Method for simultaneously determining content of components in Yang-supporting and heart-protecting paste by using UHPLC-QTRAP-MS and application of method

Abstract

The invention discloses a method for simultaneously determining the content of components in Yang-supporting and heart-protecting paste by using UHPLC-QTRAP-MS and application of the method. The method comprises the following steps: preparing a test solution and a reference solution; respectively adding into an ultra-high performance liquid chromatograph and a mass spectrometer for detection; and comparing the detection spectrum of the test sample with the detection spectrum of the reference substance, determining the characteristic peak of each component in the test sample, and calculating the content of each component according to the linear relationship between the concentration and peak area of the components in the Yang-supporting heart-protecting paste. According to the invention, the synchronous quantitative analysis method for 12 various components in the Yang-supporting and heart-protecting paste is established based on the UHPLC-QTRAP-MS technology for the first time. The method is rapid, sensitive and high in accuracy, and can provide reference for quality control of the preparation.

Description

Technical Field

[0001] The present invention relates to a method for simultaneously determining the contents of components in Zhuyang Huxin ointment by utilizing UHPLC-QTRAP-MS and application thereof, belonging to the field of component determination. Background Art

[0002] Chronic heart failure (CHF) is the terminal stage of cardiovascular disease characterized by abnormal changes in cardiac structure and / or function due to various causes, leading to impaired ventricular systolic and / or diastolic function. It has become the leading cause of cardiovascular death worldwide. According to the World Health Organization, there are over 37 million CHF patients worldwide, with a five-year mortality rate of up to 50% for moderate to severe cases. Western medicine's "Golden Triangle" and "New Quadruple" therapies are widely used in clinical practice, but they have not improved the clinical outcomes of HF. For some elderly patients, factors such as electrolyte imbalances, renal impairment, and slow heart rate limit their use. Traditional Chinese medicine (TCM), with its synergistic regulatory effects across multiple components and targets, demonstrates unique advantages in improving cardiac function and slowing disease progression. The recently published QUEST study further elevates TCM's status in the treatment of HF. Clinical studies have shown that a Qi-invigorating and blood-activating formula significantly increases patients' 6-minute walk distance, reduces NT-proBNP levels, and reduces the incidence of adverse reactions by 23% compared to Western medicine. Zhuyang Huxin Plaster is a standard formula for treating CHF at Jiangsu Provincial Hospital of Traditional Chinese Medicine. Developed by Professor Jiang Weimin of the Department of Cardiology, it draws on ancient literature and years of clinical experience. It consists of eight Chinese herbs, including Astragalus, Prepared Aconite, and Curcuma, and adheres to the therapeutic principles of "invigorating Qi, warming Yang, and promoting blood circulation and diuresis." Previous studies have shown that this formula can reduce myocardial hypertrophy by 42.7% and significantly improve left ventricular ejection fraction (LVEF) in pressure-overload rats by inhibiting the phosphorylation of GATA4, ERK1 / 2, and p38 MAPK. However, due to the complex composition of traditional Chinese medicine compounds, their quality control has been a bottleneck hindering their clinical application. The current Chinese Pharmacopoeia only provides quantitative control for individual components within individual herbs, making it difficult to fully reflect the overall quality of compound preparations. Therefore, developing analytical methods for the simultaneous determination of multiple active ingredients is crucial to ensuring the safety and stability of the efficacy of the preparation. Summary of the Invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for simultaneously determining the content of ingredients in Zhuyang Huxin Paste using UHPLC-QTRAP-MS and its application.

[0004] Technical solution: To solve the above technical problems, the present invention provides a method for simultaneously determining the content of ingredients in Zhuyang Huxin Paste using UHPLC-QTRAP-MS, comprising the following steps: (1) Preparation of test solution: Take the Zhuyang Huxin ointment, weigh it accurately, add the solvent accurately, plug it tightly, weigh it accurately, treat it with ultrasound, let it cool, weigh it accurately again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain the solution; (2) Take one or more of salidroside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, paeoniflorin, paeoniflorin, calycosin glucoside, formononetin, calycosin, benzoylhypaconitine, kaempferol, hypoaconitine, formononetin and curcumadione, and add solvent to prepare a reference solution; (3) The test solution and reference solution were added to the ultra-high performance liquid chromatography and mass spectrometer respectively for detection; (4) The detection spectrum of the test sample was compared with the detection spectrum of the reference sample. After the characteristic peaks of each component in the test sample were identified, the content of each component was calculated based on the linear relationship between the concentration and peak area of ​​the components in the Yang-Boosting Heart-Protecting Paste; the components included one or more of salidroside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, paeoniflorin, paeoniflorin, calycosin glucoside, formononetin, calycosin, benzoylhypaconitine, kaempferol, hypaconitine, formononetin or curcumadione.

[0005] Wherein, the solvent in step (1) is 50% to 100% methanol.

[0006] The detection conditions of the ultra-high performance liquid chromatography in step (3) are as follows: 0.1% formic acid-acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B; flow rate of 0.3 mL per minute; column temperature of 35°C; injection volume of 0.5-1.0 μL; Wherein, the elution gradient in step (3) is 0-2 minutes, the volume fraction of mobile phase A is 7%, and the volume fraction of mobile phase B is 93%; from 2 to 20 minutes, the volume fraction of mobile phase A changes from 7 to 14%, and the volume fraction of mobile phase B changes from 93 to 86%; from 20 to 40 minutes, the volume fraction of mobile phase A changes from 14 to 26%, and the volume fraction of mobile phase B changes from 86 to 74%; from 40 to 52 minutes, the volume fraction of mobile phase A changes from 26 to 45%, and the volume fraction of mobile phase B changes from 74 to 55%; from 52 to 65 minutes, the volume fraction of mobile phase A changes from 45 to 90%, and the volume fraction of mobile phase B changes from 55 to 10%.

[0007] The detection conditions of the mass spectrometer in step (3) are as follows: simultaneous monitoring of positive and negative ions in a multiple reaction monitoring scanning mode, an electrospray ionization source, an ionization voltage of 5500 V and -4500 V, an ion source temperature of 550°C, a spray gas of 55 psi, an auxiliary gas of 55 psi, continuous heating of the interface, nitrogen gas throughout the process, a curtain gas of 35 psi, and a collision gas of 9 psi.

[0008] The monitoring component ion pairs, declustering voltage (DP) and collision voltage (CE) of the mass spectrometer in step (3) are shown in the table below.

[0009] Mass spectrometry parameters of each component to be measured

[0010] The linear relationship between the concentration and peak area of ​​salidroside in step (4) is y1=41299.7267x1+73481.9820, and x1 is 7.165~71.65μg·mL −1 , wherein x1 is the concentration of salidroside, y1 is the peak area of ​​salidroside; the linear relationship between the concentration and peak area of ​​quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside is y2=190224.7766x2-407.4141, and x2 is 0.136~1.36μg·mL −1 , wherein x2 is the concentration of quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, y2 is the peak area of ​​quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside; the linear relationship between the concentration and peak area of ​​the paeoniflorin is y3=54485x3-35692, and x3 is 2.319~23.19μg·mL −1 , wherein x3 is the concentration of paeoniflorin, y3 is the peak area of ​​paeoniflorin; the linear relationship between the concentration and peak area of ​​paeoniflorin is y4 =74088x4+282370, and x4 is 9.28~92.80μg·mL −1 , wherein x4 is the concentration of paeoniflorin, y4 is the peak area of ​​paeoniflorin; the linear relationship between the concentration and peak area of ​​calycosin isoflavone glucoside is y5 = 4150031.6539x5 + 3736923.0769, and x5 is 0.993~9.93μg·mL −1 , where x5 is the concentration of calycosin and y5 is the peak area of ​​calycosin. The linear relationship between the concentration and peak area of ​​formononetin is y6 = 6000000x6 + 5000000, and x6 is 0.787~7.87μg·mL −1, wherein x6 is the concentration of formononetin, y6 is the peak area of ​​formononetin; the linear relationship between the concentration and peak area of ​​calycosin isoflavones is y7 =5455857.0507x7+1366153.8462, and x7 is 0.296~2.96μg·mL −1 , wherein x7 is the concentration of calycosin and y7 is the peak area of ​​calycosin; the linear relationship between the concentration and peak area of ​​benzoylhypaconitine is y8=4000000x8-4723.1, and x8 is 0.0075~0.075μg·mL −1 , wherein x8 is the concentration of benzoylhypaconitine, y8 is the peak area of ​​benzoylhypaconitine; the linear relationship between the concentration and peak area of ​​kaempferol is y9=111459x9-12685, and x9 is 0.4038~4.038μg·mL −1 , wherein x9 is the concentration of kaempferol, y9 is the peak area of ​​kaempferol; the linear relationship between the concentration and peak area of ​​​​hypaconitine is y 10 =3000000x 10 +578.26,x 10 0.0017~0.017μg·mL −1 , where x 10 is the concentration of hypoaconitine, x 10 The linear relationship between the concentration and peak area of ​​formononetin is y 11 =1371119.6948x 11 +660869.5652,x 11 0.426~4.26μg·mL −1 , where x 11 is the concentration of formononetin, y 11 The linear relationship between the concentration and peak area of ​​zedoaryldione is y 12 =9000000x 12 +54769,x 12 0.085~0.85μg·mL −1 , where x 12 is the concentration of formononetin, y 12 is the peak area of ​​formononetin.

[0011] The present invention also provides application of the method in quality control of Zhuyang Huxin ointment.

[0012] Based on ultra-high performance liquid chromatography-triple quadrupole / linear ion trap mass spectrometry (UHPLC-QTRAP-MS) technology, this invention constructs for the first time a simultaneous quantitative analysis method for the 12 ingredients (covering flavonoids, monoterpenoid glycosides, alkaloids and sesquiterpenes) in Zhuyang Huxin Paste, aiming to provide a basis for the scientific formulation of its quality standards and provide a technical reference for the multi-index quality control research of traditional Chinese medicine compound prescriptions.

[0013] Zhuyang Huxin Paste is a traditional compound paste for the treatment of chronic heart failure. It is composed of eight traditional Chinese medicines, including Astragalus, Aconite root, and Curcuma. In line with the principle that quality control of compound traditional Chinese medicines must balance the substance basis of efficacy and safety, this invention selects 12 ingredients as quality control markers. These include flavonoids (such as calycosin glucoside and formononetin), monoterpene glycosides (paeoniflorin and paeoniflorin), diester / monoester alkaloids (hypoaconitine and benzoylhypoaconitine), sesquiterpenes (curdione), and phenylethanoid glycosides (salidroside), reflecting the compound's "multi-component, multi-target" synergistic effect. Astragalus membranaceus, the main herb in this prescription, has the effects of tonifying qi and promoting yang, promoting diuresis, and strengthening the exterior. Its flavonoid component, calycosin glucoside, is not only included in the Chinese Pharmacopoeia as a quality standard for Astragalus membranaceus but has also been shown to improve cardiac function by regulating myocardial energy metabolism and inhibiting fibrosis. Formononetin, by activating the autophagic pathway, reduces cardiomyocyte apoptosis. These two synergistically enhance Astragalus membranaceus's "qi-tonifying and diuretic" effects. The toxicity-pharmacological balance of the diester alkaloids (hypoaconitine) and monoester alkaloids (benzoylhypoaconitine) contained in the prepared aconite root, serving as the auxiliary herb, is crucial for quality control. Literature indicates that hypoaconitine has a low degradation rate and high stability during decoction. Benzoylhypoaconitine, its deacetylation product, can be used to characterize both the effectiveness of the preparation process and the safety of the formulation. The hypoaconitine content detected in this invention (0.434 μg / mL) was significantly lower than that of benzoylhypoaconitine (1.160 μg / mL), consistent with the expectation that the preparation process reduces toxicity. The present invention selects these two ingredients as quality markers, ensuring the clinical safety and efficacy of the aconite-based ingredients in this prescription through a "key ingredient controls overall quality" strategy. Salidroside and kaempferol alleviate myocardial inflammatory damage by inhibiting the MAPK / ERK pathway. Total glycosides of paeonia lactiflora (TGP) can improve myocardial hypertrophy, and their effects are related to the Wnt / β-catenin signaling pathway. Quercetin disaccharide inhibits MAPK phosphorylation by scavenging ROS, synergistically combating myocardial remodeling. The synergistic effects of these ingredients reveal the molecular mechanism of the compound's "warming yang and protecting the heart" effects.

[0014] Beneficial Effects: Compared with existing technologies, this invention offers the following significant advantages: It is the first to establish a simultaneous quantitative analysis method for 12 components (flavonoids, alkaloids, and terpenes) in Zhuyang Huxin Plaster using UHPLC-QTRAP-MS technology. This method is rapid, sensitive, and highly accurate, providing a reference for quality control of this preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Selection of chromatographic columns: (A) ACQUITY UPLC HSS T3 column; (B) Eclipse Plus C18 RRHD column; (C) Poroshell EC-C18 column; Figure 2 Selection of mobile phase: (A) methanol-0.1% formic acid; (B) 0.1% formic acid acetonitrile-water; (C) 0.1% formic acid acetonitrile-0.1% formic acid; Figure 3 Selection of elution gradient: (A) Gradient 1; (B) Gradient 2; (C) Gradient 3; Figure 4 The total ion current chromatograms of blank solvent (A) and reference solution (B); Figure 5 is the total ion current chromatogram of the test solution. DETAILED DESCRIPTION

[0016] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0017] 1. Instruments: Shimadzu LC-30A ultra-high performance liquid chromatograph (Shimadzu Corporation, Japan); 4500 QTRAP triple quadrupole linear ion trap mass spectrometer (Applied Biosystems, AB Sciex, USA); Analyst ® TF 1.6.3 data acquisition workstation (AB Sciex, USA); KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., China); XPR2 / A 1 / 1000 electronic analytical balance (Mettler-Toledo, Switzerland); ME204E electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd., China); TGL-16C high-speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory, China).

[0018] 2. Reagents and drugs: Reference substances of paeoniflorin (batch number 110736-202407, purity 98.1%), calycosin glucoside (batch number 111920-201606, purity 97.6%), and formononetin (batch number 111703-201504, purity 100%) were purchased from the China Food and Drug Administration; reference substances of formononetin (batch number ST08800120MG, purity 98.0%) and calycosin glucoside (batch number ST08810120 / 6126, purity 98.0%) were purchased from Shanghai Shidande Biotechnology Co., Ltd.; reference substances of salidroside (batch number B20504, purity 98.0%) were purchased from Shanghai Shidande Biotechnology Co., Ltd. Reference substances (purity ≥98%), quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside (batch number B20528, purity ≥98%), paeoniflorin (batch number B21149, purity ≥98%), and kaempferol (batch number B21126, purity ≥98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; reference substances (benzoylhypoaconitine (batch number B412648, purity 98%), hypaconitine (batch number H464954, purity 97%), and curcumadione (batch number C418576, purity 98%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Yang-boosting heart paste was obtained from Jiangsu Provincial Hospital of Traditional Chinese Medicine (batch numbers 20240104, 20240205, and 20240508). Methanol (chromatographic grade, Thermo Fisher); acetonitrile (chromatographic grade, Thermo Fisher); formic acid (mass spectrometry grade, Aladdin); and distilled water from Watsons were used in the experiments.

[0019] 3. Preparation of reference solution: Accurately weigh appropriate amount of salidroside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, paeoniflorin, paeoniflorin, calycosin glucoside, formononetin, calycosin, benzoylhypoaconitine, kaempferol, hypoaconitine, formononetin, and curcumadione reference substances, dissolve them in methanol to prepare stock solutions. Accurately draw an appropriate amount, place it in the same volumetric flask, add methanol to the scale, and shake well to obtain a 71.65µg / mL salidroside reference solution, a 1.36µg / mL quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside reference solution, a 23.19µg / mL paeoniflorin reference solution, a 92.80µg / mL paeoniflorin reference solution, and a 9.93µg / mL calycosin reference solution. Glycoside reference solution, 7.87µg / mL formononetin reference solution, 2.96µg / mL calycosin reference solution, 0.075µg / mL benzoylhypaconitine reference solution, 4.04µg / mL kaempferol reference solution, 0.017µg / mL hypaconitine reference solution, 4.26µg / mL formononetin reference solution, 0.85µg / mL curcumadione reference solution.

[0020] 4. Preparation of test solution: Take 1 g of Zhuyang Huxin ointment, weigh it accurately, place it in a stoppered conical flask, add 20 mL of 50% methanol solution accurately, stopper it tightly, weigh it, and treat it ultrasonically (power 250 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0021] Example 1 Screening of chromatographic conditions Gradient elution was performed according to the provisions of Table 1; the flow rate was 0.3 mL per minute; the column temperature was 35°C; and the injection volume was 0.7 μL.

[0022] Table 1 Liquid phase elution procedure

[0023] Mass spectrometry conditions: Multiple reaction monitoring (MRM) scanning mode was used for simultaneous monitoring of positive and negative ions. The ion source was an electrospray ionization (ESI) source, with ionization voltages (IS) of 5500 V and -4500 V, a TEM temperature of 550°C, a spray gas (GS1) of 55 psi, an auxiliary gas (GS2) of 55 psi, a continuous interface heating, nitrogen flow throughout, a curtain gas (CUR) of 35 psi, and a collision gas (CAD) of 9 psi. The monitored ion pairs, declustering potential (DP), and collision potential (CE) are shown in Table 2.

[0024] Table 2 Mass spectrometry parameters of each component to be measured

[0025] 1. Selection of chromatographic column The 12 ingredients in Zhuyang Huxin Paste include flavonoids, alkaloids, and terpenes, with quite different properties. To achieve chromatographic separation, this experiment used 0.1% formic acid-acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B. According to the above chromatographic and mass spectrometric conditions, the ACQUITY UPLC HSS T3 column (100mm*2.1mm, 1.8μm), Eclipse Plus C18RRHD column (100mm*2.1mm, 1.8μm), and Poroshell EC-C18 column (100mm*2.1mm, 1.9μm) were compared. It was found that the ACQUITY UPLC HSS T3 column (100mm*2.1mm, 1.8μm) had a lower retention capacity for non-polar compounds than the Eclipse Plus C18 RRHD column, a longer separation time, and severe peak tailing ( Figure 1 A). Eclipse Plus C18 RRHD column (100mm*2.1mm, 1.8μm) also has some chromatographic peak tailing ( Figure 1 B), so the Poroshell EC-C18 column (100mm*2.1mm, 1.9μm) was selected. The chromatographic peak retention time is appropriate, and the peak symmetry factor of polar components (such as salidroside) is significantly better than the other two columns. It has good separation and peak shape for both polar and non-polar components ( Figure 1 C).

[0026] 2. Selection of mobile phase Using a Poroshell EC-C18 column (100mm*2.1mm, 1.9μm), the effects of mobile phases consisting of methanol, acetonitrile, and different proportions of formic acid on the separation were investigated under the above-mentioned chromatographic and mass spectrometric conditions. A gradient elution of 0.1% formic acid in acetonitrile to 0.1% formic acid was ultimately selected. The addition of formic acid shortened the retention time of flavonoids (such as quercetin disaccharide) and effectively suppressed peak tailing ( Figure 2 ).

[0027] 3. Selection of gradient program Using 0.1% formic acid acetonitrile (A)-0.1% formic acid (B) as the mobile phase, Poroshell EC-C18 (100mm*2.1mm, 1.9μm) as the chromatographic column, the flow rate was 0.3mL / min; the column temperature was 35℃; the injection volume was 0.7μL. According to the above mass spectrometry conditions, different gradient elution processes were investigated.

[0028] Among them, gradient 1:

[0029] Gradient 2:

[0030] Gradient 3:

[0031] The results are as follows Figure 3 As shown in the figure, under the gradient 3 condition, the response of each chromatographic peak is moderate and the separation is good.

[0032] 4. Selection of injection volume The mobile phase was 0.1% formic acid acetonitrile (A)-0.1% formic acid (B), the chromatographic column was Poroshell EC-C18 (100 mm*2.1 mm, 1.9 μm), the flow rate was 0.3 mL / min; the column temperature was 35°C; the injection volumes were 0.5, 0.7, and 1.0 μL, respectively, and elution was performed according to a gradient of 3. Detection was performed under the above-mentioned mass spectrometry conditions.

[0033] The results showed that the separation was good at injection volumes of 0.5~1.0 μL.

[0034] Example 2 Optimizing the ion source Using an electrospray ionization (ESI) source, we scanned each target compound in both positive and negative ion modes. We found that the positive ion mode was suitable for alkaloids such as hypaconitine and benzoylhypaconitine, some flavonoids such as calycosin and calycosin glucoside, and terpenes such as curcumadione; while the negative ion mode was suitable for saponins such as paeoniflorin and organic acids such as salidroside. Ultimately, the optimal ionization mode and characteristic ion pairs were determined. Collision energy was optimized for each target compound to achieve the optimal fragment ion abundance and improve detection sensitivity.

[0035] Example 3 Investigation of extraction conditions of test samples 1. Investigation of extraction solvent Take 1 g of Zhuyang Huxin ointment, weigh it accurately, place it in a stoppered conical flask, add 50% methanol, 70% methanol, and 20 mL of methanol accurately respectively, stopper it, weigh it, and treat it ultrasonically (power 250 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain it.

[0036] The results showed that the extraction effects were good when 50%~100% methanol was used as the extraction solvent, among which 50% methanol had the best effect.

[0037] Example 3 Specificity Investigation Accurately pipette 2 μL each of blank solvent, mixed reference solution (directly mix the reference solution and add at the same concentration), and test solution, and use octadecylsilane bonded silica gel as the filler (Poroshell EC-C18: column length 100 mm, inner diameter 2.1 mm, particle size 1.9 μm); use 0.1% formic acid in acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, and perform gradient elution as specified in Table 1; the flow rate is 0.3 mL per minute; the column temperature is 35°C; and the injection volume is 0.7 μL. Mass spectrometry conditions used multiple reaction monitoring (MRM) scanning mode with simultaneous monitoring of positive and negative ions. The ion source was an electrospray ionization source (ESI), the ionization voltage (IS) was 5500 V and -4500 V, the ion source temperature (TEM) was 550°C, the spray gas (GS1) was 55 psi, the auxiliary gas (GS2) was 55 psi, the interface was continuously heated, nitrogen was introduced throughout the process, the curtain gas (CUR) was 35 psi, and the collision gas (CAD) was 9 psi. The ion pairs, declustering voltage (DP), and collision voltage (CE) of the monitored components are shown in Table 2. The total ion current of each component is shown in Figure 4 and Figure 5 The results showed that there was no interference between the peaks of each compound, the separation effect was ideal, and the method had good specificity.

[0038] Example 4 Investigation of linear relationship Each reference solution was accurately pipetted and tested according to the conditions of Example 3. Regression was performed with the reference concentration as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 3, indicating that the components had a good linear relationship within their respective concentration ranges (r ≥ 0.9951).

[0039] Table 3 Standard curves and linear ranges of 12 chemical components

[0040] Example 5 Precision Investigation The mixed reference solution was taken and the sample was continuously injected and tested 6 times according to the conditions of Example 3. The peak area RSDs of salidroside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, paeoniflorin, paeoniflorin, isoflavone glucoside, formononetin, isoflavones, benzoylhypoaconitine, kaempferol, hypoaconitine, formononetin and curcumadione were 4.91%, 4.10%, 2.22%, 2.41%, 2.05%, 2.55%, 1.49%, 2.38%, 1.51%, 2.21%, 1.56% and 2.54%, respectively, indicating that the instrument had good precision.

[0041] Example 6 Repeatability Investigation Six portions of the mixed reference solution were sampled and measured according to the conditions of Example 3. The RSDs of salidroside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, paeoniflorin, paeoniflorin, calycosin glucoside, formononetin, calycosin, benzoylhypoaconitine, kaempferol, hypoaconitine, formononetin, and curcumadione were 4.35%, 3.81%, 2.44%, 2.96%, 3.20%, 4.66%, 4.62%, 4.54%, 3.26%, 5.38%, 3.25%, and 4.45%, respectively. This indicates that the method has good reproducibility.

[0042] Example 7 Stability Study Take 6 portions of mixed reference solution, and according to the conditions of Example 3, sample injection was performed at 0, 3, 6, 9, 12, 18, and 24 h, respectively, to measure the peak areas RSD of salidroside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, paeoniflorin, paeoniflorin, isoflavone glucoside, formononetin, isoflavones, benzoylhypoaconitine, kaempferol, hypoaconitine, formononetin, and curcumadione, respectively, and the peak areas RSD were 5.42%, 4.99%, 3.46%, 4.24%, 2.25%, 4.35%, 2.90%, 4.92%, 2.51%, 4.64%, 2.57%, and 4.73%, respectively, indicating that the solution had good stability within 24 h.

[0043] Example 8 Sample recovery test Accurately weigh 6 portions of Zhuyang Huxin Plaster (batch number 20240104) with known content of each component, 0.15 g each, and place them in a stoppered conical flask to obtain a water decoction; add an appropriate amount of the mixed reference solution at a mass ratio of 1:1 to prepare the test solution, perform injection determination according to the conditions of Example 3, and calculate the recovery rate. The results showed that the average recoveries of salidroside, quercetin-3-O-β-D-glucoside, paeoniflorin, paeoniflorin, calycosin glucoside, formononetin, calycosin, benzoylhypoaconitine, kaempferol, hypoaconitine, formononetin and curcumadione were 103.77%, 98.42%, 97.84%, 97.57%, 95.73%, 104.36%, 98.71%, 105.81%, 103.62%, 109.47%, 94.60% and 95.95%, respectively, with RSDs of 4.62%, 4.80%, 4.95%, 3.93%, 3.46%, 3.33%, 3.11%, 2.85%, 2.26%, 4.35%, 3.14% and 2.49%, respectively.

[0044] Example 9 Sample content determination 1 g of each of three different batches of Zhuyang Huxin Paste (Batch: 20240104, 20240205, 20240508) was taken, and three test solutions were prepared repeatedly according to the method under "Preparation of Test Solution". The samples were injected and measured under the conditions of Example 3, and the content was calculated. The results are shown in Table 4.

[0045] Table 4 Determination results of the content of each component (mg / g, n=3)

[0046] Table 4 shows that the contents of 12 ingredients in the three batches of Zhuyang Huxin ointment are quite different. The contents of salidroside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, paeoniflorin, paeoniflorin, calycosin glucoside, formononetin, calycosin, benzoylhypaconitine, kaempferol, hypoaconitine, formononetin, and curcumadione are 5.3 mg / g, 0.19 mg / g, 1.8 mg / g, 7.9 mg / g, 0.85 mg / g, 0.41 mg / g, 0.19 mg / g, 0.0031 mg / g, 0.25 mg / g, 0.00129 mg / g, 0.34 mg / g, and 0.054 mg / g, respectively.

Claims

1. A method for simultaneously determining the contents of ingredients in Zhuyang Huxin Paste using UHPLC-QTRAP-MS, characterized in that: The following steps are involved: (1) Preparation of test solution: Take the Zhuyang Huxin ointment, weigh it accurately, add the solvent accurately, plug it tightly, weigh it accurately, treat it with ultrasound, let it cool, weigh it accurately again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain the solution; (2) Take one or more of salidroside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, paeoniflorin, paeoniflorin, calycosin glucoside, formononetin, calycosin, benzoylhypaconitine, kaempferol, hypoaconitine, formononetin and curcumadione, and add solvent to prepare a reference solution; (3) Add the test solution and reference solution to an ultra-high performance liquid chromatograph and a mass spectrometer for detection; (4) The detection spectrum of the test sample was compared with the detection spectrum of the reference sample. After the characteristic peaks of each component in the test sample were identified, the content of each component was calculated based on the linear relationship between the concentration and peak area of ​​the components in the Yang-Boosting Heart-Protecting Paste; the components included one or more of salidroside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, paeoniflorin, paeoniflorin, calycosin glucoside, formononetin, calycosin, benzoylhypaconitine, kaempferol, hypaconitine, formononetin or curcumadione.

2. The method according to claim 1, characterized in that The solvent in step (1) is 50% to 100% methanol.

3. The method according to claim 1, characterized in that The detection conditions of the ultra-high performance liquid chromatography described in step (3) are as follows: 0.1% formic acid-acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B; flow rate of 0.3 mL per minute; column temperature of 35°C; injection volume of 0.5~1.0 μL.

4. The method according to claim 1, characterized in that In step (3), the elution gradient is 0-2 minutes, the volume fraction of mobile phase A is 7%, and the volume fraction of mobile phase B is 93%; from 2 to 20 minutes, the volume fraction of mobile phase A changes from 7 to 14%, and the volume fraction of mobile phase B changes from 93 to 86%; from 20 to 40 minutes, the volume fraction of mobile phase A changes from 14 to 26%, and the volume fraction of mobile phase B changes from 86 to 74%; from 40 to 52 minutes, the volume fraction of mobile phase A changes from 26 to 45%, and the volume fraction of mobile phase B changes from 74 to 55%; from 52 to 65 minutes, the volume fraction of mobile phase A changes from 45 to 90%, and the volume fraction of mobile phase B changes from 55 to 10%.

5. The method according to claim 1, characterized in that: The detection conditions of the mass spectrometer in step (3) are to simultaneously monitor positive and negative ions under the multiple reaction monitoring scanning mode, the ion source is an electrospray ion source, the ionization voltage is 5500 V and -4500 V, the ion source temperature is 550°C, the spray gas is 55 psi, the auxiliary gas is 55 psi, the interface is continuously heated, nitrogen is introduced throughout the process, the curtain gas is 35 psi, and the collision gas is 9 psi.

6. The method according to claim 1, characterized in that The linear relationship between the concentration and peak area of ​​salidroside in step (4) is y1=41299.7267x1+73481.9820, where x1 is 7.165~71.65μg·mL −1 , wherein x1 is the concentration of salidroside, y1 is the peak area of ​​salidroside; the linear relationship between the concentration and peak area of ​​quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside is y2=190224.7766x2-407.4141, and x2 is 0.136~1.36μg·mL −1 , wherein x2 is the concentration of quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, y2 is the peak area of ​​quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside; the linear relationship between the concentration and peak area of ​​the paeoniflorin is y3=54485x3-35692, and x3 is 2.319~23.19μg·mL −1 , wherein x3 is the concentration of paeoniflorin, y3 is the peak area of ​​paeoniflorin; the linear relationship between the concentration and peak area of ​​paeoniflorin is y4 =74088x4+282370, and x4 is 9.28~92.80μg·mL −1 , wherein x4 is the concentration of paeoniflorin, y4 is the peak area of ​​paeoniflorin; the linear relationship between the concentration and peak area of ​​calycosin isoflavone glucoside is y5 = 4150031.6539x5 + 3736923.0769, and x5 is 0.993~9.93μg·mL −1 , where x5 is the concentration of calycosin and y5 is the peak area of ​​calycosin. The linear relationship between the concentration and peak area of ​​formononetin is y6 = 6000000x6 + 5000000, and x6 is 0.787~7.87μg·mL −1 , wherein x6 is the concentration of formononetin, y6 is the peak area of ​​formononetin; the linear relationship between the concentration and peak area of ​​calycosin isoflavones is y7 =5455857.0507x7+1366153.8462, and x7 is 0.296~2.96μg·mL −1 , wherein x7 is the concentration of calycosin and y7 is the peak area of ​​calycosin; the linear relationship between the concentration and peak area of ​​benzoylhypaconitine is y8=4000000x8-4723.1, and x8 is 0.0075~0.075μg·mL −1 , wherein x8 is the concentration of benzoylhypaconitine, y8 is the peak area of ​​benzoylhypaconitine; the linear relationship between the concentration and peak area of ​​kaempferol is y9=111459x9-12685, and x9 is 0.4038~4.038μg·mL −1 , wherein x9 is the concentration of kaempferol, y9 is the peak area of ​​kaempferol; the linear relationship between the concentration and peak area of ​​​​hypaconitine is y 10 =3000000x 10 +578.26,x 10 0.0017~0.017μg·mL −1 , where x 10 is the concentration of hypoaconitine, x 10 The linear relationship between the concentration and peak area of ​​formononetin is y 11 =1371119.6948x 11 +660869.5652,x 11 0.426~4.26μg·mL −1 , where x 11 is the concentration of formononetin, y 11 The linear relationship between the concentration and peak area of ​​zedoaryldione is y 12 =9000000x 12 +54769,x 12 0.085~0.85μg·mL −1 , where x 12 is the concentration of formononetin, y 12 is the peak area of ​​formononetin.

7. Application of the method according to any one of claims 1 to 6 in the quality control of Zhuyang Huxin Paste.

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