A method for constructing a characteristic chromatogram of Zishen Yutai pills, a method for determining the content of the pills, and a detection method

Abstract

The application relates to the technical field of drug analysis and detection, in particular to a characteristic chromatogram construction method, a content determination method and a detection method of Zishen Yutai pills, and especially to a characteristic chromatogram construction method of Zishen Yutai pills, a content determination method of characteristic components and a quality detection method of one test for multiple evaluations, wherein the characteristic components include 5-hydroxymethyl furfural, strychnine acid, chlorogenic acid, cryptochlorogenic acid, strychnine and iodeside, and the strychnine acid is used as an internal reference in the quality detection method of one test for multiple evaluations. The content determination method is operable and reproducible, can accurately determine the 5-hydroxymethyl furfural, strychnine acid, chlorogenic acid, cryptochlorogenic acid, strychnine and iodeside in the Zishen Yutai pills, the one test for multiple evaluations detection method obtained on the basis can effectively save detection cost, improve detection efficiency, is accurate and reliable, and has practical value.

Description

Technical Field

[0001] This application relates to the technical field of pharmaceutical analysis and detection, and specifically relates to a method for constructing a characteristic fingerprint of Zishen Yutai Pills, a method for determining the content, and a detection method. In particular, it relates to a method for constructing a characteristic fingerprint of Zishen Yutai Pills, a method for determining the content of its characteristic components, and a quality detection method of multi-component determination by single marker. The characteristic components include 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin. In the multi-component determination by single marker quality detection, loganic acid is used as the internal reference substance. Background Art

[0002] Zishen Yutai Pills are included in the 16th volume of the "Drug Standards of the Ministry of Health of the People's Republic of China - Traditional Chinese Medicine Formulas and Preparations" (WS3-B-3113-98), and are composed of 15 Chinese herbal medicines such as Cuscuta chinensis, Rehmannia glutinosa, Codonopsis pilosula, Cornu Cervi Degelatinatum, Morinda officinalis, Lycium barbarum, Taxillus chinensis, Atractylodes macrocephala, Amomum villosum, Panax ginseng, Eucommia ulmoides, Colla Corii Asini, Polygonum multiflorum, Artemisia argyi, and Dipsacus asper. It has the effects of tonifying the kidney and spleen, replenishing qi and cultivating the primordial qi, and nourishing the blood and preventing miscarriage.

[0003] Traditional Chinese medicine compound prescriptions have the characteristics of numerous medicinal flavors and complex components. Zishen Yutai Pills are a compound prescription containing 15 Chinese herbal medicines. It is difficult to conduct overall quality control by only analyzing a single component. At present, the determination of the content of multiple target components has been widely used in the field of quality control of traditional Chinese medicines, which is a major trend in the modernization development of traditional Chinese medicines.

[0004] Lei Ting et al. disclosed a method for constructing and determining the fingerprint of Zishen Yutai Pills in a patent. The fingerprint construction method can obtain a fingerprint with characteristic peaks including the active components of multiple Chinese herbal medicines such as Dipsacus asper, Cuscuta chinensis, Panax ginseng, Lycium barbarum, Polygonum multiflorum preparatum, Atractylodes macrocephala, and Artemisia argyi. This method is for qualitative research, and the detection time of the sample is relatively long (190 minutes per injection), and it is impossible to control the quality of Zishen Yutai Pills in a short time, so it is not suitable for the quality control of Zishen Yutai Pills in actual production. Moreover, this method requires the use of an electrospray detector, which will bring additional inspection costs, limit the use scenarios, and has poor universality.

[0005] Wen Xiaoya et al. used UHPLC / Q-TOF-MS / MS technology to detect and analyze the chemical components of Zishen Yutai Pills in positive and negative ion modes, and a total of 39 chemical components in Zishen Yutai Pills were identified. Subsequently, a method for determining the characteristic fingerprint of Zishen Yutai Pills was established by high performance liquid chromatography, and the contents of 3 components, namely isochlorogenic acid C, loganin, and swertiamarin in Zishen Yutai Pills, can be determined simultaneously. However, this method requires the preparation of multiple reference substances for quantitative determination one by one, is relatively cumbersome in operation, and has relatively high inspection costs. In addition, the isochlorogenic acid C reference substance is not in the supply catalog of the National Institutes for Food and Drug Control, and it is difficult to control its quality, which may bring unnecessary inspection deviations in actual inspections.

[0006] Yang Lijuan et al. reported a method for determining the content of multiple components in Zishen Yutai Pill using reversed-phase high-performance liquid chromatography (RP-HPLC). This method can simultaneously determine the content of three components in Zishen Yutai Pill: hyperoside, verbascoside, and stilbene glycoside. However, this method requires the preparation of multiple reference standards for individual quantification, making the operation relatively cumbersome and the testing cost relatively high.

[0007] Ma Huiwen et al. reported a fingerprinting method for Zishen Yutai Pill, which can simultaneously determine five components in the pill: loganic acid, chlorogenic acid, loganin, swertiamarin, and dipsacusin VI. While this method controls the content of multiple components in Zishen Yutai Pill, loganic acid, loganin, swertiamarin, and dipsacusin VI are all derived from Dipsacus asper, and chlorogenic acid is a non-characteristic component common to many medicinal materials, thus covering a limited range of medicinal materials. Furthermore, this method requires the preparation of multiple reference standards for individual quantification, and the sample detection time is lengthy, resulting in cumbersome operation, high testing costs, and long testing time.

[0008] The methods described above all employ external standard methods to quantify one or more components in a sample. The reference standards used are often expensive, leading to high testing costs and limiting their practical application. This application, through extensive literature review, finds that the Quality Assurance System (QAMS) method can solve these problems. QAMS selects one component as an internal reference and calculates correction factors between components to simultaneously determine the content of multiple components. Therefore, it effectively reduces costs and improves testing efficiency.

[0009] Studies have found that 5-hydroxymethylfurfural, a component produced during the extraction process, has certain neurotoxicity. It can bind to human proteins, causing cumulative poisoning and abnormal growth of the colonic sac. At higher doses, it can damage human striated muscle and internal organs. Literature reports that the 5-hydroxymethylfurfural content in Rehmannia glutinosa increases approximately 20-fold after processing. Since Rehmannia glutinosa constitutes a certain proportion in Zishen Yutai Wan (a traditional Chinese medicine formula), it is necessary to thoroughly study the 5-hydroxymethylfurfural content in Zishen Yutai Wan to better understand and explore the dual nature of its drug-toxicity.

[0010] In view of this, the technical solution of this application is hereby proposed. Summary of the Invention

[0011] The purpose of this application is to provide a method for constructing a characteristic chromatogram of Zishen Yutai Pill, a method for determining its content, and a detection method. This application establishes a characteristic chromatogram of Zishen Yutai Pill using liquid chromatography, and further establishes a method for determining the content of characteristic components 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin. This method is simple to operate, has a short testing time, and high accuracy. Furthermore, this application develops a multi-evaluation quality detection method for Zishen Yutai Pill based on this method, which can save testing costs, improve testing efficiency, and is accurate and practical.

[0012] One aspect of this application provides a method for constructing a feature map of Zishen Yutai Pill, which includes the following steps:

[0013] A mixed reference solution is provided, the mixed reference solution comprising the following components: 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin.

[0014] Take the Zishen Yutai Pill, add an extraction solvent and extract to obtain the test solution.

[0015] The mixed reference solution and the test solution were subjected to high-performance liquid chromatography (HPLC) analysis to obtain chromatograms of the reference solution and the test solution, respectively.

[0016] By comparing the chromatograms of the reference standard and the test sample, a characteristic chromatogram of Zishen Yutai Pill was determined.

[0017] The conditions for the liquid chromatography analysis include:

[0018] (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume percentage of phosphoric acid in the aqueous solution of phosphoric acid is 0.05% to 0.3%.

[0019] (2) The elution process includes:

[0020] From 0 to 1 minute, the volume percentage of the mobile phase A is maintained at 5%.

[0021] Within 1 to 3 minutes, the volume percentage of the mobile phase A increased from 5% to 7%.

[0022] Within 3 to 12 minutes, the volume percentage of mobile phase A increased from 7% to 16%.

[0023] Between 12 and 28 minutes, the volume percentage of the mobile phase A increased from 16% to 25%.

[0024] In another aspect, this application provides a method for determining the content of characteristic components in Zishen Yutai Pill, which includes the following steps:

[0025] A mixed reference solution of different concentrations is provided, the mixed reference solution comprising the following components: 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin.

[0026] Different concentrations of the mixed reference standard solution were subjected to high-performance liquid chromatography (HPLC) analysis to obtain chromatograms of the reference standard. The peak areas and mass concentrations of each component were recorded, and corresponding standard curves were plotted. Regression equations for each component were then derived.

[0027] Take the Zishen Yutai Pill to be tested, add extraction solvent and extract to obtain the test solution.

[0028] The test sample solution was subjected to liquid chromatography analysis to obtain a chromatogram of the test sample. The chromatogram of the test sample was compared with the characteristic chromatogram obtained by the construction method. The characteristic peaks corresponding to 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin and swertiamarin in the chromatogram of the test sample were identified. The peak area of ​​each characteristic peak was recorded and substituted into the corresponding regression equation to calculate the mass concentration.

[0029] The conditions for the liquid chromatography analysis include:

[0030] (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume percentage of phosphoric acid in the aqueous solution of phosphoric acid is 0.05% to 0.3%.

[0031] (2) The elution process includes:

[0032] From 0 to 1 minute, the volume percentage of the mobile phase A is maintained at 5%.

[0033] Within 1 to 3 minutes, the volume percentage of the mobile phase A increased from 5% to 7%.

[0034] Within 3 to 12 minutes, the volume percentage of mobile phase A increased from 7% to 16%.

[0035] Between 12 and 28 minutes, the volume percentage of the mobile phase A increased from 16% to 25%.

[0036] The content determination method of this application measures the characteristic components including 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin. These components are involved in nine medicinal materials, including Rehmannia glutinosa, Polygonum multiflorum, Morinda officinalis, Eucommia ulmoides, Atractylodes macrocephala, Cuscuta chinensis, Artemisia argyi, Dipsacus asper, and Cuscuta chinensis. The research results are more comprehensive. Furthermore, this application is the first to control the content of 5-hydroxymethylfurfural, a toxic component in Zishen Yutai Pill, which has important reference value for the pharmacological research of Zishen Yutai Pill.

[0037] In another aspect, this application provides a method for multiple quality assessments of Zishen Yutai Pills, comprising the following steps:

[0038] A mixed reference solution is provided, the mixed reference solution comprising the following components: 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin.

[0039] The mixed reference solution was subjected to liquid chromatography analysis. The peak area and mass concentration of each component were recorded. An internal reference was selected based on the chromatographic analysis results. The relative correction factors of the remaining components relative to the internal reference were calculated.

[0040] Take the Zishen Yutai Pill to be tested, add extraction solvent and extract to obtain the test solution.

[0041] The test solution was subjected to high-performance liquid chromatography (HPLC) analysis to obtain a chromatogram of the test sample. The chromatogram of the test sample was compared with the characteristic chromatogram obtained by the construction method described above. The characteristic peaks corresponding to the characteristic components 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin in the chromatogram of the test sample were identified, and the peak areas of each characteristic peak were recorded.

[0042] The mass concentration of the characteristic component used as an internal reference in the tested Zishen Yutai Pill was calculated according to the content determination method described above. The mass concentrations of the remaining characteristic components were then calculated using the mass concentration calculation formula.

[0043] The conditions for the liquid chromatography analysis include:

[0044] (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume percentage of phosphoric acid in the aqueous solution of phosphoric acid is 0.05% to 0.3%.

[0045] (2) The elution process includes:

[0046] From 0 to 1 minute, the volume percentage of the mobile phase A is maintained at 5%.

[0047] Within 1 to 3 minutes, the volume percentage of the mobile phase A increased from 5% to 7%.

[0048] Within 3 to 12 minutes, the volume percentage of mobile phase A increased from 7% to 16%.

[0049] Between 12 and 28 minutes, the volume percentage of the mobile phase A increased from 16% to 25%.

[0050] This application develops a multi-analysis method based on the aforementioned liquid chromatography conditions. This method allows for the selection of readily available and inexpensive characteristic components, such as loganic acid, as a reference, while simultaneously determining the content of other characteristic components, such as 5-hydroxymethylfurfural, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin. The determination results show no significant difference from the external standard method. The method is accurate, reliable, and highly reproducible, significantly reducing the determination cost and enabling rapid quality testing of Zishen Yutai Pills. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 The image shows the HPLC chromatograms of the extraction of Zishen Yutai Pills with different solvents as described in section 2.2.1 of this application.

[0053] Figure 2 The image shows the HPLC chromatograms of Zishen Yutai Pills extracted at different times as described in section 2.2.2 of this application.

[0054] Figure 3 The image shows the HPLC chromatograms of Zishen Yutai Pills with different mobile phases in section 2.3.1 of this application.

[0055] Figure 4 The image shows the HPLC chromatograms of Zishen Yutai Pills at different column temperatures as described in section 2.3.2 of this application.

[0056] Figure 5 This is the HPLC chromatogram of the Zishen Yutai Pill reference standard determined in section 2.4 of this application;

[0057] Figure 6 This is the HPLC chromatogram of the Zishen Yutai Pill test sample as determined in section 2.4 of this application;

[0058] In each of the attached figures, the horizontal axis represents time (min) and the vertical axis represents the response value (mAu). Detailed Implementation

[0059] The present application is further described below with reference to the embodiments, examples, and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the protection scope of the appended claims.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0061] the term

[0062] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0063] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations encompass any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0064] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0065] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0066] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0067] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0068] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0069] In this application, weight can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.

[0070] In one aspect of this application, a method for constructing a characteristic spectrum of Zishen Yutai Pill is disclosed. The characteristic spectrum constructed by this method is highly specific, and the identified characteristic peaks include 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin. The above-mentioned components involve nine medicinal materials, including Rehmannia glutinosa, Polygonum multiflorum, Morinda officinalis, Eucommia ulmoides, Atractylodes macrocephala, Cuscuta chinensis, Artemisia argyi, Dipsacus asper, and Cuscuta chinensis (see Table 1 for details). The research results are more comprehensive and have important reference significance for the content determination and quality detection of Zishen Yutai Pill.

[0071] Table 1

[0072]

[0073]

[0074] In some embodiments, the method for constructing the characteristic spectrum of Zishen Yutai Pill includes the following steps:

[0075] A mixed reference solution is provided, comprising the following components: 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin.

[0076] Take the Zishen Yutai Pill, add an extraction solvent and extract to obtain the test solution.

[0077] The mixed reference solution and the test solution were analyzed by liquid chromatography separately to obtain the chromatograms of the reference solution and the test solution.

[0078] By comparing the chromatograms of the reference standard and the test sample, a characteristic chromatogram of Zishen Yutai Pill was determined.

[0079] The conditions for liquid chromatography analysis include:

[0080] (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphoric acid, with the volume percentage of phosphoric acid in the aqueous solution being 0.05% to 0.3%.

[0081] (2) The elution process includes:

[0082] From 0 to 1 minute, the volume percentage of mobile phase A is maintained at 5%.

[0083] Within 1 to 3 minutes, the volume percentage of mobile phase A increased from 5% to 7%.

[0084] Between 3 and 12 minutes, the volume percentage of mobile phase A increased from 7% to 16%.

[0085] Between 12 and 28 minutes, the volume percentage of mobile phase A increased from 16% to 25%.

[0086] In some implementations, high-performance liquid chromatography (HPLC) is used for liquid chromatography detection.

[0087] In some implementations, during liquid chromatography analysis, the test solution is scanned across the entire wavelength range of 200 nm to 400 nm, and the required scanning wavelength is determined by comparative analysis of the chromatograms.

[0088] In some embodiments, the wavelength for measuring loganic acid, loganin, and swertiamarin is 240 nm, the wavelength for measuring 5-hydroxymethylfurfural is 284 nm, and the wavelength for measuring cryptochlorogenic acid and chlorogenic acid is 324 nm.

[0089] In some embodiments, the conditions for liquid chromatography analysis further include at least one of the following:

[0090] (1) The chromatographic column is a C18 column.

[0091] (2) The flow rate is 0.8 mL / min to 1.2 mL / min.

[0092] (3) The column temperature is 25℃~45℃.

[0093] (4) The injection volume is 8 μL to 12 μL.

[0094] In some embodiments, the chromatographic column has a length of 50–300 mm, an inner diameter of 1.8–5 mm, and a particle size of 1.8–5 μm.

[0095] In some embodiments, the mass concentration of each component in the reference solution is independently selected from 0.16 mg / mL to 0.22 mg / mL.

[0096] In some implementations, the extraction conditions include at least one of the following:

[0097] (1) The extraction solvent is an alcohol of 20%–80% by volume, further wherein the alcohol is methanol; and / or

[0098] (2) The extraction method includes ultrasound, further wherein the power of the ultrasound is 125W to 350W and the frequency is 35kHz to 45kHz; and / or

[0099] (3) The extraction time is 15 min to 60 min.

[0100] In another aspect of this application, there is a method for determining the content of characteristic components in Zishen Yutai Pill, which is easy to operate, has good reproducibility, small measurement deviation, good stability, and high precision.

[0101] The content determination method of this application determines the characteristic components of Zishen Yutai Pill, including 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin. These components are involved in nine medicinal materials, including Rehmannia glutinosa, Polygonum multiflorum, Morinda officinalis, Eucommia ulmoides, Atractylodes macrocephala, Cuscuta chinensis, Artemisia argyi, Dipsacus asper, and Cuscuta chinensis (see Table 1 for details). The research results are more comprehensive. Furthermore, this application is the first to determine the content of 5-hydroxymethylfurfural, a toxic component in Zishen Yutai Pill, which has important reference value for the pharmacological research of Zishen Yutai Pill.

[0102] In some embodiments, the method for determining the content of characteristic components in Zishen Yutai Pills includes the following steps:

[0103] A mixed reference solution of different concentrations is provided. The mixed reference solution includes the following components: 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin.

[0104] Mixed reference standard solutions of different concentrations were analyzed by liquid chromatography to obtain chromatograms of the reference standards. The peak areas and mass concentrations of each component were recorded, and corresponding standard curves were plotted. Regression equations for each component were then derived.

[0105] Take the Zishen Yutai Pill to be tested, add extraction solvent and extract to obtain the test solution.

[0106] The test sample solution is subjected to liquid chromatography analysis to obtain the chromatogram of the test sample. The chromatogram of the test sample is compared with the characteristic chromatogram prepared by the construction method described in any of the above technical solutions. The characteristic peaks corresponding to the characteristic components 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin and swertiamarin in the chromatogram of the test sample are identified. The peak area of ​​each characteristic peak is recorded and substituted into the corresponding regression equation to calculate the mass concentration.

[0107] The conditions for liquid chromatography analysis include:

[0108] (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphoric acid, with the volume percentage of phosphoric acid in the aqueous solution being 0.05% to 0.3%.

[0109] (2) The elution process includes:

[0110] From 0 to 1 minute, the volume percentage of mobile phase A is maintained at 5%.

[0111] Within 1 to 3 minutes, the volume percentage of mobile phase A increased from 5% to 7%.

[0112] Between 3 and 12 minutes, the volume percentage of mobile phase A increased from 7% to 16%.

[0113] Between 12 and 28 minutes, the volume percentage of mobile phase A increased from 16% to 25%.

[0114] In some implementations, the obtained regression equation includes:

[0115] (1) Regression equation for 5-hydroxymethylfurfural: y1 = 74434235.17x1 - 32157.91;

[0116] (2) Regression equation of loganic acid: y2=16604976.22x2-1012.272231;

[0117] (3) Regression equation for chlorogenic acid: y3 = 31521787.40x3 - 24587.73;

[0118] (4) Regression equation for cryptochlorogenic acid: y4 = 29090004.26x4 - 24971.44;

[0119] (5) Regression equation of loganin: y5=17125581.64x5+646.7786683;

[0120] (6) Regression equation of swertiamarin: y6=15711055.7x6+9146.100111.

[0121] Understandably, the liquid chromatography analysis process in the content determination method of this application is similar to the liquid chromatography analysis process of the characteristic spectrum construction method described in any of the above technical solutions, wherein the preparation of the analyte solution is the same as the preparation of the test sample solution described in any of the above technical solutions, and will not be repeated here.

[0122] In one specific embodiment, the method for determining the content of characteristic components in Zishen Yutai Pill includes the following steps:

[0123] (1) Take appropriate amounts of 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin and swertiamarin, dissolve them in methanol, and prepare a mixed solution containing 0.1731 mg / mL, 0.2118 mg / mL, 0.1953 mg / mL, 0.2094 mg / mL, 0.2158 mg / mL and 0.2122 mg / mL of 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin and swertiamarin, respectively, to obtain a mixed reference solution;

[0124] (2) Accurately weigh 1g of Zishen Yutai Pill powder, accurately add 20mL of 50% methanol aqueous solution, seal tightly, weigh, sonicate for 30min, cool, weigh, make up the weight loss with methanol, shake well, filter through a 0.22μm microporous membrane, take the filtrate, and prepare the test solution.

[0125] (3) Take an appropriate amount of the mixed reference solution obtained in step (1), place it in a 5 mL volumetric flask, dilute it to the mark with 70% methanol, shake well, and prepare 6 mixed reference solutions of different concentrations respectively; take the 6 mixed reference solutions of different concentrations, inject 5 μL of each solution into the liquid chromatography under the liquid chromatography conditions described in any of the above technical schemes, record the peak area, and plot the standard curve with mass concentration (μg / mL) as the abscissa (X) and peak area as the ordinate (Y).

[0126] In another aspect of this application, a multi-evaluation quality testing method for Zishen Yutai Pills is disclosed. This method is based on the chromatographic analysis conditions described in any of the aforementioned technical solutions, specifically the liquid chromatography analysis method and the method for determining the content of characteristic components. The Zishen Yutai Pills to be tested are analyzed, and suitable characteristic components are selected as internal references based on the chromatographic analysis results. Then, the concentrations of the remaining characteristic components are calculated by substituting them into the relative correction factors. For example, loganic acid, which has a high content and is inexpensive and readily available, can be selected as a reference. The relative correction factors (fs / i) for 5-hydroxymethylfurfural, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin are calculated and then substituted into the mass concentration calculation equation for conversion. This method is simple to operate, can effectively save detection costs, and provides accurate and reliable results compared to the external standard method, showing promising application prospects.

[0127] In some implementations, the method for detecting Zishen Yutai Pills using a multi-evaluation approach includes the following steps:

[0128] A mixed reference solution is provided, comprising the following components: 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin.

[0129] Perform liquid chromatography analysis on the mixed reference solution, record the peak area and mass concentration of each component, select the internal reference based on the chromatographic analysis results, and calculate the relative correction factor of each other component relative to the internal reference.

[0130] Take the Zishen Yutai Pill to be tested, add extraction solvent and extract to obtain the test solution.

[0131] The test sample solution was subjected to high-performance liquid chromatography (HPLC) analysis to obtain a chromatogram of the test sample. The chromatogram of the test sample was compared with the characteristic chromatogram prepared by the construction method described in any of the above technical solutions. The characteristic peaks corresponding to the characteristic components 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin in the chromatogram of the test sample were identified, and the peak areas of each characteristic peak were recorded.

[0132] Calculate the mass concentration of the characteristic component used as an internal reference in the Zishen Yutai Pill according to the content determination method described in any of the above technical solutions, and then calculate the mass concentration of the remaining characteristic components by substituting them into the mass concentration calculation formula.

[0133] The conditions for liquid chromatography analysis include:

[0134] (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphoric acid, with the volume percentage of phosphoric acid in the aqueous solution being 0.05% to 0.3%.

[0135] (2) The elution process includes:

[0136] From 0 to 1 minute, the volume percentage of mobile phase A is maintained at 5%.

[0137] Within 1 to 3 minutes, the volume percentage of mobile phase A increased from 5% to 7%.

[0138] Between 3 and 12 minutes, the volume percentage of mobile phase A increased from 7% to 16%.

[0139] Between 12 and 28 minutes, the volume percentage of mobile phase A increased from 16% to 25%.

[0140] Understandably, the liquid chromatography analysis process in the one-test-multiple-evaluation quality detection method of this application is similar to the liquid chromatography analysis process of the characteristic spectrum construction method described in any of the above technical solutions, wherein the preparation of the analyte solution is the same as the preparation of the test sample solution described in any of the above technical solutions, and will not be repeated here.

[0141] In some implementations, a multi-point correction method is used to calculate the relative correction factors of the remaining components relative to the internal reference.

[0142] The formula for calculating the relative correction factor is: fs / i=(As×Ci) / (Ai×Cs), where As refers to the peak area of ​​the internal reference, Cs refers to the mass concentration of the internal reference, Ai refers to the peak area of ​​one of the remaining components, and Ci refers to the mass concentration of one of the remaining components.

[0143] In some implementations, the formula for calculating the mass concentration is: Ck′=(Cs′×Ak′) / (fk / i×As′); where As′ refers to the peak area of ​​the characteristic peak corresponding to the internal reference in the chromatogram of the analyte, Cs′ refers to the mass concentration of the characteristic component of the internal reference calculated according to the regression equation, Ck′ refers to the mass concentration of one of the remaining characteristic components, and Ak′ refers to the peak area of ​​one of the remaining characteristic components.

[0144] In some preferred embodiments, the internal reference is loganic acid.

[0145] In one specific embodiment, the one-test-multiple-evaluation quality testing method for Zishen Yutai Pill includes the following steps:

[0146] (1) Take appropriate amounts of 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin and swertiamarin, dissolve them in methanol, and prepare a mixed solution containing 0.1731 mg / mL, 0.2118 mg / mL, 0.1953 mg / mL, 0.2094 mg / mL, 0.2158 mg / mL and 0.2122 mg / mL of 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin and swertiamarin, respectively, to obtain a mixed reference solution;

[0147] (2) Accurately weigh 1g of Zishen Yutai Pill powder, accurately add 20mL of 50% methanol aqueous solution, seal tightly, weigh, sonicate for 30min, cool, weigh, make up the weight loss with methanol, shake well, filter through a 0.22μm microporous membrane, take the filtrate, and prepare the test solution.

[0148] (3) Take an appropriate amount of the mixed reference solution obtained in step (1), place it in a 5 mL volumetric flask, dilute it to the mark with 70% methanol, shake well, and prepare 6 mixed reference solutions of different concentrations respectively; take the 6 mixed reference solutions of different concentrations, inject 10 μL of each solution into the liquid chromatography under the liquid chromatography conditions described in any of the above technical schemes for analysis and record the peak area.

[0149] Using loganic acid as an internal reference, the relative correction factors fs / i for 5-hydroxymethylconaldehyde, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin were calculated using the following formulas with a multi-point correction method.

[0150] fs / i=(As×Ci) / (Ai×Cs), where As refers to the peak area of ​​the internal reference, Cs refers to the mass concentration of the internal reference, Ai refers to the peak area of ​​one of the remaining components, and Ci refers to the mass concentration of one of the remaining components.

[0151] Take the Zishen Yutai Pill to be tested, add extraction solvent and extract to obtain the test solution.

[0152] Perform high-performance liquid chromatography (HPLC) analysis on the test sample solution to obtain the peak area. Based on the obtained relative correction factor, substitute the values ​​into the following formulas to calculate the mass concentration.

[0153] Ck′=(Cs′×Ak′) / (fk / i×As′); where As′ refers to the peak area of ​​the characteristic peak corresponding to loganic acid in the chromatogram of the analyte, Cs′ refers to the mass concentration of the characteristic component of loganic acid calculated according to the regression equation, Ck′ refers to the mass concentration of one of the remaining characteristic components, and Ak′ refers to the peak area of ​​one of the remaining characteristic components.

[0154] The following are some specific examples.

[0155] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0156] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0157] 1. Instruments and reagents

[0158] 1.1. Instruments and Equipment

[0159] Waters e2695 high-performance liquid chromatograph (Waters Inc., USA); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 0.0001 g electronic balance (Sartorius Scientific Instruments Co., Ltd.); electric thermostatic water bath (Tianjin Tester Instrument Co., Ltd.); pure water system (Milli-Q).

[0160] 1.2. Medicines and medicinal materials (see Table 2)

[0161] Table 2 Sample Information

[0162]

[0163] 1.3. Reagents

[0164] Methanol, phosphoric acid, and water were purified water prepared in the laboratory.

[0165] 2. Development of detection methods

[0166] 2.1. Provide a control sample

[0167] Accurately weigh approximately 10 mg of 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin. Dissolve in methanol and dilute to a 10 mL volumetric flask to prepare a stock solution. Store the stock solution in a sealed container at 4°C. Take appropriate amounts of the reference standards and prepare mixed reference solutions containing 0.1731 mg / mL of 5-hydroxymethylfurfural, 0.2118 mg / mL of loganic acid, 0.1953 mg / mL of chlorogenic acid, 0.2094 mg / mL of cryptochlorogenic acid, 0.2158 mg / mL of loganin, and 0.2122 mg / mL of swertiamarin.

[0168] 2.2. Preparation of the test solution

[0169] 2.2.1. Selection of extraction solvent for test sample

[0170] Accurately weigh 1g of Zishen Yutai Pill powder and place it in a stoppered conical flask. Accurately add 20mL of water, 50% methanol, 80% methanol, and 100% methanol. Seal the flask tightly, weigh the contents, and sonicate for 30 minutes. After cooling, weigh again. Make up the lost weight with the extraction solvent, shake well, and filter to obtain the test solution. Figure 1 It can be seen that the number of chromatographic peaks in the water-extracted sample is the fewest, so water is not chosen as the extraction solvent; the peak response of the methanol-extracted sample after 40 min is significantly lower than that of 50% methanol and 80% methanol, so methanol is also not chosen as the extraction solvent; the number of chromatographic peaks and the response are basically the same when using 50% methanol and 80% methanol as extraction reagents. Considering that 50% methanol may extract more water-soluble and lipid-soluble components and can save solvent, the final extraction solvent for the test sample is 50% methanol.

[0171] 2.2.2. Selection of sample extraction time

[0172] Accurately weigh 1g of Zishen Yutai Pill powder and place it in a stoppered conical flask. Accurately add 20mL of 50% methanol, seal tightly, and weigh. Sonicate for 15min, 30min, and 45min respectively. After cooling, weigh again and replenish the lost weight with 50% methanol. Shake well and filter to obtain the test solution. Figure 2 It can be seen that the number of chromatographic peaks and the peak response are basically consistent after ultrasonic extraction for 15 min, 30 min and 45 min. Therefore, in order to take into account the extraction effect and time, the extraction time was finally determined to be 30 min.

[0173] 2.2.3. Preparation of the test sample

[0174] Based on the investigation in sections 2.2.1 to 2.2.2, the preparation method of the test sample was determined as follows: accurately weigh 1g of Zishen Yutai Pill powder (passed through a No. 4 sieve), place it in a stoppered conical flask, accurately add 20mL of 50% methanol, seal tightly, weigh, sonicate (power 250W, frequency 100kHz) for 30min, cool, weigh, make up the weight loss with methanol, shake well, and filter through a 0.22μm microporous membrane to obtain the filtrate.

[0175] 2.3. Establishment of chromatographic conditions

[0176] 2.3.1. Selection of mobile phase

[0177] Different concentrations of acid were investigated, using acetonitrile-1% phosphoric acid aqueous solution, acetonitrile-2% phosphoric acid aqueous solution, and acetonitrile-3% phosphoric acid aqueous solution as the mobile phase. Figure 3 It is known that the number of chromatographic peaks and peak responses of acetonitrile-1% phosphoric acid aqueous solution, acetonitrile-2% phosphoric acid aqueous solution, and acetonitrile-3% phosphoric acid aqueous solution are basically consistent. Considering that the higher the concentration of phosphoric acid, the greater the damage to the instrument, and at the same time, solvent can be saved, acetonitrile-1% phosphoric acid aqueous solution was chosen as the mobile phase.

[0178] 2.3.2. Selection of Column Temperature

[0179] Accurately weigh 1g of Zishen Yutai Pill powder and place it in a stoppered conical flask. Accurately add 20mL of 50% methanol, seal tightly, weigh, sonicate for 30min, cool, and weigh again. Make up the weight loss with 50% methanol, shake well, and filter to obtain the test solution. Investigate the effect of column temperature (25℃, 30℃, 35℃) on the chromatogram. Figure 4 It is known that the number of chromatographic peaks is the highest when the column temperature is 30℃, while at column temperatures of 25℃ and 35℃, two chromatographic peaks overlap at 30 min. Therefore, the column temperature of 30℃ was chosen.

[0180] 2.3.3. Established chromatographic conditions

[0181] The chromatographic column was a Waters Xselect HSS T3 (4.6×250mm, 5μm), and the operation was carried out at 30℃. The mobile phase consisted of 0.1% phosphoric acid water and acetonitrile. Gradient elution was used, and the flow rate was kept constant at 1mL / min. Loganin, loganic acid, and swertiamarin were detected at 240nm, 5-hydroxymethylfurfural was detected at 284nm, and chlorogenic acid and cryptochlorogenic acid were detected at 324nm. The injection volume was set to 10μL, and the elution program is shown in Table 3.

[0182] Table 3 Elution gradient for content determination of Zishen Yutai Pills

[0183]

[0184] 2.4. System Adaptability

[0185] Take the mixed reference solution from section 2.1 and the test solution from section 2.2.3, and inject them according to the chromatographic conditions in section 2.3.3. The detection wavelength is 240 nm. All six components showed absorption at this wavelength, and the results are as follows. Figure 5 and Figure 6 As shown, the separation between the chromatographic peaks is good. Peak 1 is 5-hydroxymethylfurfural, peak 2 is loganic acid, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 5 is loganin, and peak 6 is swertiamarin.

[0186] 2.5. Plot the standard curve

[0187] Take the mixed reference stock solution prepared under section 2.1, dilute it to a certain concentration, and inject it according to the chromatographic conditions in section 2.3.3. Plot the standard curve of reference concentration versus peak area, and obtain the regression equation. The results are shown in Table 4.

[0188] Table 4. Linearity Relationship in Content Determination

[0189]

[0190] 2.6. Methodological Examination

[0191] 2.6.1. Precision Test

[0192] Accurately pipette the mixed reference solution under section 2.1, and inject it 6 times consecutively under the chromatographic conditions of 2.3.3. Record the peak areas of the 6 components respectively, and calculate the linearity under section 2.5 (2). The results are shown in Table 5 below.

[0193] Table 5 Precision Test Table for Content Determination

[0194]

[0195]

[0196] 2.6.2. Stability Test

[0197] Accurately weigh the sample of Zishen Yutai Pill (batch number A00001M), prepare the test solution according to section 2.2.3, and inject it at 0, 2, 4, 8, 12 and 24 h under the chromatographic conditions in section 2.3.3. The results are shown in Table 6 below.

[0198] Table 6. Stability Test Table for Content Determination

[0199]

[0200] 2.6.3. Repeatability Test

[0201] Accurately weigh the sample of Zishen Yutai Pill (batch number A00001M), prepare 6 test solutions in parallel according to section 2.2.3, and inject them under the chromatographic conditions in section 2.3.3. The results are shown in Table 7.

[0202] Table 7 Repeatability Test Table for Content Determination

[0203]

[0204] 2.6.4. Recovery Test

[0205] Take approximately 0.5g of the same batch of sample (batch number A00001M) with known content, weigh 6 portions accurately, and add reference solutions with the same content as each component of Zishen Yutai Pill to each portion. Prepare 6 test solutions in parallel according to section 2.2.3, inject the samples under the chromatographic conditions in section 2.3.3, record the peak area, and calculate the recovery rate by linearity test in section 2.5. The results are shown in Tables 8 to 13.

[0206] Table 8. Recovery Rate Test Table for 5-Hydroxymethylfurfural Content Determination

[0207]

[0208] Table 9. Recovery Rate Test for Strychnosine Content Determination

[0209]

[0210]

[0211] Table 10. Chlorogenic Acid Content Determination and Recovery Rate Test Table

[0212]

[0213] Table 11 Recovery Rate Test Table for Cryptochlorogenic Acid Content Determination

[0214]

[0215] Table 12. Recovery Rate Test of Loganin Content Determination

[0216]

[0217] Table 13. Recovery Rate Test of Swertiazide Content Determination

[0218]

[0219] 2.7 Determination of sample content

[0220] Based on the established method, the content of six components in Zishen Yutai Pill was determined, and the results are shown in Table 14.

[0221] Table 14: Content Determination Table for Each Sample

[0222]

[0223]

[0224] This application establishes suitable chromatographic conditions to obtain characteristic peak data with good separation, and performs content determination based on chromatographic data. At the same time, multiple wavelengths are used for simultaneous determination, with loganic acid, loganin, and swertiamarin determined at 240 nm, 5-hydroxymethylfurfural at 284 nm, and chlorogenic acid and cryptochlorogenic acid determined at 324 nm. This successfully establishes a method for determining the content of six components in Zishen Yutai Pill.

[0225] 3. Establishment of a multi-assessment method

[0226] 3.1. Determination of relative correction factor (fs / i)

[0227] Accurately pipette 10 μL each of the mixed reference solutions I to VII under section “2.1” and inject them into the high performance liquid chromatograph. Record the peak areas of the chromatographic peaks. Using loganic acid as an internal reference, calculate the fs / i of 5-hydroxymethylconaldehyde, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin according to the following formula using the multi-point calibration method. The RSD values ​​of the fs / i of the five analytes and the internal reference were all less than 3%, as shown in Table 15.

[0228] fs / i = fs / fi = AsCi / AiCs

[0229] As represents the peak area of ​​loganic acid reference standard, and Cs represents the mass concentration of loganic acid reference standard.

[0230] Ai represents the peak area of ​​the analyte, and Ci represents the mass concentration of the analyte.

[0231] Table 15 fs / i of each component

[0232]

[0233] 3.2. Comparison of the one-measure-multiple-evaluation method and the external standard method

[0234] Take the mixed reference solution from section 2.1 and the test solution from section 2.2.3, and inject them according to the chromatographic conditions in section 2.3.3. Determine the content of six components in 26 batches of Zishen Yutai Pills using the external standard method. Calculate the content of 5-hydroxymethylfurfural, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin in the 26 batches of Zishen Yutai Pills using the fs / i calculated in section 3.1.1.

[0235] The relative error (RE) of the content of 5-hydroxymethylfurfural, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin was calculated between the one-test-multiple-evaluation method and the external standard method. The results (Tables 16-1 and 16-2) showed that the RE% of the content determination results of 26 batches of Zishen Yutai Pills obtained by the one-test-multiple-evaluation method and the external standard method was <3%, with no significant difference.

[0236] Table 16-1 Mass concentrations of six kidney-tonifying components determined by QAMS and external standard methods in 26 batches—loganic acid, 5-hydroxymethylconaldehyde, chlorogenic acid

[0237]

[0238] Table 16-2 shows the mass concentrations of six kidney-tonifying components in 26 batches determined by QAMS and external standard methods—cryptochlorogenic acid, loganin, and swertiamarin.

[0239]

[0240]

[0241] 3.3. Durability Assessment

[0242] 3.3.1. Investigation of Chromatograph and Column

[0243] This experiment investigated the effects of two liquid chromatographs, Waters e2695 and Shimadzu LC-20A, and three chromatographic columns, Waters Xselect HSS T3 (250mm×4.6mm, 5μm), Agient XDB-C18 (250mm×4.6mm, 5μm), and Phenomenex Luna C18 (250mm×4.6mm, 5μm), on fs / i. The RSD was calculated, and the results were all less than 3%, indicating that the different instruments and columns were well-suited for use. The detection results are shown in Table 17.

[0244] Table 17

[0245]

[0246] 3.3.2. Effect of different temperatures on fs / i

[0247] This experiment used a Waters e2695 chromatograph and a Waters Xselect HSS T3 column to investigate the effects of 25℃, 28℃, 30℃, and 32℃ on fs / i and calculated the RSD. The results were all less than 3%, indicating that different column temperatures had no significant effect on each component. The detection results are shown in Table 18.

[0248] Table 18

[0249]

[0250]

[0251] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0252] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0253] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic spectrum of a kidney-nourishing and fetal-nurturing pill, characterized in that, Includes the following steps: A mixed reference solution is provided, the mixed reference solution comprising the following components: 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin. Zishen Yutai Pills were extracted with an extraction solvent to obtain a test solution. The extraction solvent was 20%–80% (v / v) of methanol, and the extraction method was ultrasound. The mixed reference solution and the test solution were subjected to high-performance liquid chromatography (HPLC) analysis to obtain chromatograms of the reference solution and the test solution, respectively. By comparing the chromatograms of the reference standard and the test sample, a characteristic chromatogram of Zishen Yutai Pill was determined. The conditions for the liquid chromatography analysis include: (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume percentage of phosphoric acid in the aqueous solution of phosphoric acid is 0.05%~0.3%. (2) The elution process includes: From 0 to 1 minute, the volume percentage of the mobile phase A is maintained at 5%. Within 1 to 3 minutes, the volume percentage of mobile phase A increased from 5% to 7%. Within 3 to 12 minutes, the volume percentage of mobile phase A increased from 7% to 16%. Between 12 and 28 minutes, the volume percentage of mobile phase A increased from 16% to 25%. (3) The detection wavelength is 240 nm; (4) The chromatographic column is a C18 column.

2. The method for constructing a feature map according to claim 1, characterized in that, The conditions for the liquid chromatography analysis also include at least one of the following: (1) The flow rate is 0.8 mL / min to 1.2 mL / min. (2) The column temperature is 25℃~45℃. (3) The injection volume is 8 μL to 12 μL.

3. The method for constructing a feature map according to claim 1 or 2, characterized in that, In the reference solution, the mass concentration of each component is independently selected from 0.16 mg / mL to 0.22 mg / mL.

4. The method for constructing a feature map according to claim 1 or 2, characterized in that, The power of the ultrasound is 125W~350W and the frequency is 35kHz~45kHz.

5. The method for constructing a feature map according to claim 1 or 2, characterized in that, The extraction time is 15 min to 60 min.

6. A method for determining the content of characteristic components in a kidney-nourishing and fetal-nurturing pill, characterized in that, Includes the following steps: A mixed reference solution of different concentrations is provided, the mixed reference solution comprising the following components: 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin. Different concentrations of the mixed reference standard solution were analyzed by liquid chromatography to obtain chromatograms of the reference standard. The peak area and mass concentration of each component were recorded, and the corresponding standard curves were plotted. Regression equations for each component were obtained. Take the Zishen Yutai Pill to be tested, add an extraction solvent for extraction, and prepare a test solution. The extraction solvent is 20%~80% (v / v) of methanol, and the extraction method is ultrasound. The test sample solution is subjected to liquid chromatography analysis to obtain a chromatogram of the test sample. The chromatogram of the test sample is compared with the characteristic chromatogram prepared by the construction method according to any one of claims 1 to 5. The characteristic peaks corresponding to the characteristic components 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin and swertiamarin in the chromatogram of the test sample are identified. The peak area of ​​each characteristic peak is recorded and substituted into the corresponding regression equation to calculate the mass concentration. The conditions for the liquid chromatography analysis include: (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume percentage of phosphoric acid in the aqueous solution of phosphoric acid is 0.05%~0.3%. (2) The elution process includes: From 0 to 1 minute, the volume percentage of the mobile phase A is maintained at 5%. Within 1 to 3 minutes, the volume percentage of mobile phase A increased from 5% to 7%. Within 3 to 12 minutes, the volume percentage of mobile phase A increased from 7% to 16%. Between 12 and 28 minutes, the volume percentage of mobile phase A increased from 16% to 25%. (3) The detection wavelength is 240 nm; (4) The chromatographic column is a C18 column.

7. The content determination method according to claim 6, characterized in that, The conditions for the liquid chromatography analysis also include at least one of the following: (1) The flow rate is 0.8 mL / min to 1.2 mL / min. (2) The column temperature is 25℃~45℃. (3) The injection volume is 8 μL to 12 μL.

8. The content determination method according to claim 6 or 7, characterized in that, In the reference solution, the mass concentration of each component is independently selected from 0.16 mg / mL to 0.22 mg / mL.

9. The content determination method according to claim 6 or 7, characterized in that, The power of the ultrasound is 125W~350W and the frequency is 35kHz~45kHz.

10. The content determination method according to claim 6 or 7, characterized in that, The extraction time is 15 min to 60 min.

11. A method for multiple quality assessments of Zishen Yutai Pill, characterized in that, Includes the following steps: A mixed reference solution is provided, the mixed reference solution comprising the following components: 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin. The mixed reference solution was subjected to liquid chromatography analysis. The peak area and mass concentration of each component were recorded. An internal reference was selected based on the chromatographic analysis results. The relative correction factors of the remaining components relative to the internal reference were calculated. Take the Zishen Yutai Pill to be tested, add an extraction solvent for extraction, and prepare a test solution. The extraction solvent is 20%~80% (v / v) of methanol, and the extraction method is ultrasound. The test sample solution was subjected to high-performance liquid chromatography (HPLC) analysis to obtain a chromatogram of the test sample. The chromatogram of the test sample was compared with the characteristic chromatogram obtained by the construction method according to any one of claims 1 to 5. The characteristic peaks corresponding to the characteristic components 5-hydroxymethylfurfural, loganic acid, chlorogenic acid, cryptochlorogenic acid, loganin, and swertiamarin in the chromatogram of the test sample were identified, and the peak areas of each characteristic peak were recorded. The mass concentration of the characteristic component used as an internal reference in the Zishen Yutai Pill according to any one of claims 6 to 10 is calculated, and the mass concentration of the remaining characteristic components is calculated by substituting them into the mass concentration calculation formula. The conditions for the liquid chromatography analysis include: (1) Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume percentage of phosphoric acid in the aqueous solution of phosphoric acid is 0.05%~0.3%. (2) The elution process includes: From 0 to 1 minute, the volume percentage of the mobile phase A is maintained at 5%. Within 1 to 3 minutes, the volume percentage of mobile phase A increased from 5% to 7%. Within 3 to 12 minutes, the volume percentage of mobile phase A increased from 7% to 16%. Between 12 and 28 minutes, the volume percentage of mobile phase A increased from 16% to 25%. (3) The detection wavelength is 240 nm; (4) The chromatographic column is a C18 column.

12. The quality testing method with multiple evaluations in one test according to claim 11, characterized in that, The conditions for the liquid chromatography analysis also include at least one of the following: (1) The flow rate is 0.8 mL / min to 1.2 mL / min. (2) The column temperature is 25℃~45℃. (3) The injection volume is 8 μL to 12 μL.

13. The quality inspection method with multiple evaluations in one test according to claim 11 or 12, characterized in that, In the reference solution, the mass concentration of each component is independently selected from 0.16 to 0.22 mg / mL.

14. The quality inspection method with multiple evaluations in one test according to claim 11 or 12, characterized in that, The power of the ultrasound is 125W~350W and the frequency is 35kHz~45kHz.

15. The quality inspection method with multiple evaluations in one test according to claim 11 or 12, characterized in that, The extraction time is 15 min to 60 min.

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