions according to the method in 2.2.2. The samples were injected and measured six times according to the chromatographic conditions in 2.1. The RSDs of the peak areas of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol were calculated to be 0.54%, 0.90%, 1.10%, 0.54%, 0.98%, 1.32%, 0.24%, and 0.77%, respectively, indicating good precision of the instrument.

[0110] 2.3.4 Repeatability test

[0111] Six solutions of the same batch of test products were prepared in parallel and determined according to the chromatographic conditions in 2.1. The contents of the six samples were calculated. The RSDs of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol were 0.84%, 1.05%, 1.40%, 0.64%, 1.28%, 1.42%, 0.45%, and 1.87%, respectively, indicating that the method had good reproducibility.

[0112] 2.3.5 Stability test

[0113] The same sample was taken and the test solution was prepared according to the method in 2.2.2. The sample was injected and measured at 0, 6, 12, 24, 36, and 48 hours respectively according to the chromatographic conditions in 2.1. The RSDs of the peak areas of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol were all less than 2.0%, indicating that the sample solution was stable within 48 hours.

[0114] 2.3.6 Sample recovery test

[0115] Accurately weigh about 0.6g of the same batch of samples, a total of 6 portions, placed in a stoppered conical flask, accurately added 1mL of the mixed reference solution, and then accurately added 49mL of 70% methanol. Then, prepare the test solution according to the method under 2.2.2, and determine it according to the conditions under 2.1. Record the peak area and calculate the recovery rate. The results showed that 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin The recoveries of glycosides and paeonol were 99.78%, 97.34%, 98.01%, 97.96%, 102.18%, 97.37%, 101.58% and 97.74%, respectively, and the RSDs were 2.01%, 1.61%, 1.21%, 1.01%, 1.38%, 1.10%, 2.10% and 1.24%, respectively. The results showed that the method had good accuracy. The specific data are shown in Table 3.

[0116] Table 3 Recovery test of eight components content determination

[0117]

[0118]

[0119] 3. Discussion

[0120] 3.1 Selection of indicator components

[0121] The QDCP includes eight Chinese medicinal materials: wolfberry, chrysanthemum, Rehmannia root, wine-soaked cornus fruit, peony bark, Chinese yam, Poria cocos, and Alisma orientalis. The main components of wolfberry, Chinese yam, Poria cocos, and Alisma orientalis are polysaccharides, which are generally determined by derivatization followed by UV analysis. Although other indicator components exist, their low concentrations make them difficult to control. Therefore, the components of chrysanthemum, Rehmannia root, wine-soaked cornus fruit, and peony bark were primarily studied. The relative contents of morroniside and loganin in wine-soaked cornus fruit, paeoniflorin, paeonol, and benzoylpeoniflorin in peony bark, chlorogenic acid and 4,5-O-dicaffeoylquinic acid in chrysanthemum, and 5-hydroxymethylfurfural in Rehmannia root were relatively high, so the contents of these eight indicator components were determined.

[0122] 3.2 Preparation of test solution

[0123] The test solution preparation method of the present invention refers to the Chinese Pharmacopoeia, using reflux extraction with 70% methanol. The experiment mainly investigated the effect of different reflux times (0.5, 1, 1.5, 2, and 3 hours) on the content of the target components. The results showed that after 1 hour, the content did not increase significantly, indicating that the extraction efficiency of each target component was better after 1 hour of reflux.

[0124] 3.3 Mobile phase selection and optimization

[0125] This study compared two mobile phase systems: acetonitrile-0.3% phosphoric acid and methanol-0.3% phosphoric acid. The results showed that the morroniside peak was broadened when using acetonitrile-0.3% phosphoric acid, while the peak elution time was delayed and the resolution of some peaks was unsatisfactory when using methanol-0.3% phosphoric acid. Using a mixed acetonitrile and methanol system, after adjusting and optimizing the mobile phase and gradient program, the morroniside peak shape was improved, and the other components were well separated.

[0126] 3.4 Selection of detection wavelength

[0127] Given the complexity of traditional Chinese medicine components, further optimization and adjustment are needed on the basis of good chromatographic separation. Different substances have different ultraviolet absorption. This difference can be used to selectively set different absorption wavelengths to ensure good response while avoiding interference from miscellaneous peaks. In this experiment, the chromatographic peaks of the reference solution were collected by a diode array detector (DAD) to obtain the ultraviolet absorption graph of the index components. The maximum absorption wavelengths of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol were 284, 240, 325, 240, 230, 330, 274, and 230 nm, respectively. During data acquisition, this experiment did not set a specific wavelength channel, and directly collected 3D data. During data processing, 3D data was used and a multi-wavelength segmented integration method was adopted based on the maximum absorption wavelength of the index component. The processing wavelengths (0.00min, 284nm)(2.50min, 240nm)(3.30min, 325nm)(4.00min, 240nm)(5.50min, 230nm)(9.00min, 330nm)(11.00min, 274nm)(11.90min, 230nm) were set at different time points to extract and integrate the chromatographic peaks, thereby quickly realizing data processing and calculation of different wavelengths in different time periods under one channel. This method can avoid setting the wavelengths of multiple channels in the acquisition stage, reduce errors in instrument data acquisition, and thus improve efficiency. At the same time, it can avoid interference from some adjacent chromatographic peaks.

[0128] 3.5 Column selection

[0129] The experiment evaluated the separation effects of different chromatographic columns. Three columns from two different brands were tested: ① Waters HSS T3 C18 column (2.1×100mm, 1.8μm), ② Waters BEH C18 column (2.1×100mm, 1.8μm), and ③ Welch Xtimate C18 column (2.1×100mm, 1.8μm). The results showed that the water-resistant T3 column had better separation effect.

[0130] 3.6 Analysis of sample content results

[0131] See Figure 4 The contents of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol in 21 batches of QDCP were 0.1543-3.3554, 0.9178-2.3715, 0.0116-0.4032, 1.3095-2.7428, 0.7663-2.1391, 0.0334-0.1867, 1.9683-3.7976, and 0.2734-0.7396 mg·g, respectively. -1 The results showed that the content of chlorogenic acid and 5-hydroxymethylfurfural varied greatly, reaching 35 times and 22 times respectively; the differences of other components ranged from 1.6 to 5.6 times. The possible reasons were the quality of the medicinal materials and the differences in production process. Among them, 5-hydroxymethylfurfural is mainly the product of the Maillard reaction of polysaccharide components during the processing of Rehmannia glutinosa under high temperature heating or long-term storage. Different processing temperatures will significantly affect the content of 5-hydroxymethylfurfural; the polysaccharide content and component ratio in Rehmannia glutinosa from different origins vary, which may also affect the formation of 5-HMF; chlorogenic acid is one of the main active ingredients of chrysanthemum, and different origins and processing techniques will also affect the level of chlorogenic acid.

[0132] In this study, a UPLC-DAD method was established to simultaneously determine the contents of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin, and paeonol in Qiju Dihuang concentrated pills (concentrated pills). The multi-wavelength segmented integration method was used, and its accuracy and effectiveness were demonstrated through methodological validation. This method can provide a reference for the quality control and evaluation of Qiju Dihuang concentrated pills (concentrated pills).

[0133] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that falls within the scope defined by the claims of the present invention falls within the scope of protection of the present invention.

Claims

1. A method for determining the content of Qiju Dihuang concentrated pills, characterized in that: The content determination method comprises the steps of simultaneously determining the contents of 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin and paeonol in Qijudihuang concentrated pills by ultra-high performance liquid chromatography; The chromatographic conditions of the ultra-high performance liquid chromatography method include: acetonitrile as mobile phase A, methanol as mobile phase B, 0.3% phosphoric acid solution as mobile phase C, and gradient elution conditions are: The chromatographic conditions also include: the detection wavelength is multi-wavelength segmented integration, and data processing is performed at 284 nm at 0.00 minute, 240 nm at 2.50 minute, 325 nm at 3.30 minute, 240 nm at 4.00 minute, 230 nm at 5.50 minute, 330 nm at 9.00 minute, 274 nm at 11.00 minute, and 230 nm at 11.90 minute; the chromatographic column is filled with octadecylsilane bonded silica gel; The content determination method further comprises the preparation of the Qiju Dihuang concentrated pill solution: extracting the Qiju Dihuang concentrated pill with 70% methanol by heating under reflux and filtering, and obtaining the filtrate.

2. The method for determining the content of Qiju Dihuang concentrated pills according to claim 1, wherein: The chromatographic conditions also include a mobile phase flow rate of 0.2-0.4 mL / min and a column temperature of 30-40°C.

3. The method for determining the content of Qiju Dihuang concentrated pills according to claim 1, wherein: The calculation of the content determination is to calculate the content of the component to be measured by external standard method or standard curve method.

4. The method for determining the content of Qiju Dihuang concentrated pills according to claim 1, wherein: In the preparation of the Qiju Dihuang concentrated pill solution, the amount of 70% methanol added is 40-60 mL, and the heating reflux time is 0.5-2 hours.

5. The method for determining the content of Qiju Dihuang concentrated pills according to claim 4, wherein: The preparation of the Qiju Dihuang concentrated pill solution comprises the following steps: taking 0.5-0.7 g of the Qiju Dihuang concentrated pills, adding 40-60 mL of 70% methanol, sealing and weighing, heating and reflux for 0.5-1.5 hours, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking and filtering, and taking the filtrate to obtain the Qiju Dihuang concentrated pill solution.

6. The method for determining the content of Qiju Dihuang concentrated pills according to claim 1, characterized in that: The heating reflux time is 1 h.

7. The method for determining the content of Qiju Dihuang concentrated pills according to claim 1, characterized in that: The content determination method also includes the preparation of a reference solution: dissolving 5-hydroxymethylfurfural, morroniside, chlorogenic acid, loganin, paeoniflorin, 4,5-O-dicaffeoylquinic acid, benzoylpeoniflorin and paeonol reference substances in 70% methanol to prepare a mixed reference solution.

8. The method for determining the content of Qiju Dihuang concentrated pills according to claim 7, characterized in that: The concentrations of the components in the mixed reference solution were: 5-hydroxymethylfurfural 4.69-234.53 μg / mL, morroniside 2.43-121.52 μg / mL, chlorogenic acid 1.12-56.09 μg / mL, loganin 2.81-140.33 μg / mL, paeoniflorin 2.55-127.64 μg / mL, 4,5-O-dicaffeoylquinic acid 1.17-58.60 μg / mL, benzoylpeoniflorin 2.22-110.75 μg / mL, and paeonol 4.93-246.25 μg / mL.

9. The method for determining the content of Qiju Dihuang concentrated pills according to claim 1, characterized in that: The chromatographic column is a Waters HSS T3 C18 chromatographic column with a specification of 2.1×100 mm and a filler particle size of 1.8 μm.

10. Use of the content determination method according to any one of claims 1 to 9 in the field of quality control of Qiju Dihuang Concentrated Pills.

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