A fingerprint detection method for Shengmai injection

Abstract

The present invention discloses a method for detecting the fingerprint of Shengmai injection, which comprises: preparing a test solution, preparing a reference solution, and measuring with a high performance liquid chromatograph, taking the reference peak retention time and peak area as 1, and establishing a reference spectrum and a fingerprint of Shengmai injection. A detection method for the fingerprint of Shengmai injection is established by using UPLC-TQ-MS / MS, which can simultaneously detect 19 effective components such as ginsenosides, ophiopogon saponins, and schizandra lignins, and can comprehensively and accurately detect the quality of Shengmai injection, providing a scientific basis for ensuring clinical effectiveness.

Description

Technical Field

[0001] The invention relates to a quality detection method for a traditional Chinese medicine preparation, and in particular to a fingerprint detection method for a Shengmai injection. Background Art

[0002] Shengmai injection is made of red ginseng, ophiopogon and schisandra chinensis through modern scientific technology. It replenishes qi and nourishes yin, restores the pulse and consolidates the body. It is used for patients with qi and yin deficiency, palpitations, shortness of breath, cold limbs, sweating, pulse cessation, myocardial infarction, cardiogenic shock, septic shock and other symptoms. Ginseng contains a variety of ginsenosides, ophiopogon contains a variety of ophiopogon saponins, and schisandra chinensis contains a variety of lignin compounds. At present, the detection method of Shengmai injection mainly focuses on the detection of ginsenosides, and fails to fully detect the quality of Shengmai injection.

[0003] Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method that can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations. Therefore, in order to more comprehensively and effectively control the quality of Shengmai injection and ensure the safety and efficacy of clinical medication, it is necessary to research and design an HPLC fingerprint determination method that can objectively, accurately and comprehensively detect the quality of Shengmai injection based on existing technologies. Summary of the invention

[0004] Purpose of the invention: In order to solve the shortcomings of the prior art, the present invention provides a fingerprint spectrum detection method with high precision, good repeatability and stability, which can comprehensively and accurately evaluate the quality of Shengmai injection, thereby more effectively ensuring the clinical safety and effectiveness of the finished product of Shengmai injection.

[0005] Technical solution: In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0006] A method for detecting fingerprint spectrum of Shengmai injection, the method comprising the following steps:

[0007] (1) Preparation of test solution: Take different batches of Shengmai injection, dilute them, and filter them through a 0.22 μm microporous filter membrane to obtain Shengmai injection test solution;

[0008] (2) Preparation of mixed reference solution: accurately weigh ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, Re, Rg1, Rg2, Rh1, ophiopogon saponins A, B, C, D, schisandrin A, schisandrin B, schisandrin C, schisandrin, schisandrin A and schisandrin B, dissolve them in methanol to obtain a reference solution;

[0009] (3) Determination of HPLC fingerprint of Shengmai injection: Accurately pipette the test solution of Shengmai injection in step (1) and the mixed reference solution in step (2), inject into high performance liquid chromatograph, and record the chromatogram;

[0010] (4) Export the fingerprint of Shengmai injection obtained in step (3) and import it into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A; select the chromatographic peaks existing in the chromatograms of different batches of Shengmai injection as common peaks; generate a reference fingerprint of Shengmai injection using the average value calculation method, calculate the relative retention time and relative peak area of ​​each common peak; and mark the chemical composition of the peaks in the reference fingerprint according to the retention time of the chromatogram of the mixed reference solution.

[0011] As a preferred embodiment, the above-mentioned Shengmai injection fingerprint detection method, step (1) preparation of the test solution: preparation of the test solution: take 13 batches of Shengmai injection, dilute them, and pass them through a 0.22μm microporous filter membrane to obtain the Shengmai injection test solution.

[0012] As a preferred embodiment, the fingerprint spectrum detection method of Shengmai injection described above, step (2) preparation of mixed reference solution: accurately weigh ginsenosides Rb1, Rb2, Rc, Rd, Rg3, Rh2, Re, Rg1, Rh1, ophiopogon saponins A, B, C, D, schisandrin A, schisandrin B, schisandrin C, schisandrin, schisandrin A and schisandrin B, add methanol to dissolve, and prepare reference mixed solutions with concentrations of 1.02, 1.67, 1.56, 1.43, 1.78, 2.51, 2.05, 3.51, 2.56, 1.89, 1.77, 2.67, 2.90, 3.42, 3.62, 3.34, 2.68, 2.42, 2.73 mg / ml respectively.

[0013] As a preferred embodiment, the above-mentioned Shengmai injection fingerprint detection method is characterized in that the chromatographic conditions described in step (3) are: Acquity BEH C18 chromatographic column, size 100mm×2.1mm, 1.7μm, chromatographic separation is performed at an automatic injection temperature of 4°C; Q-TOF / MS analysis is performed in positive mode and negative mode, using full scan mode, column temperature 40°C, mobile phase 0.1% formic acid water as phase A solvent-acetonitrile as phase B solvent, flow rate 0.3mL / min; gradient elution program: 0-3.0min, 3%-25%B; 3-4min, 25%-45%B; 4-10min, 45%-95%B; 10-13min, 95%B; 13-17min, 95%-3%B; 17-20min, 3%B; injection volume is 5μL;

[0014] Mass spectrometry conditions: capillary voltage 3 kV, sample cone temperature 55 °C, source temperature 100 °C, desolvation temperature 100 °C, cone gas flow 50 L / h, desolvation gas N2 flow 800 L / h, mass range m / z 50-1200 Da.

[0015] The gradient elution conditions used were as follows: gradient elution program: 0-3.0 min, 3%-25% B; 3-4 min, 25%-45% B; 4-10 min, 45%-95% B; 10-13 min, 95% B; 13-17 min, 95%-3% B; 17-20 min, 3% B.

[0016] There are 19 common peaks in Shengmai injection, namely, ophiopogonin A, ophiopogonin B, ginsenoside Rh2, ginsenoside Re, ginsenoside Rg1, ophiopogonin C, ginsenoside Rh1, ophiopogonin D,

[0017] Ginsenoside Rb3, ginsenoside Rc, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rd, ginsenoside Rg3, schisandrin, schisandrin A, schisandrin B, schisandrin A, schisandrin B, schisandrin C.

[0018] The fingerprint spectrum detection method of Shengmai injection provided by the present invention has the following advantages compared with the prior art:

[0019] (1) The present invention uses UPLC-TQ-MS / MS to establish a detection method for the fingerprint of Shengmai injection, which can simultaneously detect 19 active ingredients such as ginsenosides, ophiopogon saponins, and Schisandra lignins, and can comprehensively and accurately detect the quality of Shengmai injection, providing a scientific basis for ensuring clinical effectiveness.

[0020] (2) Methodological proof shows that the detection method of the HPLC fingerprint of Shengmai injection of the present invention has high accuracy, high stability and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the HPLC-UV (204nm) fingerprint of Shengmai Injection.

[0022] Figure 2 This is the HPLC-UV (204nm) reference spectrum of Shengmai injection (R). DETAILED DESCRIPTION

[0023] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0024] Example 1

[0025] Experimental Materials:

[0026] All 13 batches (S1-S13) of Shengmai injection were provided by Changshu Leiyunshang Pharmaceutical Co., Ltd. Ginsenosides, Ophiopogon japonicus saponins, Schisandrin A, Schisandrin B, Schisandrin C, Schisandrin, Schisandrin A and Schisandrin B were provided by Shanghai Yuanye Biotechnology Co., Ltd. Acetonitrile was provided by Tianjin Tianli Chemical Reagent Co., Ltd.

[0027] A method for detecting fingerprint spectrum of Shengmai injection, characterized in that the method comprises the following steps:

[0028] (1) Preparation of test solution: Take 13 batches of Shengmai injection, dilute it 10 times with methanol, and filter it through a 0.22 μm microporous filter to obtain 13 batches of Shengmai injection test solution.

[0029] (2) Preparation of mixed reference solution: Accurately weigh ginsenosides Rb1, Rb2, Rc, Rd, Rg3, Rh2, Re, Rg1, Rh1, ophiopogon saponins A, B, C, D, schisandrin A, schisandrin B, schisandrin C, schisandrin, schisandrin A and schisandrin B, add methanol to dissolve and prepare reference mixed solutions with concentrations of 1.02, 1.67, 1.56, 1.43, 1.78, 2.51, 2.05, 3.51, 2.56, 1.89, 1.77, 2.67, 2.90, 3.42, 3.62, 3.34, 2.68, 2.42, 2.73 mg / ml, respectively.

[0030] (3) Determination of HPLC fingerprint of Shengmai injection: accurately aspirate the test solution of Shengmai injection in step (1) and the mixed reference solution in step (2), inject into high performance liquid chromatograph, and record the chromatogram; the chromatographic conditions are: AcquityBEH C18 chromatographic column, specification 100mm×2.1mm, 1.7μm, chromatographic separation is performed at an automatic injection temperature of 4°C; Q-TOF / MS analysis is performed in positive mode and negative mode, using full scan mode, column temperature 40°C, mobile phase 0.1% formic acid water as phase A solvent-acetonitrile as phase B solvent, flow rate 0.3mL / min; gradient elution program is: 0~3.0min, 3%~25%B; 3~4min, 25%~45%B; 4~10min, 45%~95%B; 10~13min, 95%B; 13~17min, 95%~3%B; 17~20min, 3%B; injection volume is 5μL;

[0031] The mass spectrometry conditions were as follows: capillary voltage 3 kV, sample cone temperature 55 °C, source temperature 100 °C, desolvation temperature 100 °C, cone gas flow 50 L / h, desolvation gas N2 flow 800 L / h, mass range m / z 50-1200 Da.

[0032] (4) Export the fingerprint of Shengmai injection obtained in step (3) and import it into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A; select the chromatographic peaks existing in the chromatograms of different batches of Shengmai injection as common peaks; generate a reference fingerprint of Shengmai injection using the average value calculation method, calculate the relative retention time and relative peak area of ​​each common peak; and mark the chemical composition of the peaks in the reference fingerprint according to the retention time of the chromatogram of the mixed reference solution.

[0033] There are 19 common peaks in Shengmai injection, namely, ophiopogonin A, ophiopogonin B, ginsenoside Rh2, ginsenoside Re, ginsenoside Rg1, ophiopogonin C, ginsenoside Rh1, ophiopogonin D,

[0034] Ginsenoside Rb3, ginsenoside Rc, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rd, ginsenoside Rg3, schisandrin, schisandrin A, schisandrin B, schisandrin A, schisandrin B, schisandrin C.

[0035] Example 2: Methodological Investigation

[0036] 1. Precision test

[0037] Accurately weigh 10 ml of Shengmai injection (S1), prepare the Shengmai injection test solution according to the method described in Example 1, continuously inject 6 times according to the chromatographic conditions described in Example 1, record the chromatogram, and import it into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (National Pharmacopoeia Committee 2012A Edition). The calculated similarities are all above 0.99, indicating that the instrument has good precision.

[0038] 2. Repeatability test

[0039] Accurately weigh 10 ml of Shengmai injection (S1), prepare the Shengmai injection test solution according to the method described in Example 1, inject according to the chromatographic conditions described in Example 1, record the chromatogram, and import it into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (National Pharmacopoeia Committee 2012A Edition). The calculated similarities are all above 0.99, indicating that the method provided by the present invention has good repeatability.

[0040] 3. Stability test

[0041] Accurately weigh 10 ml of Shengmai injection (S1), prepare the Shengmai injection test solution according to the method described in Example 1, inject the sample according to the chromatographic conditions described in Example 1, and inject the sample at 0, 2, 4, 8, 12, and 24 h respectively, and import it into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (National Pharmacopoeia Committee 2012A Edition), and the calculated similarities are all above 0.99, indicating that the sample determined by this method is stable within 24 h.

[0042] Example 3 Similarity Analysis

[0043] 13 different batches of Shengmai injection test solution were taken, sampled according to the method in Example 1, and the chromatogram was recorded. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (Chinese Pharmacopoeia Committee 2012A Edition) was introduced, and the similarity calculation results showed that the similarity of the 13 batches of Shengmai injection samples was 0.951-0.990, and the similarity was good, as shown in Table 1.

[0044] Table 1 Similarity evaluation results of 13 batches of Shengmai injection

[0045]

[0046]

Claims

1. A method for detecting fingerprints of Shengmai injection, characterized in that: The method comprises the following steps: (1) Preparation of test solution: Take different batches of Shengmai injection, dilute them, and filter them through a 0.22 μm microporous filter membrane to obtain the Shengmai injection test solution; (2) Preparation of mixed reference solution: accurately weigh ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, Re, Rg1, Rg2, Rh1, ophiopogon saponins A, B, C, D, schisandrin A, schisandrin B, schisandrin C, schisandrin, schisandrin A and schisandrin B, dissolve them in methanol to obtain a reference solution; (3) Determination of HPLC fingerprint of Shengmai injection: Accurately pipette the test solution of Shengmai injection in step (1) and the mixed reference solution in step (2), inject them into the high performance liquid chromatograph, and record the chromatogram; (4) Exporting the fingerprint of Shengmai injection obtained in step (3) and importing it into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A; selecting the chromatographic peaks existing in the chromatograms of different batches of Shengmai injection as common peaks; generating a reference fingerprint of Shengmai injection by using the average value calculation method, calculating the relative retention time and relative peak area of ​​each common peak; and marking the chemical components of the peaks in the reference fingerprint according to the retention time of the chromatogram of the mixed reference solution; Step (3) Chromatographic conditions: Acquity BEH C18 column, size 100 mm × 2.1 mm, 1.7 μm, chromatographic separation at an automatic injection temperature of 4°C; Q-TOF / MS analysis was performed in positive mode and negative mode, using full scan mode, column temperature 40°C, mobile phase 0.1% formic acid water as phase A solvent-acetonitrile as phase B solvent, flow rate 0.3 mL / min; gradient elution, gradient elution conditions: 0~3.0 min, 3%~25% B; 3~4 min, 25%~45% B; 4~10 min, 45%~95% B; 10~13 min, 95% B; 13~17 min, 95%~3% B; 17~20 min, 3% B, injection volume 5 μL; Mass spectrometry conditions: capillary voltage 3 kV, sample cone temperature 55 °C, source temperature 100 °C, desolvation temperature 100 °C, cone gas flow 50 L / h, desolvation gas N2 flow 800 L / h, mass range m / z 50~1200 Da.

2. The fingerprint detection method of Shengmai injection according to claim 1, characterized in that: Step (1) Preparation of test solution: Take 13 batches of Shengmai injection, dilute them, and filter them through a 0.22 μm microporous filter membrane to obtain the Shengmai injection test solution.

3. The fingerprint detection method of Shengmai injection according to claim 1, characterized in that: Step (2) Preparation of mixed reference solution: Accurately weigh ginsenosides Rb1, Rb2, Rc, Rd, Rg3, Rh2, Re, Rg1, Rh1, ophiopogon saponins A, B, C, D, schisandrin A, schisandrin B, schisandrin C, schisandrin, schisandrin A and schisandrin B, add methanol to dissolve, and prepare reference mixed solutions with concentrations of 1.02, 1.67, 1.56, 1.43, 1.78, 2.51, 2.05, 3.51, 2.56, 1.89, 1.77, 2.67, 2.90, 3.42, 3.62, 3.34, 2.68, 2.42, 2.73 mg / ml, respectively.

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