Fingerprint construction and detection method for highly water-soluble components in Shenqi Fuzheng Injection

Abstract

This invention relates to a method for constructing and detecting fingerprint spectra of highly water-soluble components in Shenqi Fuzheng Injection. The fingerprint spectra construction method includes the following steps: First, solid-phase extraction of Shenqi Fuzheng Injection is performed using a solid-phase extraction column with a non-polar divinylphenyl neutral polymer adsorbent as the packing material, followed by centrifugation to remove impurities using a suitable solvent; then, a test sample is prepared using amide-bonded hybrid silica gel as the packing material under suitable mobile phase conditions; finally, the test sample is analyzed by high-performance liquid chromatography (HPLC) using amide-bonded ethylene-bridged hybrid silica gel as the packing material under suitable mobile phase conditions to construct the fingerprint spectra. Based on this, the invention achieves the analysis and detection of highly water-soluble components in Shenqi Fuzheng Injection. The method is simple, effective, and provides stable and reproducible analytical results, which is beneficial for monitoring, detecting, and evaluating the quality of highly water-soluble components in the finished Shenqi Fuzheng Injection product.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and in particular to a method for constructing and detecting fingerprint spectra of highly water-soluble components in Shenqi Fuzheng Injection. Background Technology

[0002] Shenqi Fuzheng Injection is an intravenous infusion preparation made from Astragalus membranaceus and Codonopsis pilosula. It has the effects of strengthening the body's resistance, replenishing qi and nourishing deficiency, and promoting blood circulation and removing blood stasis. Shenqi Fuzheng Injection is the first pure traditional Chinese medicine intravenous infusion approved for marketing. Long-term clinical research and practice have shown that Shenqi Fuzheng Injection has the effect of protecting the hematopoietic system function and improving immune function in cancer patients, making it an effective adjuvant therapy for cancer patients. In addition, Shenqi Fuzheng Injection also has certain therapeutic effects on the elderly and weak, women with menstrual disorders, qi deficiency, and blood deficiency.

[0003] Shenqi Fuzheng Injection is made from two medicinal herbs, Astragalus membranaceus and Codonopsis pilosula. However, the production process involves many steps, diverse chemical components, and complex pharmacodynamic material basis, making its quality control and evaluation difficult. In particular, there is a lack of simple and effective methods for the analysis and characterization of the highly water-soluble components in Shenqi Fuzheng Injection.

[0004] Chinese patents such as CN1899362A, CN101352478A, CN102621244A, and CN103808840A all involve the construction of fingerprint spectra for Shenqi Fuzheng Injection. However, the characteristic components in their fingerprint spectra are all glycosides, saponins, and phenolic compounds, and they do not involve highly water-soluble components such as sugars, polyols, nucleosides, and proline in Shenqi Fuzheng Injection. Since highly water-soluble components are also effective components in Shenqi Fuzheng Injection, analyzing and monitoring these components, and establishing fingerprint spectra and detection methods for highly water-soluble components in Shenqi Fuzheng Injection, is of great significance for improving the quality control and safety of Shenqi Fuzheng Injection, in order to ensure the consistency of injection quality and efficacy. Summary of the Invention

[0005] Based on this, the present invention provides a method for constructing a fingerprint spectrum of highly water-soluble components in Shenqi Fuzheng Injection. This fingerprint spectrum construction method establishes a fingerprint spectrum for highly water-soluble components in Shenqi Fuzheng Injection, enabling accurate analysis of the quality of highly water-soluble components in Shenqi Fuzheng Injection, and the analysis results are reproducible. Simultaneously, the present invention also provides a method for detecting highly water-soluble components in Shenqi Fuzheng Injection using this fingerprint spectrum.

[0006] In a first aspect, the present invention provides a method for constructing a fingerprint spectrum of a highly water-soluble component in Shenqi Fuzheng Injection, comprising the following steps:

[0007] Dissolve glucose, sucrose, and fructose reference standards in solvent A to prepare reference solution A;

[0008] Sorbitol, mannose, and sucrose reference standards were dissolved in solvent B to prepare reference solution B.

[0009] The sample of Shenqi Fuzheng Injection was subjected to solid-phase extraction. The solid-phase extraction column used was filled with a neutral polymer adsorbent of non-polar divinylphenyl. The eluent was collected, and then the eluent was evaporated to dryness. The residue was mixed with a mixture of water and acetonitrile, centrifuged, and the supernatant was collected.

[0010] The supernatant was separated from the reference solution A by high performance liquid chromatography. The separated liquid corresponding to the retention time of the characteristic peak of the reference solution A was collected from the supernatant. After removing the solvent, it was dissolved in a mixture of water and acetonitrile to prepare the test solution.

[0011] The test solution and reference solution B were subjected to high performance liquid chromatography to obtain the fingerprint spectrum of the highly water-soluble components in the Shenqi Fuzheng injection.

[0012] The conditions for separation by high performance liquid chromatography include:

[0013] The chromatographic column is packed with amide-bonded hybrid silica gel;

[0014] The mobile phase includes mobile phase A1, mobile phase B1 and mobile phase C1, wherein mobile phase A1 is an acetonitrile solution containing triethylamine or a methanol solution containing triethylamine, mobile phase B1 is an aqueous solution containing triethylamine, and mobile phase C1 is a mixed solution of acetonitrile and water containing ammonium acetate or a mixed solution of acetonitrile and water containing ammonium formate.

[0015] The conditions for detection by high performance liquid chromatography include:

[0016] The chromatographic column was packed with amide-bonded ethylene-bridged hybrid silica gel.

[0017] The mobile phase includes mobile phase A2 and mobile phase B2, wherein mobile phase A2 is a mixed solution of acetonitrile, methanol, and acetonitrile-water containing ammonium acetate or a mixed solution of acetonitrile-water containing ammonium formate, and mobile phase B2 is an aqueous solution containing ammonium acetate or an aqueous solution containing ammonium formate.

[0018] In one embodiment, in the high-performance liquid chromatography separation conditions, mobile phase A1 is an acetonitrile solution of triethylamine with a volume fraction of 0.01% to 2%, mobile phase B1 is an aqueous solution of triethylamine with a volume fraction of 0.01% to 2%, and mobile phase C1 is an acetonitrile-water mixed solution of ammonium acetate with a concentration of 5 to 40 mmol / L, wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (0.8 to 1.2):(0.8 to 1.2).

[0019] In one embodiment, the high-performance liquid chromatography (HPLC) separation conditions involve gradient elution of the mobile phase, wherein the gradient elution includes:

[0020] From 0 to 5 min, the volume percentage of mobile phase A1 was maintained at 90%, the volume percentage of mobile phase B1 was maintained at 10%, and the volume percentage of mobile phase C1 was maintained at 0%.

[0021] Over a period of 5–5.1 min, the volume percentage of mobile phase A1 changed from 90% to 50%, the volume percentage of mobile phase B1 changed from 10% to 50%, and the volume percentage of mobile phase C1 remained at 0%.

[0022] 5.1–11 min, the volume percentage of mobile phase A1 is maintained at 50%, the volume percentage of mobile phase B1 is maintained at 50%, and the volume percentage of mobile phase C1 is maintained at 0%.

[0023] Over 11–11.5 min, the volume percentage of mobile phase A1 changed from 50% to 0%, the volume percentage of mobile phase B1 changed from 50% to 0%, and the volume percentage of mobile phase C1 changed from 0% to 100%.

[0024] From 11.5 to 20 min, the volume percentage of mobile phase A1 remained at 0%, the volume percentage of mobile phase B1 remained at 0%, and the volume percentage of mobile phase C1 changed from 100% to 0%.

[0025] In one embodiment, the conditions for separation by high-performance liquid chromatography further include:

[0026] An electro-fogging detector was used for detection, with a column temperature of 20℃~40℃ and a flow rate of 0.5~1.5mL / min.

[0027] In one embodiment, in the high performance liquid chromatography detection conditions, mobile phase A2 is an acetonitrile-water mixed solution of ammonium acetate with a concentration of 1-10 mmol / L, wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (97-99):(1-3), and mobile phase B2 is an aqueous solution of ammonium acetate with a concentration of 5-40 mmol / L.

[0028] In one embodiment, the high-performance liquid chromatography (HPLC) detection conditions involve gradient elution of the mobile phase, wherein the gradient elution includes:

[0029] From 0 to 5 min, the volume percentage of mobile phase A2 was maintained at 95%, and the volume percentage of mobile phase B2 was maintained at 5%.

[0030] Over 5–25 minutes, the volume percentage of mobile phase A2 changed from 95% to 85%, and the volume percentage of mobile phase B2 changed from 5% to 15%.

[0031] Over 25–30 minutes, the volume percentage of mobile phase A2 changed from 85% to 65%, and the volume percentage of mobile phase B2 changed from 15% to 35%.

[0032] For 30–35 min, the volume percentage of mobile phase A2 is maintained at 65%, and the volume percentage of mobile phase B2 is maintained at 35%.

[0033] Over 35–36 minutes, the volume percentage of mobile phase A2 changed from 65% to 95%, and the volume percentage of mobile phase B2 changed from 35% to 5%.

[0034] For 36–45 min, the volume percentage of mobile phase A2 is maintained at 95%, and the volume percentage of mobile phase B2 is maintained at 5%.

[0035] In one embodiment, the conditions for detection by high-performance liquid chromatography further include:

[0036] An electro-fogging detector was used for detection, with a column temperature of 40℃~80℃ and a flow rate of 0.2~0.4mL / min.

[0037] In one embodiment, water is used as the eluent in the solid-phase extraction.

[0038] In one embodiment, during the mixing of the resulting distillation residue with a mixture of water and acetonitrile, the volume ratio of water to acetonitrile is (0.1–1):(0.1–1); and / or

[0039] The centrifugation conditions include: a rotation speed of 10,000–16,000 rpm and a time of 5–30 min; and / or

[0040] During the dissolution process using a mixture of water and acetonitrile, the volume ratio of water to acetonitrile is (0.8–1.2):(0.8–1.2).

[0041] In one embodiment, solvent A is a mixture of water and acetonitrile in a volume ratio of (0.8-1.2):(0.8-1.2); solvent B is a mixture of water and acetonitrile in a volume ratio of (0.8-1.2):(0.8-1.2).

[0042] In one embodiment, the fingerprint spectrum of the water-soluble components in the Shenqi Fuzheng Injection contains 10 characteristic peaks, which include characteristic peaks of fructose, sorbitol, mannose, glucose, proline, inositol and sucrose.

[0043] A second aspect of the present invention provides a method for detecting highly water-soluble components in Shenqi Fuzheng Injection, comprising the following steps:

[0044] The sample of Shenqi Fuzheng Injection to be tested was subjected to solid-phase extraction. The solid-phase extraction column used was filled with a neutral polymer adsorbent of non-polar divinylphenyl. The eluent was collected, and then the eluent was evaporated to dryness. The residue was mixed with a mixture of water and acetonitrile, centrifuged, and the supernatant was collected.

[0045] The supernatant was separated from the reference solution A by high performance liquid chromatography. The separated liquid corresponding to the retention time of the characteristic peak of the reference solution A was collected from the supernatant. After removing the solvent, it was dissolved in a mixture of water and acetonitrile to prepare the sample solution to be tested.

[0046] The sample solution to be tested is detected by high performance liquid chromatography to obtain a chromatogram of the highly water-soluble components in the Shenqi Fuzheng Injection. The chromatogram is then compared with the fingerprint chromatogram obtained by the fingerprint chromatogram construction method for the highly water-soluble components in the Shenqi Fuzheng Injection described in the first aspect.

[0047] The conditions for separation by high performance liquid chromatography include:

[0048] The chromatographic column is packed with amide-bonded hybrid silica gel;

[0049] The mobile phase includes mobile phase A1, mobile phase B1 and mobile phase C1, wherein mobile phase A1 is an acetonitrile solution containing triethylamine or a methanol solution containing triethylamine, mobile phase B1 is an aqueous solution containing triethylamine, and mobile phase C1 is a mixed solution of acetonitrile and water containing ammonium acetate or a mixed solution of acetonitrile and water containing ammonium formate.

[0050] The conditions for detection by high performance liquid chromatography include:

[0051] The chromatographic column was packed with amide-bonded ethylene-bridged hybrid silica gel.

[0052] The mobile phase includes mobile phase A2 and mobile phase B2, wherein mobile phase A2 is a mixed solution of acetonitrile, methanol, and acetonitrile-water containing ammonium acetate or a mixed solution of acetonitrile-water containing ammonium formate, and mobile phase B2 is an aqueous solution containing ammonium acetate or an aqueous solution containing ammonium formate.

[0053] In one embodiment, in the high-performance liquid chromatography separation conditions, mobile phase A1 is an acetonitrile solution of triethylamine with a volume fraction of 0.01% to 2%, mobile phase B1 is an aqueous solution of triethylamine with a volume fraction of 0.01% to 2%, and mobile phase C1 is an acetonitrile-water mixed solution of ammonium acetate with a concentration of 5 to 40 mmol / L, wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (0.8 to 1.2):(0.8 to 1.2).

[0054] In one embodiment, the high-performance liquid chromatography (HPLC) separation conditions involve gradient elution of the mobile phase, wherein the gradient elution includes:

[0055] From 0 to 5 min, the volume percentage of mobile phase A1 was maintained at 90%, the volume percentage of mobile phase B1 was maintained at 10%, and the volume percentage of mobile phase C1 was maintained at 0%.

[0056] Over a period of 5–5.1 min, the volume percentage of mobile phase A1 changed from 90% to 50%, the volume percentage of mobile phase B1 changed from 10% to 50%, and the volume percentage of mobile phase C1 remained at 0%.

[0057] 5.1–11 min, the volume percentage of mobile phase A1 is maintained at 50%, the volume percentage of mobile phase B1 is maintained at 50%, and the volume percentage of mobile phase C1 is maintained at 0%.

[0058] Over 11–11.5 min, the volume percentage of mobile phase A1 changed from 50% to 0%, the volume percentage of mobile phase B1 changed from 50% to 0%, and the volume percentage of mobile phase C1 changed from 0% to 100%.

[0059] From 11.5 to 20 min, the volume percentage of mobile phase A1 remained at 0%, the volume percentage of mobile phase B1 remained at 0%, and the volume percentage of mobile phase C1 changed from 100% to 0%.

[0060] In one embodiment, the conditions for separation by high-performance liquid chromatography further include:

[0061] An electro-fogging detector was used for detection, with a column temperature of 20℃~40℃ and a flow rate of 0.5~1.5mL / min.

[0062] In one embodiment, in the high performance liquid chromatography detection conditions, mobile phase A2 is an acetonitrile-water mixed solution of ammonium acetate with a concentration of 1-10 mmol / L, wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (97-99):(1-3), and mobile phase B2 is an aqueous solution of ammonium acetate with a concentration of 5-40 mmol / L.

[0063] In one embodiment, the high-performance liquid chromatography (HPLC) detection conditions involve gradient elution of the mobile phase, wherein the gradient elution includes:

[0064] From 0 to 5 min, the volume percentage of mobile phase A2 was maintained at 95%, and the volume percentage of mobile phase B2 was maintained at 5%.

[0065] Over 5–25 minutes, the volume percentage of mobile phase A2 changed from 95% to 85%, and the volume percentage of mobile phase B2 changed from 5% to 15%.

[0066] Over 25–30 minutes, the volume percentage of mobile phase A2 changed from 85% to 65%, and the volume percentage of mobile phase B2 changed from 15% to 35%.

[0067] For 30–35 min, the volume percentage of mobile phase A2 is maintained at 65%, and the volume percentage of mobile phase B2 is maintained at 35%.

[0068] Over 35–36 minutes, the volume percentage of mobile phase A2 changed from 65% to 95%, and the volume percentage of mobile phase B2 changed from 35% to 5%.

[0069] For 36–45 min, the volume percentage of mobile phase A2 is maintained at 95%, and the volume percentage of mobile phase B2 is maintained at 5%.

[0070] In one embodiment, the conditions for detection by high-performance liquid chromatography further include:

[0071] An electro-fogging detector was used for detection, with a column temperature of 40℃~80℃ and a flow rate of 0.2~0.4mL / min.

[0072] In one embodiment, water is used as the eluent in the solid-phase extraction.

[0073] In one embodiment, during the mixing of the resulting distillation residue with a mixture of water and acetonitrile, the volume ratio of water to acetonitrile is (0.1–1):(0.1–1); and / or

[0074] The centrifugation conditions include: a rotation speed of 10,000–16,000 rpm and a time of 5–30 min; and / or

[0075] During the dissolution process using a mixture of water and acetonitrile, the volume ratio of water to acetonitrile is (0.8–1.2):(0.8–1.2).

[0076] During the research process, this invention discovered the following difficulties in the analysis and characterization of highly water-soluble components in Shenqi Fuzheng Injection: (1) The excipients in Shenqi Fuzheng Injection mainly include sodium chloride, disodium EDTA, and sodium metabisulfite, which can interfere with the analysis of highly water-soluble components; (2) It is difficult to distinguish between sugar components, polyol components, and excipient salts in Shenqi Fuzheng Injection. To address these difficulties, we established the following analytical method:

[0077] First, this invention utilizes a solid-phase extraction column with a neutral polymer adsorbent of nonpolar divinylphenyl as the packing material to perform solid-phase extraction of Shenqi Fuzheng Injection, followed by centrifugation with a suitable solvent to remove impurities, thus effectively enriching the target compound to be analyzed. Then, using amide-bonded hybrid silica gel as the packing material, under suitable mobile phase conditions, the target compound is desalted and enriched to prepare a test sample. Finally, using amide-bonded ethylene-bridged hybrid silica gel as the packing material, under suitable mobile phase conditions, the test sample is analyzed by high-performance liquid chromatography (HPLC) to construct a fingerprint spectrum.

[0078] Based on this, the present invention establishes a fingerprint spectrum and detection method for highly water-soluble components in Shenqi Fuzheng Injection, realizing the analysis and detection of highly water-soluble components in Shenqi Fuzheng Injection. Moreover, the method is simple to operate, effective, and the analysis results can be stably reproduced, which is beneficial for the monitoring, detection and quality evaluation of highly water-soluble components in the finished product of Shenqi Fuzheng Injection. Attached Figure Description

[0079] Figure 1 The reference fingerprint spectrum of the highly water-soluble components of Shenqi Fuzheng Injection is shown, with common characteristic peaks 1 to 10 marked from left to right, namely: common peak 1, common peak 2, fructose, sorbitol, mannose, glucose, common peak 7, proline, inositol and sucrose.

[0080] Figure 2 Fingerprint chromatograms of the strongly water-soluble components of the Shenqi Fuzheng Injection test sample and their reference standards. Detailed Implementation

[0081] The fingerprinting method and detection method for the highly water-soluble components in the Astragalus and Ginseng Injection of the present invention will be further described in detail below with reference to specific embodiments. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention.

[0082] 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 invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0083] As used herein, the terms “and / or,” “or / and,” and “and / or” may include any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all the related listed items.

[0084] In this invention, terms such as "first aspect" and "second aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, terms such as "first" and "second" serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on the quantity.

[0085] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0086] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0087] Unless otherwise specified, the percentage content involved in this invention refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0088] Unless otherwise specified, all percentage concentrations mentioned in this invention refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0089] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument.

[0090] In this invention, "strong water-soluble components in Shenqi Fuzheng Injection" refers to components such as sugars, polyols, nucleosides, and proline in Shenqi Fuzheng Injection.

[0091] This invention provides a method for constructing a fingerprint spectrum of highly water-soluble components in Shenqi Fuzheng Injection, comprising the following steps:

[0092] Dissolve glucose, sucrose, and fructose reference standards in solvent A to prepare reference solution A;

[0093] Sorbitol, mannose, and sucrose reference standards were dissolved in solvent B to prepare reference solution B.

[0094] The sample of Shenqi Fuzheng Injection was subjected to solid-phase extraction. The solid-phase extraction column used was filled with a neutral polymer adsorbent of non-polar divinylphenyl. The eluent was collected, and then the eluent was evaporated to dryness. The residue was mixed with a mixture of water and acetonitrile, centrifuged, and the supernatant was collected.

[0095] The supernatant was separated from the reference solution A by high performance liquid chromatography. The separated liquid corresponding to the retention time of the characteristic peak of the reference solution A was collected from the supernatant. After removing the solvent, it was dissolved in a mixture of water and acetonitrile to prepare the test solution.

[0096] The test solution and reference solution B were subjected to high performance liquid chromatography to obtain the fingerprint spectrum of the highly water-soluble components in the Shenqi Fuzheng injection.

[0097] The conditions for separation by high performance liquid chromatography include:

[0098] The chromatographic column is packed with amide-bonded hybrid silica gel;

[0099] The mobile phase includes mobile phase A1, mobile phase B1 and mobile phase C1, wherein mobile phase A1 is an acetonitrile solution containing triethylamine or a methanol solution containing triethylamine, mobile phase B1 is an aqueous solution containing triethylamine, and mobile phase C1 is a mixed solution of acetonitrile and water containing ammonium acetate or a mixed solution of acetonitrile and water containing ammonium formate.

[0100] The conditions for detection by high performance liquid chromatography include:

[0101] The chromatographic column was packed with amide-bonded ethylene-bridged hybrid silica gel.

[0102] The mobile phase includes mobile phase A2 and mobile phase B2, wherein mobile phase A2 is a mixed solution of acetonitrile, methanol, and acetonitrile-water containing ammonium acetate or a mixed solution of acetonitrile-water containing ammonium formate, and mobile phase B2 is an aqueous solution containing ammonium acetate or an aqueous solution containing ammonium formate.

[0103] In one example, the high-performance liquid chromatography (HPLC) separation conditions include a column length of 100 mm to 250 mm, an inner diameter of 3 mm to 5 mm, and a packing particle size of 1 μm to 5 μm. Further, the column is BEH Amide, 150 mm × 4.6 mm, 3.5 μm.

[0104] In one example, under the conditions of the high-performance liquid chromatography (HPLC) separation, mobile phase A1 is an acetonitrile solution of triethylamine with a volume fraction of 0.01%–2%, mobile phase B1 is an aqueous solution of triethylamine with a volume fraction of 0.01%–2%, and mobile phase C1 is an acetonitrile-water mixture of ammonium acetate with a concentration of 5–40 mmol / L, wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixture is (0.8–1.2):(0.8–1.2). Further, mobile phase A1 is an acetonitrile solution of triethylamine with a volume fraction of 0.03%–0.07%, mobile phase B1 is an aqueous solution of triethylamine with a volume fraction of 0.03%–0.07%, and mobile phase C1 is an acetonitrile-water mixture of ammonium acetate with a concentration of 8–12 mmol / L. Furthermore, mobile phase A1 is an acetonitrile solution of triethylamine with a volume fraction of 0.05%, mobile phase B1 is an aqueous solution of triethylamine with a volume fraction of 0.05%, and mobile phase C1 is a mixed solution of ammonium acetate in acetonitrile and water with a concentration of 10 mmol / L. Additionally, the volume ratio of acetonitrile to water in the mixed solution is 1:1.

[0105] In one example, the high-performance liquid chromatography (HPLC) separation conditions involve gradient elution of the mobile phase, wherein the gradient elution includes:

[0106] From 0 to 5 min, the volume percentage of mobile phase A1 was maintained at 90%, the volume percentage of mobile phase B1 was maintained at 10%, and the volume percentage of mobile phase C1 was maintained at 0%.

[0107] Over a period of 5–5.1 min, the volume percentage of mobile phase A1 changed from 90% to 50%, the volume percentage of mobile phase B1 changed from 10% to 50%, and the volume percentage of mobile phase C1 remained at 0%.

[0108] 5.1–11 min, the volume percentage of mobile phase A1 is maintained at 50%, the volume percentage of mobile phase B1 is maintained at 50%, and the volume percentage of mobile phase C1 is maintained at 0%.

[0109] Over 11–11.5 min, the volume percentage of mobile phase A1 changed from 50% to 0%, the volume percentage of mobile phase B1 changed from 50% to 0%, and the volume percentage of mobile phase C1 changed from 0% to 100%.

[0110] From 11.5 to 20 min, the volume percentage of mobile phase A1 remained at 0%, the volume percentage of mobile phase B1 remained at 0%, and the volume percentage of mobile phase C1 changed from 100% to 0%.

[0111] In one example, the separation conditions for the high-performance liquid chromatography (HPLC) further include: detection using an electro-fogging detector, a column temperature of 20°C–40°C, and a flow rate of 0.5–1.5 mL / min. Further, the column temperature is 28°C–32°C, and the flow rate is 0.7–0.9 mL / min. Even further, the column temperature is 30°C, and the flow rate is 0.8 mL / min.

[0112] In one example, the conditions for separation by high-performance liquid chromatography further include: an injection volume of 10–50 μL. More specifically, the injection volume is 15–25 μL. Even more specifically, the injection volume is 20 μL.

[0113] In one example, during the high-performance liquid chromatography separation process, collecting the separation liquid in the supernatant corresponding to the retention time of the characteristic peak of the reference solution A means collecting the separation liquid within ±0.5 min of the retention time of the characteristic peak of the reference solution A.

[0114] In one example, the high-performance liquid chromatography (HPLC) detection conditions include a column length of 50 mm to 150 mm, an inner diameter of 1.5 mm to 3 mm, and a packing particle size of 1 μm to 3 μm. Further, the column is BEH Amide, 100 mm × 2.1 mm, 1.7 μm.

[0115] In one example, under the conditions of the high-performance liquid chromatography (HPLC) detection, mobile phase A2 is an acetonitrile-water mixture of ammonium acetate with a concentration of 1–10 mmol / L, wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixture is (97–99):(1–3), and mobile phase B2 is an aqueous solution of ammonium acetate with a concentration of 5–40 mmol / L. Further, mobile phase A2 is an acetonitrile-water mixture of ammonium acetate with a concentration of 4–6 mmol / L, and mobile phase B2 is an aqueous solution of ammonium acetate with a concentration of 18–22 mmol / L. Even further, mobile phase A2 is an acetonitrile-water mixture of ammonium acetate with a concentration of 5 mmol / L, and mobile phase B2 is an aqueous solution of ammonium acetate with a concentration of 20 mmol / L. Additionally, the volume ratio of acetonitrile to water in the acetonitrile-water mixture is 99:1.

[0116] In one example, the conditions for detection by high-performance liquid chromatography (HPLC) involve elution of the mobile phase using a gradient elution method, wherein the gradient elution includes:

[0117] From 0 to 5 min, the volume percentage of mobile phase A2 was maintained at 95%, and the volume percentage of mobile phase B2 was maintained at 5%.

[0118] Over 5–25 minutes, the volume percentage of mobile phase A2 changed from 95% to 85%, and the volume percentage of mobile phase B2 changed from 5% to 15%.

[0119] Over 25–30 minutes, the volume percentage of mobile phase A2 changed from 85% to 65%, and the volume percentage of mobile phase B2 changed from 15% to 35%.

[0120] For 30–35 min, the volume percentage of mobile phase A2 is maintained at 65%, and the volume percentage of mobile phase B2 is maintained at 35%.

[0121] Over 35–36 minutes, the volume percentage of mobile phase A2 changed from 65% to 95%, and the volume percentage of mobile phase B2 changed from 35% to 5%.

[0122] For 36–45 min, the volume percentage of mobile phase A2 is maintained at 95%, and the volume percentage of mobile phase B2 is maintained at 5%.

[0123] In one example, the detection conditions for the high-performance liquid chromatography (HPLC) further include: detection using an electrospray detector, a column temperature of 40℃~80℃, and a flow rate of 0.2~0.4 mL / min. Further, the column temperature is 55℃~65℃, and the flow rate is 0.3~0.4 mL / min. Even further, the column temperature is 60℃, and the flow rate is 0.35 mL / min.

[0124] In one example, water is used as the eluent in the solid-phase extraction.

[0125] In one example, the solid-phase extraction (SPE) uses a solid-phase extraction column with a specification of 3–20 mL / 100–500 mg and a packing material particle size of 20 μm–80 μm. Further, the SPE column has a specification of 4–8 mL / 180–220 mg and a packing material particle size of 40 μm–50 μm. Even further, the SPE column has a specification of 6 mL / 200 mg and a packing material particle size of 45 μm.

[0126] In one example, during the mixing of the resulting distillate with a mixture of water and acetonitrile, the volume ratio of water to acetonitrile was (0.1–1):(0.1–1). Further, the volume ratio of water to acetonitrile was 1:1.

[0127] In one example, the centrifugation conditions include: a rotation speed of 10,000–16,000 rpm and a time of 5–30 min. Further, the rotation speed is 13,500–14,500 rpm and the time is 5–15 min. Even further, the rotation speed is 14,000 rpm and the time is 10 min.

[0128] In one example, during the dissolution process using a mixture of water and acetonitrile, the volume ratio of water to acetonitrile is (0.8–1.2):(0.8–1.2). Further, the volume ratio of water to acetonitrile is 1:1.

[0129] In one example, solvent A is a mixture of water and acetonitrile in a volume ratio of (0.8–1.2):(0.8–1.2); solvent B is a mixture of water and acetonitrile in a volume ratio of (0.8–1.2):(0.8–1.2). Further, solvent A is a mixture of water and acetonitrile in a volume ratio of 1:1; solvent B is a mixture of water and acetonitrile in a volume ratio of 1:1.

[0130] In one example, reference solution A contains 0.1–1 mg of glucose, sucrose, and fructose reference standards per 1 mL. Further, reference solution A contains 0.2–0.6 mg of glucose, sucrose, and fructose reference standards per 1 mL. Even further, reference solution A contains 0.4 mg of glucose, sucrose, and fructose reference standards per 1 mL.

[0131] In one example, reference solution B contains 0.01–0.05 mg of sorbitol reference standard, 0.01–0.05 mg of mannose reference standard, and 0.1–0.5 mg of sucrose reference standard per 1 mL. Further, reference solution B contains 0.01–0.03 mg of sorbitol reference standard, 0.01–0.03 mg of mannose reference standard, and 0.1–0.3 mg of sucrose reference standard per 1 mL. Even further, reference solution B contains 0.02 mg of sorbitol reference standard, 0.02 mg of mannose reference standard, and 0.2 mg of sucrose reference standard per 1 mL.

[0132] In one example, the fingerprint spectrum of the water-soluble components in the Shenqi Fuzheng Injection contains 10 characteristic peaks, which include characteristic peaks of fructose, sorbitol, mannose, glucose, proline, inositol and sucrose.

[0133] Furthermore, the information of 10 characteristic peaks in the fingerprint spectrum of the water-soluble components in the Shenqi Fuzheng Injection is shown in Table 1 below:

[0134] Table 1

[0135]

[0136]

[0137] This invention also provides a method for detecting highly water-soluble components in Shenqi Fuzheng Injection, comprising the following steps:

[0138] The sample of Shenqi Fuzheng Injection to be tested was subjected to solid-phase extraction. The solid-phase extraction column used was filled with a neutral polymer adsorbent of non-polar divinylphenyl. The eluent was collected, and then the eluent was evaporated to dryness. The residue was mixed with a mixture of water and acetonitrile, centrifuged, and the supernatant was collected.

[0139] The supernatant was separated from the reference solution A by high performance liquid chromatography. The separated liquid corresponding to the retention time of the characteristic peak of the reference solution A was collected from the supernatant. After removing the solvent, it was dissolved in a mixture of water and acetonitrile to prepare the sample solution to be tested.

[0140] The sample solution to be tested was subjected to high performance liquid chromatography to obtain a chromatogram of the highly water-soluble components in the Shenqi Fuzheng Injection. The chromatogram was then compared with the fingerprint chromatogram obtained by the above-mentioned fingerprint chromatogram construction method for highly water-soluble components in Shenqi Fuzheng Injection.

[0141] The conditions for separation by high performance liquid chromatography include:

[0142] The chromatographic column is packed with amide-bonded hybrid silica gel;

[0143] The mobile phase includes mobile phase A1, mobile phase B1 and mobile phase C1, wherein mobile phase A1 is an acetonitrile solution containing triethylamine or a methanol solution containing triethylamine, mobile phase B1 is an aqueous solution containing triethylamine, and mobile phase C1 is a mixed solution of acetonitrile and water containing ammonium acetate or a mixed solution of acetonitrile and water containing ammonium formate.

[0144] The conditions for detection by high performance liquid chromatography include:

[0145] The chromatographic column was packed with amide-bonded ethylene-bridged hybrid silica gel.

[0146] The mobile phase includes mobile phase A2 and mobile phase B2, wherein mobile phase A2 is a mixed solution of acetonitrile, methanol, and acetonitrile-water containing ammonium acetate or a mixed solution of acetonitrile-water containing ammonium formate, and mobile phase B2 is an aqueous solution containing ammonium acetate or an aqueous solution containing ammonium formate.

[0147] Understandably, the preparation of the test sample solution, the separation conditions of the high-performance liquid chromatography (HPLC) method, and the detection conditions of the HPLC method are consistent with the test sample solution, the separation conditions of the HPLC method, and the detection conditions of the HPLC method in the fingerprint chromatogram construction method, and will not be repeated here.

[0148] The following are specific examples.

[0149] The information of the 27 batches of Shenqi Fuzheng Injection samples used in the examples is shown in Table 2 below.

[0150] Table 2

[0151] serial number batch number serial number batch number S1 180208 S14 190104 S2 180209 S15 190112 S3 180501 S16 190113 S4 180502 S17 190120 S5 180708 S18 190130 S6 180805 S19 190131 S7 180819 S20 190135 S8 180901 S21 190137 S9 180908 S22 190144 S10 181027 S23 190407 S11 181112 S24 190416 S12 181213 S25 190421 S13 190101 S26 190605 S27 Non-conforming samples

[0152] Example 1: Establishment of a comparative fingerprint of the highly water-soluble components of Shenqi Fuzheng Injection

[0153] Instruments: Thermo Fisher Scientific REACTI-THERM TS-18824 nitrogen blowing system (Thermo Fisher Scientific, USA); Thermo UltiMate 3000 liquid chromatograph (Thermo Fisher Scientific, USA, Diode Array Detector, Chromeleon 7 workstation); Waters BEH Amide (100mm × 2.1mm, 1.7μm); Waters XBridge Amide (150mm × 4.6mm, 3.5μm).

[0154] Reagents: Acetonitrile was chromatographic grade (Merck, Germany), formic acid was chromatographic grade (SIGMA-ALDAICH, USA), ultrapure water (produced from Synergy UV ultrapure water system), ammonium acetate was chromatographic grade (Honeywell, Fluka, USA), triethylamine was chromatographic grade (Fisher Chemical, USA), and other reagents were all analytical grade (Sinopharm Chemical Reagent Co., Ltd.).

[0155] Chromatographic conditions: Amide-bonded ethylidene-bridged hybrid silica gel was used as the packing material; a Waters BEH Amide column (100 mm × 2.1 mm, 1.7 μm) was used; mobile phase A was 5 mmol / L ammonium acetate acetonitrile-water (99:1) solution, and mobile phase B was 20 mmol / L ammonium acetate solution. Gradient elution was performed according to the specifications in Table 3, with a flow rate of 0.35 mL / min; detection was performed using an electro-fogging detector; column temperature was 60 °C.

[0156] Table 3

[0157]

[0158]

[0159] Preparation of reference solution A: Take appropriate amounts of glucose, sucrose and fructose reference standards, accurately weigh them, and add acetonitrile-water (1:1) to prepare a mixed reference solution containing 0.4 mg of glucose, sucrose and fructose per 1 mL.

[0160] Preparation of reference solution B: Take appropriate amounts of sorbitol, mannose and sucrose reference standards, accurately weigh them, and add acetonitrile-water (1:1) to prepare a mixed reference solution containing 0.02 mg of sorbitol, 0.02 mg of mannose and 0.2 mg of sucrose per 1 mL.

[0161] Preparation of test solutions: Take 15 mL of samples numbered S1 to S26 respectively, add them to the pre-treated solid-phase extraction column (solid-phase extraction column with non-polar divinylphenyl neutral polymer adsorbent as packing material, specification: 6 mL / 200 mg, 45 μm. Pre-washed sequentially with 6 mL methanol and 10 mL water), elute with 10 mL water, collect the eluent, evaporate to dryness; transfer the residue to a 5 mL volumetric flask, add water to the mark, and shake well. Accurately pipette 0.5 mL into a centrifuge tube, accurately add 0.5 mL acetonitrile, shake well; centrifuge at 14000 rpm for 10 minutes, collect the supernatant, purify online under the following chromatographic conditions, and inject 20 μL; simultaneously inject reference solution A for retention time localization.

[0162] The chromatographic column was a Waters XBridge Amide column (150 mm × 4.6 mm, 3.5 μm) with amide-bonded hybrid silica gel as the packing material. The mobile phase consisted of 0.05% triethylamine acetonitrile as mobile phase A, 0.05% triethylamine solution as mobile phase B, and 10 mmol / L ammonium acetate acetonitrile-water (1:1) solution as mobile phase C. Gradient elution was performed according to the specifications in Table 4, with a flow rate of 0.8 mL / min. The column was detected by an electro-fogging detector at a temperature of 30 °C.

[0163] Table 4

[0164] Time (min) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0～5 90→90 10→10 0→0 5～5.1 90→50 10→50 0→0 5.1～11 50→50 50→50 0→0 11～11.5 50→0 50→0 0→100 11.5～20 0→0 0→0 100→0

[0165] Collect the eluent of reference solution A within the retention time range (-0.5 to +0.5 minutes) in a clean glass container, dry it with nitrogen, and accurately add 200 μL of acetonitrile-water (1:1) to dissolve it, thus obtaining the test solution.

[0166] Determination method: Accurately pipette 2 μL of reference solution B and test solution into the liquid chromatograph, measure and record the chromatographic peaks within 2-45 minutes.

[0167] Establishment of the comparative feature map:

[0168] The chromatograms of samples numbered S1 to S26 were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition," and the average method was used to generate the control characteristic chromatogram (see...). Figure 1 The similarity of samples S1 to S26 was all above 0.96.

[0169] Characteristic peak identification:

[0170] The retention times of trisaccharides (glucose, sucrose, and fructose), sorbitol, mannitol, inositol, and proline standards were compared to confirm the attribution of characteristic peaks. The results are as follows: Figure 2 As shown.

[0171] Example 2: Methodological investigation of fingerprinting of highly water-soluble components in Shenqi Fuzheng Injection

[0172] (1) Instrument precision assessment

[0173] Sample S20 was prepared according to the aforementioned method for preparing the test solution. Six consecutive injections were performed to examine the precision of the fingerprint chromatogram. The chromatogram from the precision experiment was imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" to calculate the similarity, obtaining the fingerprint chromatograms of the strongly water-soluble components of the Shenqi Fuzheng Injection. The results showed that the similarity was all above 0.98, as shown in Table 5. This result indicates that the instrument precision is high.

[0174] Table 5

[0175] Group 1st time 2nd time 3rd time 4th 5th 6th Comparison chart Similarity 0.992 0.989 0.987 0.990 0.988 0.987 1.000

[0176] (2) Fingerprint repeatability test

[0177] Sample S20 was prepared according to the aforementioned method for preparing the test solution. Six parallel test samples were prepared, and the changes in the fingerprint chromatogram were examined. The repeatability chromatograms were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" to calculate the similarity. The results showed that the similarity was above 0.98, indicating that the fingerprint chromatogram method established in this invention has good repeatability (Table 6).

[0178] Table 6

[0179] Group First 2nd 3rd 4th 5th 6th Comparison chart Similarity 0.988 0.992 0.985 0.994 0.990 0.989 1.000

[0180] (3) Fingerprint stability test

[0181] Take sample number S20 and prepare a test sample according to the aforementioned test sample solution preparation method. Store at room temperature and measure the test sample solution at 0, 4, 8, 16, 24, 36 and 48 hours respectively. Import the stability test chromatogram into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" and calculate the similarity. The similarity is greater than 0.98, indicating that the test sample solution of Shenqi Fuzheng Injection is stable within 24 hours (Table 7).

[0182] Table 7

[0183] time 0h 4h 8h 16h 24h 36h 48h Similarity 0.990 0.986 0.988 0.993 0.990 0.987 0.986

[0184] (4) System suitability test

[0185] Take sample number S20 and prepare a test sample according to the aforementioned test sample solution preparation method. The chromatographic peaks of the test sample solution are well separated within 20 minutes, with 10 common peaks and no missing peaks.

[0186] Example 3: Fingerprint Detection of Strongly Water-Soluble Components in Shenqi Fuzheng Injection

[0187] The test samples were prepared according to the aforementioned test sample solution preparation method. Under the aforementioned HPLC analysis conditions, the fingerprint chromatograms of the highly water-soluble components of each batch of Shenqi Fuzheng Injection test samples in Table 8 were determined. The experimental chromatograms of the test samples were then imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" to calculate the similarity. The results are shown in Table 8.

[0188] Table 8

[0189] serial number Similarity serial number Similarity serial number Similarity S1 0.977 S2 0.977 S3 0.974 S4 0.980 S5 0.976 S6 0.981 S7 0.983 S8 0.982 S9 0.992 S10 0.970 S11 0.983 S12 0.988 S13 0.972 S14 0.969 S15 0.991 S16 0.981 S17 0.970 S18 0.984 S19 0.980 S20 0.986 S21 0.989 S22 0.989 S23 0.973 S24 0.971 S25 0.980 S26 0.981 S27 0.813

[0190] The fingerprint chromatogram method for the highly water-soluble components of Shenqi Fuzheng Injection constructed according to this invention showed that the main chromatographic peaks of the test solution eluted within 20 minutes, with 10 common peaks and no missing peaks. Table 8 shows that the similarity between the HPLC chromatogram of the qualified batch of Shenqi Fuzheng Injection and the control chromatogram was above 0.96, while a batch of unqualified sample S27 had a similarity of only 0.813. This result indicates that the method constructed in this invention can effectively monitor, detect, and evaluate Shenqi Fuzheng Injection.

[0191] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments 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.

[0192] The embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of 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 fingerprint of a strong water-soluble component in Shenqi Fuzheng injection, characterized in that, The method comprises the following steps: A reference solution A is prepared by dissolving glucose, sucrose and fructose into solvent A; A reference solution B is prepared by dissolving sorbitol, mannose and sucrose into solvent B; The sample of the injection is subjected to solid phase extraction, a non-polar divinylphenyl neutral polymer adsorbent is used as the filler of the solid phase extraction column, the eluent is collected, and then the eluent is evaporated to dryness, the residue is mixed with a mixture of water and acetonitrile, centrifuged, and the supernatant is collected; The supernatant is subjected to high performance liquid chromatography separation with the reference solution A, the separated liquid corresponding to the retention time of the characteristic peak of the reference solution A in the supernatant is collected, and after the solvent is removed, the separated liquid is dissolved in a mixture of water and acetonitrile to prepare a test sample solution; The test sample solution is subjected to high performance liquid chromatography detection with the reference solution B to obtain the fingerprint of the strong water-soluble components in the injection; The separation conditions of the high performance liquid chromatography comprise: The chromatographic column uses amide-bonded hybrid silica gel as the filler; The mobile phase comprises mobile phase A1, mobile phase B1 and mobile phase C1, wherein the mobile phase A1 is a triethylamine-containing acetonitrile solution or a triethylamine-containing methanol solution, the mobile phase B1 is a triethylamine-containing water solution, and the mobile phase C1 is an ammonium acetate-containing acetonitrile water mixture solution or an ammonium formate-containing acetonitrile water mixture solution; The detection conditions of the high performance liquid chromatography comprise: The chromatographic column uses amide-bonded ethylene bridge hybrid silica gel as the filler; The mobile phase comprises mobile phase A2 and mobile phase B2, wherein the mobile phase A2 is acetonitrile, methanol, an ammonium acetate-containing acetonitrile water mixture solution or an ammonium formate-containing acetonitrile water mixture solution, and the mobile phase B2 is an ammonium acetate-containing water solution or an ammonium formate-containing water solution; In the separation conditions of the high performance liquid chromatography, the mobile phase A1 is a triethylamine acetonitrile solution with a volume fraction of 0.01%-2%, the mobile phase B1 is a triethylamine water solution with a volume fraction of 0.01%-2%, the mobile phase C1 is an ammonium acetate acetonitrile water mixture solution with a concentration of 5-40 mmol / L, and the volume ratio of acetonitrile to water in the acetonitrile water mixture solution is (0.8-1.2):(0.8-1.2). The gradient elution of the mobile phase comprises: 0-5 min, the volume percentage of the mobile phase A1 is kept at 90%, the volume percentage of the mobile phase B1 is kept at 10%, and the volume percentage of the mobile phase C1 is kept at 0%; 5-5.1 min, the volume percentage of the mobile phase A1 changes from 90% to 50%, the volume percentage of the mobile phase B1 changes from 10% to 50%, and the volume percentage of the mobile phase C1 is kept at 0%; 5.1-11 min, the volume percentage of the mobile phase A1 is kept at 50%, the volume percentage of the mobile phase B1 is kept at 50%, and the volume percentage of the mobile phase C1 is kept at 0%; 11-11.5 min, the volume percentage of the mobile phase A1 changes from 50% to 0%, the volume percentage of the mobile phase B1 changes from 50% to 0%, and the volume percentage of the mobile phase C1 changes from 0% to 100%; 11.5-20 min, the volume percentage of the mobile phase A1 is kept at 0%, the volume percentage of the mobile phase B1 is kept at 0%, and the volume percentage of the mobile phase C1 changes from 100% to 0%. In the detection condition of the high performance liquid chromatography, the mobile phase A2 is an acetonitrile-water mixed solution of ammonium acetate with a concentration of 1-10 mmol / L, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (97-99):(1-3), and the mobile phase B2 is an aqueous solution of ammonium acetate with a concentration of 5-40 mmol / L. In the detection condition of the high performance liquid chromatography, the gradient elution of the mobile phase comprises: 0-5 min, the volume percentage of the mobile phase A2 is kept at 95%, and the volume percentage of the mobile phase B2 is kept at 5%; 5-25 min, the volume percentage of the mobile phase A2 changes from 95% to 85%, and the volume percentage of the mobile phase B2 changes from 5% to 15%; 25-30 min, the volume percentage of the mobile phase A2 changes from 85% to 65%, and the volume percentage of the mobile phase B2 changes from 15% to 35%; 30-35 min, the volume percentage of the mobile phase A2 is kept at 65%, and the volume percentage of the mobile phase B2 is kept at 35%; 35-36 min, the volume percentage of the mobile phase A2 changes from 65% to 95%, and the volume percentage of the mobile phase B2 changes from 35% to 5%; 36-45 min, the volume percentage of the mobile phase A2 is kept at 95%, and the volume percentage of the mobile phase B2 is kept at 5%. The fingerprint spectrum of the strong water-soluble components in the Shenqi Fuzheng injection contains 10 characteristic peaks, and the 10 characteristic peaks include the characteristic peaks of fructose, sorbitol, mannose, glucose, proline, inositol and sucrose.

2. The method for constructing the fingerprint of the strong water-soluble components in Shenqi Fuzheng Injection according to claim 1, characterized in that, The separation condition of the high performance liquid chromatography further comprises: The detection is performed by using an electro-spray detector, the column temperature is 20-40 DEG C, and the flow rate is 0.5-1.5 mL / min.

3. The method for constructing the fingerprint of the strong water-soluble components in Shenqi Fuzheng Injection according to claim 1, characterized in that, The detection condition of the high performance liquid chromatography further comprises: The column temperature is 40-80 DEG C, and the flow rate is 0.2-0.4 mL / min.

4. The method for constructing the fingerprint of the strong water-soluble components in Shenqi Fuzheng Injection according to claim 1, characterized in that, The eluent used in the solid phase extraction is water.

5. The method for constructing the fingerprint of the strong water-soluble components in Shenqi Fuzheng Injection according to claim 1, characterized in that, The volume ratio of water to acetonitrile in the mixing of the obtained residue with the mixture of water and acetonitrile is (0.1-1):(0.1-1).

6. The method for constructing the fingerprint of the strong water-soluble components in Shenqi Fuzheng Injection according to claim 1, characterized in that, The centrifugation conditions include a rotation speed of 10000-16000 rpm and a time of 5-30 min.

7. The method according to claim 1, wherein the method is characterized in that, The volume ratio of water to acetonitrile in the mixing of the obtained residue with the mixture of water and acetonitrile is (0.8-1.2):(0.8-1.2).

8. The method for constructing the fingerprint of the strong water-soluble components in the Shenqi Fuzheng injection according to claim 1, characterized in that, The solvent A is a mixture of water and acetonitrile with a volume ratio of (0.8-1.2):(0.8-1.2), and the solvent B is a mixture of water and acetonitrile with a volume ratio of (0.8-1.2):(0.8-1.2).

9. A method for detecting a strong water-soluble component in Shenqi Fuzheng Injection, characterized in that, The method comprises the following steps: The sample of the Shenqi Fuzheng injection to be detected is subjected to solid phase extraction, a non-polar divinylphenyl neutral polymer adsorbent is used as the filler of the solid phase extraction column, the eluent is collected, and then the eluent is evaporated to dryness, the obtained residue is mixed with a mixture of water and acetonitrile, centrifuged, and the supernatant is taken; The supernatant is subjected to high performance liquid chromatography separation with a reference solution A, the separated liquid corresponding to the retention time of the characteristic peak of the reference solution A in the supernatant is collected, and after removal of the solvent, the obtained sample solution to be detected is prepared by mixing with a mixture of water and acetonitrile; The sample solution to be detected is subjected to high performance liquid chromatography detection to obtain the atlas of the strong water-soluble components in the Shenqi Fuzheng injection, and the atlas is compared with the fingerprint atlas obtained by the method for constructing the fingerprint atlas of the strong water-soluble components in the Shenqi Fuzheng injection according to any one of claims 1-8. The high performance liquid chromatography separation conditions include: The chromatographic column uses amide bond hybrid silica gel as the filler; The mobile phase includes mobile phase A1, mobile phase B1 and mobile phase C1, wherein the mobile phase A1 is a triethylamine acetonitrile solution or a triethylamine methanol solution, the mobile phase B1 is a triethylamine water solution, and the mobile phase C1 is an ammonium acetate acetonitrile water mixed solution or an ammonium formate acetonitrile water mixed solution; The high performance liquid chromatography detection conditions include: The chromatographic column uses amide bond hybrid silica gel as the filler; The mobile phase includes mobile phase A2 and mobile phase B2, wherein the mobile phase A2 is acetonitrile, methanol, an ammonium acetate acetonitrile water mixed solution or an ammonium formate acetonitrile water mixed solution, and the mobile phase B2 is an ammonium acetate water solution or an ammonium formate water solution; In the high performance liquid chromatography separation conditions, the mobile phase A1 is a triethylamine acetonitrile solution with a volume fraction of 0.01%-2%, the mobile phase B1 is a triethylamine water solution with a volume fraction of 0.01%-2%, and the mobile phase C1 is an ammonium acetate acetonitrile water mixed solution with a concentration of 5-40 mmol / L, and the volume ratio of acetonitrile to water in the acetonitrile water mixed solution is (0.8-1.2):(0.8-1.2). In the separation condition of the high performance liquid chromatography, the mobile phase is eluted in a gradient elution manner, and the gradient elution comprises: 0-5 min, the volume percentage of the mobile phase A1 is kept at 90%, the volume percentage of the mobile phase B1 is kept at 10%, and the volume percentage of the mobile phase C1 is kept at 0%; 5-5.1 min, the volume percentage of the mobile phase A1 changes from 90% to 50%, the volume percentage of the mobile phase B1 changes from 10% to 50%, and the volume percentage of the mobile phase C1 is kept at 0%; 5.1-11 min, the volume percentage of the mobile phase A1 is kept at 50%, the volume percentage of the mobile phase B1 is kept at 50%, and the volume percentage of the mobile phase C1 is kept at 0%; 11-11.5 min, the volume percentage of the mobile phase A1 changes from 50% to 0%, the volume percentage of the mobile phase B1 changes from 50% to 0%, and the volume percentage of the mobile phase C1 changes from 0% to 100%; 11.5-20 min, the volume percentage of the mobile phase A1 is kept at 0%, the volume percentage of the mobile phase B1 is kept at 0%, and the volume percentage of the mobile phase C1 changes from 100% to 0%. In the detection condition of the high performance liquid chromatography, the mobile phase A2 is an acetonitrile-water mixed solution of ammonium acetate with a concentration of 1-10 mmol / L, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (97-99):(1-3), and the mobile phase B2 is an aqueous solution of ammonium acetate with a concentration of 5-40 mmol / L. In the detection condition of the high performance liquid chromatography, the mobile phase is eluted in a gradient elution manner, and the gradient elution comprises: 0-5 min, the volume percentage of the mobile phase A2 is kept at 95%, and the volume percentage of the mobile phase B2 is kept at 5%; 5-25 min, the volume percentage of the mobile phase A2 changes from 95% to 85%, and the volume percentage of the mobile phase B2 changes from 5% to 15%; 25-30 min, the volume percentage of the mobile phase A2 changes from 85% to 65%, and the volume percentage of the mobile phase B2 changes from 15% to 35%; 30-35 min, the volume percentage of the mobile phase A2 is kept at 65%, and the volume percentage of the mobile phase B2 is kept at 35%; 35-36 min, the volume percentage of the mobile phase A2 changes from 65% to 95%, and the volume percentage of the mobile phase B2 changes from 35% to 5%; 36-45 min, the volume percentage of the mobile phase A2 is kept at 95%, and the volume percentage of the mobile phase B2 is kept at 5%.

10. The method for detecting the strong water-soluble components in the Shenqi Fuzheng injection according to claim 9, characterized in that, The separation condition of the high performance liquid chromatography further comprises: The detection is performed by using an electro-spray detector, the column temperature is 20-40 DEG C, and the flow rate is 0.5-1.5 mL / min.

11. The method for detecting the strong water-soluble components in the Shenqi Fuzheng injection according to claim 9, characterized in that, The detection condition of the high performance liquid chromatography further comprises: The detection is performed by using an electro-spray detector, the column temperature is 40-80 DEG C, and the flow rate is 0.2-0.4 mL / min.

12. The method for detecting the strong water-soluble components in the Shenqi Fuzheng injection according to any one of claims 9-11, characterized in that, In the solid phase extraction, the eluent used is water.

13. The method for detecting the strong water-soluble components in the Shenqi Fuzheng injection according to any one of claims 9-11, characterized in that, The volume ratio of water to acetonitrile in the mixing of the obtained residue with the mixture of water and acetonitrile is (0.1-1):(0.1-1).

14. The method for detecting the strong water-soluble components in the Shenqi Fuzheng injection according to any one of claims 9-11, characterized in that, The centrifugation conditions include a rotation speed of 10000-16000 rpm and a time of 5-30 min.

15. The method for detecting the strong water-soluble components in the Shenqi Fuzheng injection according to any one of claims 9-11, characterized in that, The volume ratio of water to acetonitrile in the mixing of the obtained residue with the mixture of water and acetonitrile is (0.8-1.2):(0.8-1.2).

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