UPLC fingerprint spectrum detection method of Xuebijing injection

Abstract

The invention provides a UPLC (Ultra Performance Liquid Chromatography) fingerprint spectrum detection method of Xuebijing injection, and belongs to the technical field of quality analysis and detection of traditional Chinese medicines. According to the method, the conditions of ultra-high performance liquid chromatography detection are controlled, accurate detection of the Xuebijing injection fingerprint spectrum is achieved, and the method has the advantages of being good in precision, high in detection speed, multiple in identified component and the like. 20 common peaks are determined according to the obtained UPLC fingerprint spectrum of the Xuebijing injection, five representative components (a peak 10, a peak 12, a peak 13, a peak 15 and a peak 17) are identified, medicinal material attribution is performed on the 20 common peaks, and the 20 common peaks can be used for characterizing components from medicinal materials of safflower, radix paeoniae rubra, ligusticum wallichii, angelica sinensis and salvia miltiorrhiza. And full coverage of the Xuebijing injection prescription medicinal materials is realized, and the overall quality control principle of the traditional Chinese medicine is embodied.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality analysis and detection of traditional Chinese medicines, and particularly relates to a UPLC fingerprint detection method for Xuebijing injection. Background Art

[0002] Xuebijing Injection is a traditional Chinese medicine compound consisting of five herbs: safflower, red peony root, ligusticum chuanxiong, salvia miltiorrhiza, and angelica sinensis. It has the effects of promoting blood circulation and removing blood stasis, clearing away heat and detoxifying, and promoting qi and blood circulation. It is suitable for conditions such as sepsis, systemic inflammatory response syndrome, multiple organ dysfunction syndrome, and novel coronavirus infection. Its clinical application is currently becoming increasingly widespread.

[0003] Xuebijing Injection, as a traditional Chinese medicine compound, has a relatively complex chemical composition. Controlling a single component is no longer sufficient for overall quality control. Fingerprinting, a key analytical method for evaluating the quality consistency and stability of traditional Chinese medicines (TCMs), decoction pieces, extracts, and preparations, can more comprehensively reflect the multi-component information of TCMs and is therefore of great significance for improving TCM quality control.

[0004] Currently, there are studies using high-performance liquid chromatography (HPLC) or ultra-performance liquid chromatography (UPLC) to establish fingerprint detection methods for Xuebijing Injection. HPLC methods require significant time and mobile phase consumption, resulting in low resolution and sensitivity. Compared to HPLC, UPLC offers advantages such as enhanced resolution and sensitivity, high efficiency, and excellent stability. However, existing UPLC methods also have a detection time of 60 minutes and a large RSD. They identify 39 common peaks and four phenolic acid compounds, resulting in a small number of identified components and a single type. The common peaks are not assigned to medicinal materials, and therefore do not fully cover the medicinal materials in the Xuebijing Injection prescription. Summary of the Invention

[0005] The present invention aims to provide a UPLC fingerprint detection method for Xuebijing injection. The detection method provided by the present invention has a short detection time, good precision, can identify multiple components, and can attribute common peaks to medicinal materials.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a UPLC fingerprint detection method for Xuebijing injection, comprising the following steps:

[0008] (1) Mixing paeoniflorin with methanol to obtain a reference solution;

[0009] (2) Mixing Xuebijing injection with water to obtain a test solution;

[0010] (3) The reference solution and the test solution obtained in steps (1) and (2) are respectively subjected to ultra-high performance liquid chromatography detection to obtain the UPLC fingerprint of Xuebijing injection; the conditions for the ultra-high performance liquid chromatography detection include: the chromatographic column is a Waters ACQUITY UPLC BEH C18 chromatographic column; the mobile phase A is methanol; the mobile phase B is 0.1% phosphoric acid; the gradient elution, the gradient elution conditions are: the volume fraction of the mobile phase A increases from 2% to 60% from 0 to 30 min, the volume fraction of the mobile phase A increases from 60% to 90% from 30 to 31 min, the volume fraction of the mobile phase A is 90% from 31 to 33 min, the volume fraction of the mobile phase A decreases from 90% to 2% from 33 to 34 min, and the volume fraction of the mobile phase A is 2% from 34 to 40 min; the flow rate is 0.3 mL / min; the column temperature is 34 to 36 ° C; the detection wavelength is 280 nm, and the injection volume is 1 μL.

[0011] Preferably, the concentration of the reference solution in step (1) is 0.58-0.62 mg / mL.

[0012] Preferably, the concentration of the reference solution in step (1) is 0.60 mg / mL.

[0013] Preferably, in step (2), the volume ratio of Xuebijing injection to water is 1:(1.8-2.2).

[0014] Preferably, in step (2), the volume ratio of Xuebijing injection to water is 1:2.

[0015] Preferably, the column temperature during ultra-high performance liquid chromatography detection in step (3) is 35°C.

[0016] Preferably, the UPLC fingerprint of the Xuebijing injection obtained in step (3) has 20 common peaks.

[0017] Preferably, the relative retention times of the 20 common peaks are: peak 1 0.16, peak 2 0.17, peak 3 0.20, peak 4 0.21, peak 5 0.36, peak 6 0.49, peak 7 0.51, peak 8 0.59, peak 9 0.77, peak 10 0.88, peak 11 0.95, peak 12 1.00, peak 13 1.08, peak 14 1.11, peak 15 1.44, peak 16 1.52, peak 17 1.66, peak 18 1.87, peak 19 1.95, and peak 20 2.00.

[0018] Preferably, among the 20 common peaks, peak No. 10 is hydroxysafflor yellow A, peak No. 12 is paeoniflorin, peak No. 13 is ferulic acid, peak No. 15 is ligustrin I, and peak No. 17 is salvianolic acid B.

[0019] Preferably, among the 20 common peaks, peak No. 1 belongs to safflower, red peony root, Chuanxiong, Chinese angelica and salvia miltiorrhiza, peak No. 2 belongs to safflower, red peony root, Chuanxiong and Chinese angelica, peak No. 3 belongs to red peony root, peak No. 4 belongs to safflower, red peony root, Chuanxiong, Chinese angelica and salvia miltiorrhiza, peak No. 5 belongs to salvia miltiorrhiza, peak No. 6 belongs to safflower, red peony root, Chuanxiong, Chinese angelica and salvia miltiorrhiza, peak No. 7 belongs to safflower, peak No. 8 belongs to safflower, red peony root, Chuanxiong, Angelica sinensis and Salvia miltiorrhiza, Peak No. 9 belongs to Carthamus tinctorius, Peak No. 10 belongs to Carthamus tinctorius, Peak No. 11 belongs to Carthamus tinctorius, Chuanxiong and Angelica sinensis, Peak No. 12 belongs to Paeonia lactiflora, Peak No. 13 belongs to Chuanxiong and Angelica sinensis, Peak No. 14 belongs to Carthamus tinctorius, Peak No. 15 belongs to Chuanxiong and Angelica sinensis, Peak No. 16 belongs to Chuanxiong and Angelica sinensis, Peak No. 17 belongs to Salvia miltiorrhiza, Peak 18 belongs to Carthamus tinctorius, Peak 19 belongs to Salvia miltiorrhiza, and Peak 20 belongs to Paeonia lactiflora.

[0020] The present invention provides a UPLC fingerprint detection method for Xuebijing injection, comprising the following steps: (1) mixing paeoniflorin with methanol to obtain a reference solution; (2) mixing Xuebijing injection with water to obtain a test solution; (3) subjecting the reference solution and the test solution obtained in steps (1) and (2) to ultra-high performance liquid chromatography detection, respectively, to obtain a UPLC fingerprint of Xuebijing injection; the conditions for the ultra-high performance liquid chromatography detection include: the chromatographic column is Waters ACQUITY UPLC BEH C18 chromatographic column; mobile phase A is methanol; mobile phase B is 0.1% phosphoric acid; gradient elution, the gradient elution conditions are: the volume fraction of mobile phase A increases from 2% to 60% from 0 to 30 min, the volume fraction of mobile phase A increases from 60% to 90% from 30 to 31 min, the volume fraction of mobile phase A is 90% from 31 to 33 min, the volume fraction of mobile phase A decreases from 90% to 2% from 33 to 34 min, and the volume fraction of mobile phase A is 2% from 34 to 40 min; the flow rate is 0.3 mL / min; the column temperature is 34 to 36°C; the detection wavelength is 280 nm, and the injection volume is 1 μL. The present invention controls the conditions for ultra-high performance liquid chromatography detection to achieve accurate detection of the fingerprint spectrum of Xuebijing Injection, and has the advantages of good precision, fast detection speed, and multiple identification components. According to the obtained UPLC fingerprint spectrum of Xuebijing Injection, 20 common peaks are determined, 5 representative components (peak 10, peak 12, peak 13, peak 15 and peak 17) are identified, and the 20 common peaks are attributed to medicinal materials. The 20 common peaks can characterize the components derived from the medicinal materials of Carthamus tinctorius, Paeonia lactiflora, Ligusticum chuanxiong, Angelica sinensis and Salvia miltiorrhiza, thereby achieving full coverage of the prescribed medicinal materials of Xuebijing Injection and reflecting the overall quality control principle of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a comparison chart of the chromatographic peaks of Xuebijing Injection at different detection wavelengths;

[0022] Figure 2This is a comparison of chromatographic peaks of Xuebijing injection with different mobile phases;

[0023] Figure 3 This is a comparison of chromatographic peaks of Xuebijing Injection with different gradient elution procedures;

[0024] Figure 4 This is a comparison chart of the effects of different chromatographic columns on the chromatographic peaks of Xuebijing Injection;

[0025] Figure 5 This is a comparison chart of the effects of different column temperatures on the chromatographic peaks of Xuebijing Injection;

[0026] Figure 6 This is a comparison chart of the effects of different flow rates on the chromatographic peaks of Xuebijing Injection;

[0027] Figure 7 This is a comparison chart of the effects of diluted and undiluted samples on the chromatographic peaks of Xuebijing Injection;

[0028] Figure 8 This is the fingerprint chromatogram of Xuebijing injection in Example 1;

[0029] Figure 9 This is the chromatogram overlay of the precision and intermediate precision of Xuebijing Injection;

[0030] Figure 10 This is the reference fingerprint of Xuebijing Injection;

[0031] Figure 11 This is the fingerprint overlay of 23 batches of Xuebijing injection;

[0032] Figure 12 This is a comparative chromatogram of Xuebijing injection and extracts of various medicinal materials (safflower, red peony root, angelica, salvia miltiorrhiza, and ligusticum chuanxiong);

[0033] Figure 13 The chromatograms are compared between Xuebijing injection (test solution) and negative preparations (negative solution lacking safflower, negative solution lacking red peony root, negative solution lacking Chuanxiong, negative solution lacking angelica, negative solution lacking salvia miltiorrhiza, and negative solution lacking Chuanxiong and angelica);

[0034] Figure 14 TIC and UV chromatograms in positive and negative ion modes for the qualitative analysis of Xuebijing Injection using high-resolution mass spectrometry.

[0035] Figure 15 Chromatogram for peak identification of the test solution of Xuebijing injection;

[0036] Figure 16 The spectra of the reference solution of each component and the spectra of the corresponding components in the test solution. DETAILED DESCRIPTION

[0037] The application provides a UPLC fingerprint detection method of Xuebijing injection, which comprises the following steps:

[0038] (1) mixing paeoniflorin with methanol to obtain a reference solution;

[0039] (2) mixing Xuebijing injection with water to obtain a test solution;

[0040] (3) performing UPLC detection on the reference solution and the test solution obtained in steps (1) and (2) respectively to obtain the UPLC fingerprint of Xuebijing injection.

[0041] The application mixes paeoniflorin with methanol to obtain a reference solution.

[0042] In the application, the concentration of the reference solution is preferably 0.58-0.62 mg / mL. As an embodiment, the concentration of the reference solution can be specifically 0.58 mg / mL, 0.59 mg / mL, 0.60 mg / mL, 0.61 mg / mL or 0.62 mg / mL. The application uses paeoniflorin as a reference and controls the concentration thereof, the retention time of which is moderate and the separation is good, and the detection accuracy can be further improved.

[0043] The application mixes Xuebijing injection with water to obtain a test solution.

[0044] In the application, the volume ratio of Xuebijing injection to water is preferably 1:(1.8-2.2). As an embodiment, the volume ratio of Xuebijing injection to water can be specifically 1:1.8, 1:1.9, 1:2, 1:2.1 or 1:2.2. The application directly samples after diluting Xuebijing injection with water, controls the volume ratio of Xuebijing injection to water, and the chromatographic peak shape can be optimized, and the detection accuracy is further improved.

[0045] In the application, the mixing is preferably shaking.

[0046] After mixing, the application preferably performs microporous filter membrane filtration on the mixed solution to obtain a test solution.

[0047] In the application, the pore size of the microporous filter membrane is preferably 0.22 μm.

[0048] After obtaining the reference solution and the test solution, the application performs UPLC detection on the reference solution and the test solution respectively to obtain the UPLC fingerprint of Xuebijing injection.

[0049] In the present invention, the conditions for ultra-high performance liquid chromatography detection include: a Waters ACQUITY UPLC BEH C18 chromatographic column; mobile phase A is methanol; mobile phase B is 0.1% phosphoric acid (volume percentage); gradient elution, the gradient elution conditions are: the volume fraction of mobile phase A increases from 2% to 60% from 0 to 30 min, the volume fraction of mobile phase A increases from 60% to 90% from 30 to 31 min, the volume fraction of mobile phase A is 90% from 31 to 33 min, the volume fraction of mobile phase A decreases from 90% to 2% from 33 to 34 min, and the volume fraction of mobile phase A is 2% from 34 to 40 min; the flow rate is 0.3 mL / min; the column temperature is 34 to 36° C., preferably 35° C.; the detection wavelength is 280 nm, and the injection volume is 1 μL.

[0050] In the present invention, the specifications of the chromatographic column are preferably: 100 mm×2.1 mm, 1.7 μm.

[0051] In the present invention, the theoretical plate number during ultra-high performance liquid chromatography detection is preferably ≥10,000, calculated based on the paeoniflorin peak.

[0052] The present invention controls the conditions for ultra-high performance liquid chromatography detection to achieve accurate detection of the fingerprint spectrum of Xuebijing injection, and has the advantages of good precision, fast detection speed, etc.

[0053] The present invention preferably selects chromatographic peaks with good peak shape, separation degree > 1.5, peak area > 6000 and being the effective ingredients of Xuebijing Injection from the obtained UPLC fingerprint of Xuebijing Injection, and numbering them from left to right to obtain common peaks.

[0054] In the present invention, the UPLC fingerprint of the obtained Xuebijing injection preferably has 20 common peaks.

[0055] In the present invention, the relative retention times of the 20 common peaks are preferably: peak 1 0.16, peak 2 0.17, peak 3 0.20, peak 4 0.21, peak 5 0.36, peak 6 0.49, peak 7 0.51, peak 8 0.59, peak 9 0.77, peak 10 0.88, peak 11 0.95, peak 12 1.00, peak 13 1.08, peak 14 1.11, peak 15 1.44, peak 16 1.52, peak 17 1.66, peak 18 1.87, peak 19 1.95, peak 20 2.00, wherein peak 12 is a reference peak.

[0056] The UPLC fingerprint of Xuebijing Injection obtained by the detection method of the present invention was processed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software, and compared with the control fingerprint, the similarity was greater than 0.9.

[0057] The present invention preferably uses paeoniflorin as a reference substance, takes 23 batches of Xuebijing injection samples for detection, calibrates 20 common peaks according to the obtained spectrum, and uses the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Version)" software to process the chromatographic data of the 23 batches of Xuebijing injection samples. The "median" method is used, the time window width is 0.10, and multi-point correction of known chromatographic peaks is performed to perform MARK peak matching on the chromatographic peaks of the fingerprints of the 23 batches of Xuebijing injection samples to generate a control fingerprint spectrum.

[0058] The present invention preferably uses ultra-high performance liquid chromatography combined with high-resolution mass spectrometry data to confirm 5 components among the 20 common peaks through retention time and PDA scanning analysis of chromatographic peaks compared with reference substances (hydroxysafflor yellow A, ferulic acid, paeoniflorin, ligusticum lactone I, and salvianolic acid B).

[0059] In the present invention, when confirming the components in the common peak, the ultra-high performance liquid chromatography conditions are the same as those in the above-mentioned UPLC fingerprint detection method for Xuebijing injection, except that methanol-0.1% formic acid is used as the mobile phase.

[0060] In the present invention, when confirming the components in the common peak, the conditions of high-resolution mass spectrometry are preferably: heated electrospray ionization source (H-ESI), scanning mode is Full MS / dd MS2 positive ion scanning and Full MS / dd MS2 negative ion scanning. Ion source parameters: sheath gas flow rate: 48 arb, auxiliary gas flow rate: 11 arb, purge gas flow rate 5 arb, spray voltage 3.2 kV (+) and 2.8 kV (-), ion transfer tube temperature 350 ° C, auxiliary gas heating temperature 300 ° C. In positive ion mode, the primary scanning range is m / z 100-1500, in negative ion mode, the primary scanning range is m / z 100-1500, the resolution is 35000, and the AGC target is 10 6 , the maximum injection time is 100ms; the secondary scanning resolution is 17500, and the AGC target is 10 5 , the maximum injection time is 50ms and the SteppedNCE is 20%.

[0061] In the present invention, among the 20 common peaks, peak No. 10 is hydroxysafflor yellow A, peak No. 12 is paeoniflorin, peak No. 13 is ferulic acid, peak No. 15 is ligustrin I, and peak No. 17 is salvianolic acid B.

[0062] The present invention preferably detects safflower medicinal material extract, salvia miltiorrhiza medicinal material extract, red peony root medicinal material extract, angelica medicinal material extract, Chuanxiong medicinal material extract, safflower negative injection, red peony root negative injection, Chuanxiong negative injection, angelica negative injection, salvia miltiorrhiza negative injection, Chuanxiong and Angelica negative injection with the test solution according to the chromatographic conditions in the above-mentioned UPLC fingerprint detection method of Xuebijing injection, and calibrates the ownership of 20 common peaks in the fingerprint of the test solution through the retention time of the chromatographic peaks and PDA scanning analysis.

[0063] As an embodiment, the preparation method of the safflower medicinal material extract is: take 5g of safflower medicinal material powder, add 40mL of 30% (volume concentration) ethanol, weigh the weight, heat and reflux for 1h, cool and make up the lost weight with 30% ethanol, filter, and obtain the safflower medicinal material extract.

[0064] As an embodiment, the preparation method of the Danshen medicinal material extract, the red peony root medicinal material extract, the Chinese angelica medicinal material extract, and the Chuanxiong medicinal material extract is: take 5g each of Danshen medicinal material, red peony root medicinal material, Chinese angelica medicinal material, and Chuanxiong medicinal material powder, add 50mL of water, weigh the weight, heat and reflux for 2h, cool and make up the lost weight with water, filter, and obtain each medicinal material extract.

[0065] In the present invention, among the 20 common peaks, peak 1 belongs to safflower, red peony root, Chuanxiong, Chinese angelica and salvia miltiorrhiza, peak 2 belongs to safflower, red peony root, Chuanxiong and Chinese angelica, peak 3 belongs to red peony root, peak 4 belongs to safflower, red peony root, Chuanxiong, Chinese angelica and salvia miltiorrhiza, peak 5 belongs to salvia miltiorrhiza, peak 6 belongs to safflower, red peony root, Chuanxiong, Chinese angelica and salvia miltiorrhiza, peak 7 belongs to safflower, peak 8 belongs to safflower, red peony root, Chuanxiong and Chinese angelica. , Angelica and Salvia miltiorrhiza, Peak No. 9 belongs to Carthamus tinctorius, Peak No. 10 belongs to Carthamus tinctorius, Peak No. 11 belongs to Carthamus tinctorius, Chuanxiong and Angelica, Peak No. 12 belongs to Paeonia lactiflora, Peak No. 13 belongs to Chuanxiong and Angelica, Peak No. 14 belongs to Carthamus tinctorius, Peak No. 15 belongs to Chuanxiong and Angelica, Peak No. 16 belongs to Chuanxiong and Angelica, Peak No. 17 belongs to Salvia miltiorrhiza, Peak 18 belongs to Carthamus tinctorius, Peak 19 belongs to Salvia miltiorrhiza, and Peak 20 belongs to Paeonia lactiflora.

[0066] The detection method of the present invention determines 20 common peaks, identifies the representative components of each prescription medicinal material, and identifies 5 representative components (peak 10, peak 12, peak 13, peak 15 and peak 17) from the 20 common peaks; the 20 common peaks are attributed to the medicinal materials. The 20 common peaks can characterize the components derived from the medicinal materials safflower, red peony root, Chuanxiong, angelica and salvia miltiorrhiza, achieving full coverage of the prescription medicinal materials of Xuebijing Injection and reflecting the overall quality control principles of traditional Chinese medicine.

[0067] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0068] The instruments used in the present invention are as follows: Waters ACQUITY UPLC H-Class ultra-high performance liquid chromatograph, PDA ultraviolet detector (Waters Corporation), Empower™ 3 software data processing system; Waters ACQUITY UPLC I-Class ultra-high performance liquid chromatograph, PDA ultraviolet detector (Waters Corporation), Empower™ 3 software data processing system; Thermo Fisher U3000-QE liquid chromatograph high-resolution mass spectrometer; electronic analytical balances (Mettler Toledo MS204TS / 02, Mettler Toledo XSE105DU, both from Mettler, Switzerland); Master Touch-DUVF water purifier (Shanghai Hetai Company); Elma Schmidbauer GmbH / P300H ultrasonic cleaner (Elma, Germany).

[0069] The samples used in the present invention are as follows: Xuebijing injection (batch number: 2304082, 2304112, 2306162, 2306161, 2306151, 2306152, 2305151, 2305152, 2305162, 2305161, 2304172, 2304171, 2304181, 2304182, 2303181, 2303171, 2303172, 2303182, 2302162, 2302151, 2302152, 2302161, 2305301); Carthamus tinctorius negative sample solution; Paeonia lactiflora negative sample solution; Ligusticum chuanxiong negative sample solution; Angelica sinensis negative sample solution; Salvia miltiorrhiza negative sample solution; Ligusticum chuanxiong and Angelica sinensis double negative sample solution; Carthamus tinctorius medicinal material (batch number: Z01001230101); Paeonia lactiflora medicinal material (batch number: Z00601230301); Angelica sinensis medicinal material (batch number: Z00901230102); Ligusticum chuanxiong medicinal material (batch number: Z00701230301); Salvia miltiorrhiza medicinal material (batch number: Z00801230201). All of the above samples were provided by Tianjin Hongri Pharmaceutical Co., Ltd.

[0070] Test Example 1

[0071] Selection of detection wavelength

[0072] (1) Mixing paeoniflorin with methanol to obtain a reference solution with a paeoniflorin concentration of 0.60 mg / mL;

[0073] (2) Accurately measure 5 mL of Xuebijing injection and place it in a 10 mL volumetric flask. Dilute to the mark with water, shake well, and filter through a microporous filter membrane to obtain the test solution.

[0074] (3) Accurately aspirate 1 μL of the reference solution and the test solution, respectively, and inject them into the ultra-high performance liquid chromatograph for ultra-high performance liquid chromatography detection to obtain the UPLC fingerprint of Xuebijing Injection; the conditions for ultra-high performance liquid chromatography detection are as follows: the chromatographic column is a Waters ACQUITY UPLC BEH C18 chromatographic column; the mobile phase A is methanol; the mobile phase B is 0.1% phosphoric acid; the gradient elution is as shown in Table 1; the flow rate is 0.3 mL / min; the column temperature is 35°C; the detection wavelengths are 210 nm, 280 nm, 330 nm and 403 nm, respectively; the injection volume is 1 μL, and the theoretical plate number is not less than 10,000 calculated based on the paeoniflorin peak.

[0075] Table 1 Gradient elution program in Test Example 1

[0076] Time (min) Mobile phase A (%) Mobile phase B (%) 0～30 2→60 98→40 30～31 60→90 40→10 31～33 90 10 33～34 90→2 10→98 34～40 2 98

[0077] The chromatogram obtained in Test Example 1 is as follows Figure 1 As shown. Figure 1 As can be seen from the figure, the 280nm detection wavelength can more comprehensively reflect the chemical components in Xuebijing Injection, with good separation of the chromatographic peaks and a stable baseline. Therefore, the detection wavelength of 280nm was selected.

[0078] Test Example 2

[0079] Choice of mobile phase

[0080] Methanol-0.1% (volume concentration) phosphoric acid, methanol-0.1% (volume concentration) formic acid and methanol-0.1% (volume concentration) acetic acid were selected as mobile phases, the detection wavelength was 280 nm, and other detection conditions were the same as those in Test Example 1. The chromatogram obtained was as follows: Figure 2 As shown. Figure 2 As can be seen from the figure, methanol-0.1% phosphoric acid provides the best separation results. Not only is the baseline stable, but the selected target peaks are also essentially separated, meeting the required resolution. Therefore, methanol-0.1% phosphoric acid was selected as the mobile phase.

[0081] Test Example 3

[0082] Selection of gradient elution program

[0083] The detection was performed according to the gradient elution program in Table 2, the detection wavelength was 280 nm, and other detection conditions were the same as those in Test Example 1. The chromatogram obtained was as follows: Figure 3 As shown. Figure 3 As can be seen from the figure, gradient elution program 2 achieves the goal of a stable baseline and good separation of the analytes. Therefore, gradient elution program 2 is selected as the gradient elution program.

[0084] Table 2 Different gradient elution programs

[0085]

[0086]

[0087] Test Example 4

[0088] Column selection

[0089] Waters CSHTM Fluoro-Phenyl (2.1*100mm, 1.7μm), WatersACQUITY UPLC HSS T3 (2.1*100mm, 1.8μm), Thermo Hypersil TMGold aQ (2.1*100mm, 1.9μm), Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HD(2.1*100mm, 1.8μm), Waters ACQUITY BEH C18(2.1*100mm, 1.7μm), Waters CORTECS T3 (2.1*100mm, 1.6μm), Agilent ZORBAX SB-C18 Rapid Resolution (2.1*100mm, 1.8μm) chromatographic column, detection wavelength is 280nm, other conditions are as in Test Example 1, the chromatogram obtained is as follows Figure 4 As shown. Figure 4 As can be seen, Waters ACQUITY BEH C18 (2.1*100mm, 1.7μm) has a better separation effect. Therefore, the chromatographic column is selected as Waters ACQUITY BEH C18 (2.1*100mm, 1.7μm).

[0090] Test Example 5

[0091] Selection of column temperature

[0092] The column temperatures were selected at 30°C, 35°C and 40°C respectively, the detection wavelength was 280nm, and the other conditions were tested according to Test Example 1. The chromatograms obtained were as follows: Figure 5 As shown. Figure 5 As can be seen from the figure, a column temperature of 35°C achieves a stable baseline and good separation of the analytes. Therefore, the column temperature of 35°C was selected.

[0093] Test Example 6

[0094] Flow rate selection

[0095] The flow rates were 0.28 mL / min, 0.30 mL / min, and 0.32 mL / min, respectively, and the detection wavelength was 280 nm. Other conditions were tested according to Test Example 1. The chromatograms obtained were as follows: Figure 6 As shown. Figure 6 As can be seen from the figure, the flow rate of 0.30 mL / min has a better overall separation effect. Therefore, the flow rate of 0.30 mL / min is selected.

[0096] Based on test examples 1 to 6, the chromatographic conditions for UPLC fingerprint detection of Xuebijing injection were determined to be: the chromatographic column was a Waters ACQUITY UPLC BEH C18 chromatographic column; mobile phase A was methanol; mobile phase B was 0.1% phosphoric acid; gradient elution, and the gradient elution conditions were: the volume fraction of mobile phase A increased from 2% to 60% from 0 to 30 min, the volume fraction of mobile phase A increased from 60% to 90% from 30 to 31 min, the volume fraction of mobile phase A was 90% from 31 to 33 min, the volume fraction of mobile phase A decreased from 90% to 2% from 33 to 34 min, and the volume fraction of mobile phase A was 2% from 34 to 40 min; the flow rate was 0.3 mL / min; the column temperature was 35°C; and the detection wavelength was 280 nm.

[0097] Test Example 7

[0098] Dilution volume investigation

[0099] Take 5 mL of Xuebijing injection, accurately measure it, put it into a 10 mL volumetric flask, add water to dilute to the scale, shake well, and filter it with a microporous filter membrane to obtain the test solution 1. Take Xuebijing injection, filter it with a microporous filter membrane to obtain the test solution 2. Test solutions 1 and 2 were injected and compared according to the detection method in Test Example 1 and the detection wavelength was 280 nm. The chromatograms obtained are as follows: Figure 7 As shown. Figure 7 It can be seen that the peak shape of the chromatographic peak of test solution 1 (especially hydroxysafflor yellow A) is obviously better than that of test solution 2. Therefore, the preparation method of the test solution is determined as follows: take Xuebijing injection, accurately measure 5 mL, place it in a 10 mL volumetric flask, add water to dilute to the scale, shake well, and filter with a microporous filter membrane to obtain.

[0100] Example 1

[0101] A UPLC fingerprint detection method for Xuebijing injection is:

[0102] (1) Paeoniflorin was mixed with methanol to obtain a reference solution with a paeoniflorin concentration of 0.60 mg / mL;

[0103] (2) Xuebijing Injection was precisely measured at 5 mL, placed in a 10 mL volumetric flask, diluted to the calibration mark with water, shaken well, and filtered with a microporous filter to obtain a test solution;

[0104] (3) The reference solution and the test solution were each precisely taken at 1 μL and injected into an ultra-high performance liquid chromatograph for ultra-high performance liquid chromatography detection to obtain the UPLC fingerprint of Xuebijing Injection. The conditions for ultra-high performance liquid chromatography detection were as follows: the chromatographic column was a Waters ACQUITY UPLC BEH C18 chromatographic column; the mobile phase A was methanol; the mobile phase B was 0.1% phosphoric acid; gradient elution was performed with a gradient elution program of 0-30 min, the volume fraction of the mobile phase A rising from 2% to 60%, 30-31 min, the volume fraction of the mobile phase A rising from 60% to 90%, 31-33 min, the volume fraction of the mobile phase A being 90%, 33-34 min, the volume fraction of the mobile phase A falling from 90% to 2%, and 34-40 min, the volume fraction of the mobile phase A being 2%; the flow rate was 0.3 mL / min; the column temperature was 35°C; the detection wavelength was 280 nm; the injection amount was 1 μL; and the theoretical plate number calculated based on the paeoniflorin peak was not less than 10,000.

[0105] The chromatogram obtained in Example 1 is shown in Figure 8 The 20 chromatographic peaks with good peak shape, separation degree > 1.5, peak area > 6000, and being effective components in Xuebijing Injection were selected and numbered from left to right.

[0106] Methodology verification

[0107] According to the determined chromatographic conditions, Xuebijing Injection (batch number: 2304112) was selected for methodology verification, and the precision, repeatability, stability, and intermediate precision of the method were tested.

[0108] (1) Precision test

[0109] The test solution was prepared according to the method in Example 1, 1 μL of the test solution was precisely taken, and 6 injections were successively performed for determination. The relative retention time and relative peak area of the main chromatographic peaks 1-20 are shown in Tables 3 and 4.

[0110] Table 3 Relative retention time precision results of Xuebijing Injection (N = 6)

[0111]

[0112] Table 4 Relative peak area precision results of Xuebijing injection (N=6)

[0113]

[0114]

[0115] It can be seen from Tables 3 and 4 that the RSDs of the relative retention times and relative peak areas of the main chromatographic peaks are both less than 3.2%, which meets the technical requirements of fingerprint analysis and the method has good precision.

[0116] (2) Repeatability test

[0117] Six test solutions of Xuebijing injection were prepared according to the method in Example 1 and injected for analysis. The relative retention times and relative peak areas of the main chromatographic peaks 1 to 20 are shown in Tables 5 and 6.

[0118] Table 5 Relative retention time repeatability results of Xuebijing injection (N=6)

[0119]

[0120] Table 6 Relative peak area repeatability results of Xuebijing injection (N=6)

[0121]

[0122] It can be seen from Tables 5 and 6 that the RSDs of the relative retention times and relative peak areas of the main chromatographic peaks are both less than 4.8%, indicating that the method has good repeatability.

[0123] (3) Stability test

[0124] The test solution of Xuebijing injection was prepared according to the method in Example 1, and the samples were injected and analyzed at 0, 2, 4, 8, 12, and 24 hours. The relative retention time and relative peak area results of the main chromatographic peaks 1 to 20 are shown in Tables 7 and 8.

[0125] Table 7 Relative retention time stability results of Xuebijing injection test solution

[0126]

[0127]

[0128] Table 8 Relative peak area stability results of Xuebijing injection test solution

[0129]

[0130] As can be seen from Tables 7 and 8, the RSDs of the relative retention times and relative peak areas of the main chromatographic peaks were both less than 4.8%, indicating that the test solution had good stability within 24 h.

[0131] (4) Intermediate precision test

[0132] Different analysts, different test dates, different instruments, for the same batch of samples, 6 test solutions were prepared according to the method in Example 1, and the relative retention time and relative peak area RSDs of the main chromatographic peaks 1 to 20 were examined. The results are shown in Tables 9 and 10. The similarity between the intermediate precision spectrum and the reference spectrum was calculated, and the results are shown in Table 11. The overlay diagram of the precision and intermediate precision chromatograms of Xuebijing Injection is shown in Figure 11. Figure 9 shown.

[0133] Table 9 Relative retention time results of Xuebijing injection under intermediate precision

[0134]

[0135]

[0136] Table 10 Relative peak area results under intermediate precision of Xuebijing injection

[0137]

[0138] Table 11 Similarity results of intermediate precision of Xuebijing injection

[0139] Serial number Similarity Repeatability -1 0.999 Repeatability -2 0.999 Repeatability -3 0.999 Repeatability -4 0.999 Repeatability -5 0.999 Repeatability -6 0.999 Intermediate precision -1 0.998 Intermediate precision-2 0.998 Intermediate precision-3 0.998 Intermediate precision -4 0.998 Intermediate precision -5 0.998 Intermediate precision -6 0.999 RSD (N=12) 0.1％

[0140] As can be seen from Tables 9 to 11, the RSDs of the relative retention times and relative peak areas of the main chromatographic peaks 1 to 20 are all less than 4.0%. The intermediate precision of the detection method of the present invention is good.

[0141] Establishment of control fingerprint

[0142] Paeoniflorin with moderate retention time and good separation was selected as a reference. 23 batches of Xuebijing injection samples were taken for determination. Based on the obtained spectra, 20 common peaks were calibrated. The chromatographic data of the 23 batches of Xuebijing injection samples were processed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Version)" software. The "median" method was used with a time window width of 0.10. Through multi-point correction of known chromatographic peaks, the chromatographic peaks of the fingerprints of the 23 batches of Xuebijing injection samples were MARK matched to generate a reference fingerprint, as shown in Figure 2. Figure 10 shown.

[0143] Test Example 8

[0144] 23 batches of Xuebijing injection samples were taken and tested according to the detection method in Example 1. The chromatographic data of the 23 batches of samples were processed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Version)" software. Compared with the control fingerprint, the 23 batches of sample chromatograms showed 20 common peaks corresponding to the control fingerprint, numbered from left to right. The similarity of the 23 batches of samples was greater than 0.9. The results are shown in Table 12. The superposition of the fingerprints of 23 batches of Xuebijing injection is shown in Figure 12. Figure 11 shown.

[0145] Table 1223 Similarity Data of Xuebijing Injection

[0146]

[0147]

[0148] Attribution of common peaks in the fingerprint of Xuebijing injection

[0149] Take 5g of safflower powder, accurately weigh it in a stoppered conical flask, then accurately add 40mL of 30% (volume concentration) ethanol, weigh it, heat and reflux for 1h, let it cool and make up the lost weight with 30% ethanol, filter it, and get the safflower extract; take 5g each of salvia miltiorrhiza powder, red peony root powder, angelica root powder, and ligusticum chuanxiong powder, accurately weigh it in a stoppered conical flask, then accurately add 50mL of water, weigh it, heat and reflux for 2h, let it cool and make up the lost weight with water, filter it, and get the extracts of each medicinal material. Separately, take the negative injection solution lacking each medicinal material, filter it directly, and analyze it according to the chromatographic conditions in Example 1. According to the retention time of the chromatographic peak and the PDA scanning analysis, the attribution results of the 20 common peaks marked in the fingerprint are shown in Table 13, and the corresponding chromatograms are shown in Table 13. Figure 12 、 Figure 13 .

[0150] Table 13 Chromatographic peak attribution information

[0151]

[0152]

[0153] Identification of common peaks

[0154] Combining high-resolution mass spectrometry data, we identified five components among the 20 common peaks. By comparing the retention times of the chromatographic peaks with those of the reference substance and using PDA scanning analysis, we ultimately confirmed the five common peak components. Peak 10 is hydroxysafflor yellow A, Peak 12 is paeoniflorin, Peak 13 is ferulic acid, Peak 15 is ligustrin I, and Peak 17 is salvianolic acid B. The specific research process is as follows:

[0155] (1) High-resolution mass spectrometry identification results

[0156] Chromatographic conditions: The same as those in Example 1, except that the phosphoric acid in methanol-0.1% phosphoric acid was replaced with formic acid in view of the special requirements of mass spectrometry for the mobile phase.

[0157] Mass spectrometry conditions: heated electrospray ionization source (H-ESI), scanning mode: FullMS / ddMS2 positive ion scan and FullMS / ddMS2 negative ion scan. Ion source parameters: sheath gas flow rate: 48 arb, auxiliary gas flow rate: 11 arb, purge gas flow rate 5 arb, spray voltage 3.2 kV (+) and 2.8 kV (-), ion transfer tube temperature 350 ° C, auxiliary gas heating temperature 300 ° C. In positive ion mode, the primary scan range is m / z 100-1500, and in negative ion mode, the primary scan range is m / z 100-1500, the resolution is 35000, and the AGC target is 1*10 6 , the maximum injection time is 100ms; the secondary scanning resolution is 17500, and the AGC target is 1*10 5 , the maximum injection time is 50ms and the SteppedNCE is 20%.

[0158] The qualitative analysis of Xuebijing injection was performed using high-resolution mass spectrometry under the above chromatographic and mass spectrometric conditions. The total ion current (TIC) and UV chromatograms in positive / negative ion mode are shown in Figure 2. Figure 14 The results of mass spectrometry data analysis are shown in Table 14.

[0159] Table 14 Mass spectrometry analysis of compounds in Xuebijing injection

[0160]

[0161] Note: * indicates that the retention time and fragment ions are consistent with those of the reference substance.

[0162] (2) Identification results of reference substances under liquid chromatography conditions

[0163] The test solution was prepared according to the method in Example 1 and sampled for detection. At the same time, the reference solution was sampled for detection and the components were identified. The results showed that among the 20 common peaks, peak 10: hydroxysafflor yellow A; peak 12: paeoniflorin; peak 13: ferulic acid; peak 15: ligustrin I; peak 17: salvianolic acid B. Figure 15 The spectra of each component reference solution and the spectra of each corresponding component in the test solution are shown in Figure 16 .

[0164] In summary, the detection method provided by the present invention has the advantages of good precision, fast detection speed, and multiple identification components, and is of great significance for the overall quality control of Xuebijing Injection.

[0165] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A UPLC fingerprint detection method for Xuebijing injection, comprising the following steps: (1) Mixing paeoniflorin with methanol to obtain a reference solution; (2) Mixing Xuebijing injection with water to obtain a test solution; (3) The reference solution and the test solution obtained in steps (1) and (2) are respectively subjected to ultra-high performance liquid chromatography detection to obtain the UPLC fingerprint of Xuebijing injection; the conditions for the ultra-high performance liquid chromatography detection include: the chromatographic column is a Waters ACQUITY UPLC BEH C18 chromatographic column; the mobile phase A is methanol; the mobile phase B is 0.1% phosphoric acid; the gradient elution, the gradient elution conditions are: the volume fraction of the mobile phase A increases from 2% to 60% from 0 to 30 min, the volume fraction of the mobile phase A increases from 60% to 90% from 30 to 31 min, the volume fraction of the mobile phase A is 90% from 31 to 33 min, the volume fraction of the mobile phase A decreases from 90% to 2% from 33 to 34 min, and the volume fraction of the mobile phase A is 2% from 34 to 40 min; the flow rate is 0.3 mL / min; the column temperature is 34 to 36 ° C; the detection wavelength is 280 nm, and the injection volume is 1 μL.

2. The UPLC fingerprint detection method for Xuebijing injection according to claim 1, characterized in that: The concentration of the reference solution in step (1) is 0.58-0.62 mg / mL.

3. The UPLC fingerprint detection method for Xuebijing injection according to claim 2, characterized in that: The concentration of the reference solution in step (1) is 0.60 mg / mL.

4. The UPLC fingerprint detection method for Xuebijing injection according to claim 1, characterized in that: In the step (2), the volume ratio of Xuebijing injection to water is 1:(1.8-2.2).

5. The UPLC fingerprint detection method for Xuebijing injection according to claim 4, characterized in that: In step (2), the volume ratio of Xuebijing injection to water is 1:

2.

6. The UPLC fingerprint detection method for Xuebijing injection according to claim 1, characterized in that: The column temperature during ultra-high performance liquid chromatography detection in step (3) is 35°C.

7. The UPLC fingerprint detection method for Xuebijing injection according to claim 1, characterized in that: The UPLC fingerprint of the Xuebijing injection obtained in step (3) has 20 common peaks.

8. The UPLC fingerprint detection method for Xuebijing injection according to claim 7, characterized in that: The relative retention times of the 20 common peaks are: peak 1 0.16, peak 2 0.17, peak 3 0.20, peak 4 0.21, peak 5 0.36, peak 6 0.49, peak 7 0.51, peak 8 0.59, peak 9 0.77, peak 10 0.88, peak 11 0.95, peak 12 1.00, peak 13 1.08, peak 14 1.11, peak 15 1.44, peak 16 1.52, peak 17 1.66, peak 18 1.87, peak 19 1.95, and peak 20 2.

00.

9. The UPLC fingerprint detection method for Xuebijing injection according to claim 7, characterized in that: Among the 20 common peaks, peak 10 is hydroxysafflor yellow A, peak 12 is paeoniflorin, peak 13 is ferulic acid, peak 15 is ligustrin I, and peak 17 is salvianolic acid B.

10. The UPLC fingerprint detection method for Xuebijing Injection according to claim 7, characterized in that: Among the 20 common peaks, peak 1 belongs to safflower, red peony root, Chuanxiong, Chinese angelica and salvia miltiorrhiza, peak 2 belongs to safflower, red peony root, Chuanxiong and Chinese angelica, peak 3 belongs to red peony root, peak 4 belongs to safflower, red peony root, Chuanxiong, Chinese angelica and salvia miltiorrhiza, peak 5 belongs to salvia miltiorrhiza, peak 6 belongs to safflower, red peony root, Chuanxiong, Chinese angelica and salvia miltiorrhiza, peak 7 belongs to safflower, peak 8 belongs to safflower, red peony root, Chuanxiong and Chinese angelica and Salvia miltiorrhiza, peak No. 9 belongs to Carthamus tinctorius, peak No. 10 belongs to Carthamus tinctorius, peak No. 11 belongs to Carthamus tinctorius, Chuanxiong and Angelica sinensis, peak No. 12 belongs to Paeonia lactiflora, peak No. 13 belongs to Chuanxiong and Angelica sinensis, peak No. 14 belongs to Carthamus tinctorius, peak No. 15 belongs to Chuanxiong and Angelica sinensis, peak No. 16 belongs to Chuanxiong and Angelica sinensis, peak No. 17 belongs to Salvia miltiorrhiza, peak No. 18 belongs to Carthamus tinctorius, peak No. 19 belongs to Salvia miltiorrhiza, and peak No. 20 belongs to Paeonia lactiflora.

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