A method for simultaneous determination of multiple components in Huazhuo Sanjie Chubi prescription based on UHPLC-QqQ-MS / MS technology

Abstract

The present invention discloses a method for simultaneously determining the contents of multiple components in Huazhuo Sanjie Chubi Recipe based on UHPLC-QqQ-MS / MS technology. The method can simultaneously determine the contents of 28 components in Huazhuo Sanjie Chubi Recipe, fill the gap in the determination of the contents of chemical components in Huazhuo Sanjie Chubi Recipe, and provide a scientific basis for the quality control research of Huazhuo Sanjie Chubi Recipe. The method provided by the present invention is simple to operate, has a short analysis time, can complete the quantification of 28 components in only 17 minutes, has good precision, repeatability and stability, and has the characteristics of strong specificity and high sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for simultaneously determining the contents of multiple components in a Huazhuo Sanjie Chubi prescription based on UHPLC-QqQ-MS / MS technology. Background Art

[0002] The prescription for Huazhuo Sanjie Chubi is composed of Atractylodes lancea, Smilax glabra, Phellodendron chinense, Coix seed, Sarcandra chinensis, Radix Dianthus, Herba Lysimachiae, Herba Cat's Claw, Verbena officinalis, and Cyathula capitata. The whole prescription takes into account both the symptoms and the root cause, uses both cold and warm effects, and strengthens the body and eliminates evil. It has the functions of invigorating the spleen, promoting dampness and turbidity, clearing away heat, removing phlegm and activating blood circulation. It has achieved satisfactory results in the clinical treatment of chronic gouty arthritis, and has advantages over Tongfengning in improving patients' blood uric acid and the TCM symptoms of chronic gout.

[0003] However, there is currently a lack of systematic research on the material basis of the efficacy of Huazhuo Sanjie Chubi Formula, and there is no research on the quality control of multiple components of Huazhuo Sanjie Chubi Formula. Therefore, establishing a method for determining the content of multiple components in Huazhuo Sanjie Chubi Formula is of great significance to improving the quality control of Huazhuo Sanjie Chubi Formula. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the object of the present invention is to provide a method for simultaneously determining the contents of multiple components in the Huazhuo Sanjie Chubi prescription based on UHPLC-QqQ-MS / MS technology. This method can simultaneously determine the contents of 28 components in the Huazhuo Sanjie Chubi prescription, and has a short detection time and high accuracy, providing a scientific basis for subsequent quality control research.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for simultaneously determining the contents of multiple components in Huazhuo Sanjie Chubi prescription based on UHPLC-QqQ-MS / MS technology comprises the following steps:

[0007] (1) extracting the decoction pieces of each herbal medicine in the Huazhuo Sanjie Chubi prescription with water, adding 0.1% formic acid water-acetonitrile solution to the extract, mixing thoroughly, centrifuging, filtering, and adding a mixed internal standard solution to obtain a test solution; wherein the Huazhuo Sanjie Chubi prescription is prepared from the following raw materials in weight ratio: 1-3 parts of Atractylodes lancea, 2-8 parts of Smilax glabra, 1-3 parts of Phellodendron chinense, 2-6 parts of Coix seeds, 2-4 parts of Sarcandra chinensis, 1-3 parts of Radix Dipterocarpa, 2-4 parts of Lysimachia chinensis, 2-4 parts of Radix Cat's Claw, 1-3 parts of Verbena officinalis, and 1-3 parts of Cyathula officinalis;

[0008] (2) Ultra-high performance liquid chromatography-tandem mass spectrometry was used to determine the contents of multiple components in the test solution; the chromatographic conditions used in the ultra-high performance liquid chromatography were: Thermo Accucore aQ column; acetonitrile was used as mobile phase A, 0.1% formic acid aqueous solution was used as mobile phase B, and the gradient elution program was: 0-0.1 min, 10% A; 0.1-3.0 min, 10% A→13% A; 3.0-8.0 min, 13% A; 8.0-8.5 min, 13% A→17% A; 8.5-11.0 min, 17% A; 11.0-13.0 min, 17% A→70% A; 13.0-14.0 min, 70% A→2% A; 14.0-15.0 min, 2% A; 15.0-15.5 min, 2% A→10% A; 15.5-17.0 min, 10% A.

[0009] Preferably, in step (1), the volume ratio of 0.1% formic acid water to acetonitrile in the 0.1% formic acid water-acetonitrile solution is 9:1.

[0010] Preferably, in step (1), the mixed internal standard solution is a mixed solution of puerarin apiglycoside, strychnine and 2-hydroxycinnamic acid.

[0011] Preferably, in step (1), the extraction is performed by heating and reflux extraction, and the extraction is performed 1-3 times, each time for 1-2 hours.

[0012] Preferably, in step (1), the volume ratio of the extract to the 0.1% formic acid water-acetonitrile solution is 1:1.

[0013] Preferably, in step (2), the chromatographic conditions adopted by the ultra-high performance liquid chromatography also include: a flow rate of 0.2 mL / min; an injection volume of 1-2 μL; and a column temperature of 45°C.

[0014] Preferably, in step (2), the mass spectrometry conditions are: using a HESI ion source, positive and negative ion switching mode: sheath gas: 40, auxiliary gas: 10; spray voltage: 3.8 (+) / 2.8 (-) kV; ion transfer tube temperature: 320 ° C; nebulizer temperature: 350 ° C; dwell time: 7.3 ms; carrier gas is N2; collision gas is Ar; scanning mode: MRM, Q1 resolution: 7000; Q3 resolution: 7000; CID gas: 1.5 mTorr.

[0015] The present invention also provides application of the method in quality control of Huazhuo Sanjie Chubi prescription.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] Based on UHPLC-QqQ-MS / MS technology, the present invention establishes a method for determining the contents of multiple components in the Huazhuo Sanjie Chubi recipe for the first time. Through the optimization of chromatographic mass spectrometry conditions, this method can simultaneously determine the contents of 28 components in the Huazhuo Sanjie Chubi recipe, filling the gap in the determination of the contents of chemical components in the Huazhuo Sanjie Chubi recipe and providing a scientific basis for the quality control research of the Huazhuo Sanjie Chubi recipe.

[0018] The method provided by the present invention is simple to operate and has a short analysis time. The quantification of 28 components can be completed in only 17 minutes. The method has good precision, repeatability and stability, and has the characteristics of strong specificity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 MRM chromatograms of blank solution (A), mixed reference solution (B) and test solution (C);

[0020] Figure 2 This is the investigation result of the chromatographic column Acquity UPLC@BEH;

[0021] Figure 3 This is the result of the investigation of the chromatographic column Accucore 150-Amide-HILIC;

[0022] Figure 4 This is the result of the Accucore aQ test on the chromatographic column;

[0023] Figure 5 This is the result of the investigation of the mobile phase methanol-pure water;

[0024] Figure 6 This is the result of the investigation of the mobile phase acetonitrile-pure water;

[0025] Figure 7 The mobile phase was acetonitrile-0.1% formic acid aqueous solution;

[0026] Figure 8 This is the result of the investigation at a flow rate of 0.1 mL / min;

[0027] Fig. 9 This is the result of the investigation at a flow rate of 0.2 mL / min;

[0028] Fig.10 This is the result of the investigation at a flow rate of 0.3 mL / min;

[0029] Figure 11-13 The results were investigated for different gradient elution programs. DETAILED DESCRIPTION

[0030] In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. However, the implementation methods of the present invention are not limited to the following embodiments.

[0031] Embodiment 1:

[0032] 1.1 Materials and reagents

[0033] Acetonitrile, methanol, and pure water were of mass spectrometry grade and purchased from Merck, Germany; formic acid was of HPLC grade and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the remaining reagents were of analytical grade; the reference substances citric acid (batch number: 111679-201602), phellodendronine hydrochloride (batch number: 111895-201805), verbascoside (batch number: 11530-201914), and jatrorrhizine hydrochloride (batch number: 110733-202110) were purchased from the China Food and Drug Administration; catechins (batch number: S01HB191501), quercetin (batch number: Y13N 11Q130724), caffeic acid (batch number: M28HB183194), protocatechuic acid (batch number: O23HB197783), verbenaside (batch number: Z30A10S587300), isoschaftoside (batch number: P13D11S134210), taxifolin (batch number: C15J11Q107526), ​​isofraquinone (batch number: M27GB141526), ​​rutin (batch number: D13HB202526), ​​vitexin rhamnoside (batch number: Y27A9H60174), isoquercitrin (batch number: X29O11Y1289 70), astilbin (batch number: D26GB172582), kaempferol (batch number: Y11N11H130735), demethylene berberine (batch number: M19GB148924), quercetin (batch number: M23IB210619), rosmarinic acid (batch number: Y06A9K67402), cyasterone (batch number: A27GB158923), berberine hydrochloride (batch number: S01A10K94340), ferulic acid (batch number: G13S11L124423), irisin (batch number: Y17N9L75483), limonin (Batch number: H23J9K65962), isoastilbin (Batch number: P22J9F53283), trigonelline hydrochloride (Batch number: Z19A11H121866), puerarin apiglycoside (internal standard 1, batch number: J13HB173936), and strychnine (internal standard 3, batch number: MUST-17110210) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; 2-hydroxycinnamic acid (internal standard 2, batch number: H1134255) and mulberry yellow (batch number: I2223417) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. All of the above reference substances were HPLC grade.The medicinal materials Atractylodes lancea, Smilax glabra, Phellodendron chinense, Coix seed, Sarcandra chinensis, Cyathula capitata, Lysimachia chinensis, Cat's Claw, Verbena officinalis, and Cyathula capitata were all purchased from Zhicheng Pharmacy on Shangjie Street, Minhou County, Fuzhou, with batch numbers 221018003, 221014, 221004, 22112601, 21062307, 2202018119, 2209019005, 22031208, 221001, and 211201. The above ten medicinal materials were identified by Professor Huang Zehao of the School of Pharmacy of Fujian University of Chinese Medicine and all met the standards of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0034] 2 Methods and Results

[0035] 2.1 Solution preparation

[0036] 2.1.1 Reference substances and internal standard solutions Weigh the appropriate amount of the above 28 reference substances accurately, place them in a volumetric flask, dissolve them with methanol and make up to volume to obtain a reference substance mother solution of about 100 μg / mL. Accurately pipette an appropriate amount of internal standard substance mother solution, dilute it with 0.1% formic acid water-acetonitrile (9:1, v / v) solution to a mixed internal standard solution of 500 ng / mL; then accurately pipette an appropriate amount of the mother solution of the component to be tested, add an appropriate amount of the mixed internal standard solution, and dilute it with 0.1% formic acid water-acetonitrile (9:1, v / v) solution to a mixed reference substance solution of 1 μg / mL.

[0037] 2.1.2 Test solution According to the prescription amount (ratio of Atractylodes: Smilax glabra: Phellodendron chinense: Coix seeds: Sarcandra chinensis: Radix Dioscoreae: Herba Lysimachiae: Herba Cat's claws: Verbena: Cyathula officinalis = 1:3:1:3:3:2:3:3:2, 115 g / prescription in total), weigh 10 pieces of medicinal materials, put them in a round-bottom flask, add 12 times the amount of water, shake well, soak for 30 min, heat and reflux in an electric heating mantle for 3 times, each time for 2 h, filter while hot, combine the filtrate, let it stand and cool, concentrate the extract to 1 L, accurately pipette an appropriate amount of the extract, add an equal volume of 0.1% formic acid water-acetonitrile (9:1, v / v) solution, mix thoroughly, and simmer for 10 min. The mixture was centrifuged at 000 rpm / min for 10 min, the supernatant was taken, filtered through a 0.22 μm microporous membrane, diluted 2.5 times, and an equal volume of the mixed internal standard solution was added to obtain the test solution.

[0038] 2.2 Determination conditions

[0039] 2.2.1 Chromatographic conditions Chromatographic column: Thermo Accucore aQ column (2.1×100mm, 2.6μm); mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B), gradient elution (0-0.1min, 10% A; 0.1-3.0min, 10% A→13% A; 3.0-8.0min, 13% A; 8.0-8.5min, 13% A→17% A; 8.5-11.0min, 17% A; 11.0-13.0min, 17% A→70% A; 13.0-14.0min, 70% A→2% A; 14.0-15.0min, 2% A; 15.0-15.5min, 2% A→10% A; 15.5-17.0min, 10% A); flow rate: 0.2mL / min; injection volume: 2μL; column temperature: 45℃.

[0040] 2.2.2 Mass spectrometry conditions HESI ion source, positive and negative ion switching mode: sheath gas: 40, auxiliary gas: 10; spray voltage: 3.8 (+) / 2.8 (-) kV; ion transfer tube temperature: 320 ° C; nebulizer temperature: 350 ° C; dwell time: 7.3 ms; carrier gas: N2; collision gas: Ar; scan mode: MRM, Q1 resolution: 7000; Q3 resolution: 7000; CID gas:

[0041] 1.5mTorr; The mass spectrometry optimization parameters of each component to be tested are shown in Table 1, and the MRM chromatograms of the mixed reference and test solution are shown in Figure 1 .

[0042] Table 1 Mass spectrometry optimization parameters for each component to be analyzed and internal standard

[0043]

[0044]

[0045] 2.3 Linear regression equation and quantitative limit investigation Accurately measure an appropriate amount of the mixed reference solution, dilute it step by step with pure methanol, and add an equal volume of internal standard solution to obtain 13 standard solutions of different concentrations. The standard solution was sampled and measured, and a linear regression was performed with the peak area ratio of the component to the internal standard as the ordinate and the concentration of the component as the abscissa. The mass concentration of each component when the signal-to-noise ratio was 10 and 3 was used as the quantitative limit (LOQ) and detection limit (LOD), respectively, as shown in Table 2.

[0046] Table 2 Linear equations, detection limits and quantification limits of each component to be tested

[0047]

[0048]

[0049] 2.4 Precision test: Take an appropriate amount of the mixed reference substance mother solution and prepare three concentration gradients of low, medium and high within the linear range. Under the same LC-MS conditions, the samples were injected continuously for 6 times on the same day to determine the intra-day precision. The inter-day precision was investigated by continuous measurement for 3 days, and the peak area of ​​each component was recorded and the RSD was calculated. The results are shown in Table 3. The RSDs were all less than 6.38%, indicating that the instrument had good precision.

[0050] Table 3 Precision test results

[0051]

[0052]

[0053] 2.5 Repeatability test The same extract sample was accurately aspirated, and 6 test solutions were prepared according to the same method in "2.1.2". Under the same chromatographic and mass spectrometric conditions, the samples were continuously injected and measured on the same day. The average content and RSD value of each component were calculated based on the peak area and linear equation of the target component. The results are shown in Table 4. The RSDs were all less than 6.08%, indicating that the method had good repeatability.

[0054] 2.6 Stability test: Precisely weigh the medicinal pieces according to the prescribed amount, prepare the test solution in the same way as in “2.1.2”, and inject the sample for determination at 0, 2, 4, 8, 12, 24, and 48 h after preparation. Record the peak area of ​​each component and calculate the RSD. The results are shown in Table 4. The RSDs were all less than 6.17%, indicating that the test solution was stable within 48 h.

[0055] Table 4 Repeatability and stability test results

[0056]

[0057]

[0058] 2.7 Investigation of sample recovery rate Accurately pipette an appropriate amount of extract with known content into 9 parallel portions, accurately add appropriate amounts of low, medium and high concentration reference solutions respectively, prepare the test solutions in the same way as in "2.1.2", inject and measure respectively, and calculate the average recovery rate and RSD. The results are shown in Table 5.

[0059] Table 5. Sample recovery test results

[0060]

[0061]

[0062]

[0063] 2.8 Content Determination The medicinal pieces were accurately weighed according to the prescribed amount, and 6 parallel portions were prepared as test solution (S1-S6) according to the method in "2.1.2". The contents of 28 components in the samples were calculated according to the internal standard method. The results are shown in Table 6.

[0064] Table 6 Determination results of the content of each component

[0065]

[0066] 3 Optimization of chromatographic conditions

[0067] 3.1 Optimization of chromatographic columns

[0068] The chromatographic columns used were Acquity UPLC@BEH (specifications: 100×2.1 mm, 1.7 μm), Accucore150-Amide-HILIC (specifications: 100×2.1 mm, 2.6 μm), and Accucore aQ (specifications: 150×2.1 mm, 2.6 μm). Other test conditions were the same as in Example 1. The results are shown in FIG. Figure 2-4 As shown in the figure, the Accucore aQ column has better separation of the target peaks, high column efficiency and shortest time.

[0069] 3.2 Screening of mobile phase

[0070] At the same time, different mobile phase systems including acetonitrile-pure water, methanol-pure water, and acetonitrile-0.1% formic acid aqueous solution were investigated. The experimental results are as follows Figure 5-7 As shown, the peak capacity of the acetonitrile system is the best, and the overall peak response is higher than that of the methanol system. The chromatographic separation is improved after adding formic acid to the aqueous phase, and the peak shape is sharp. A better separation effect can be obtained using 0.1% formic acid aqueous solution.

[0071] 3.3 Optimization of flow rate

[0072] The present invention investigated three different flow rates of 0.1 mL / min, 0.2 mL / min, and 0.3 mL / min. Figure 8-10 As shown, at a flow rate of 0.2 mL / min, the peak shape is optimal and the separation degree of each component can reach basic separation.

[0073] 3.4 Optimization of column temperature

[0074] The present invention investigates three column temperatures of 25°C, 35°C and 45°C. At a column temperature of 45°C, the peak elution time is the fastest, the molecular diffusion degree of each component is high, and the separation effect is the best.

[0075] 3.5 Optimization of gradient elution program

[0076] Elution gradient①:

[0077] Chromatographic column: Thermo Accucore aQ column (2.1×100mm, 2.6μm); mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B), gradient elution (0-0.1min, 10% A; 0.1-3.0min, 10% A→13% A; 3.0-8.0min, 13% A; 8.0-8.5min, 13% A→25% A; 8.5-11.0min, 25% A→28% A; 11.0-13.0min, 28% A→90% A; 13.0-14.0min, 90% A→10% A; 14.0-17.0min, 10% A); flow rate: 0.2mL / min; injection volume: 2μL; column temperature: 45℃. Fig.11 As shown, under this elution condition, some compounds interfered in the ion channel and did not achieve baseline separation, so the elution ratio was further adjusted.

[0078] Elution gradient②:

[0079] Column: Thermo Accucore aQ column (2.1×100mm, 2.6μm); mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B), gradient elution (0-0.1min, 10% A; 0.1-3.0min, 10% A→13% A; 3.0-8.0min, 13% A; 8.0-8.5min, 13% A→23% A; 8.5-11.0min, 23% A; 11.0-13.0min, 23% A→80% A; 13.0-14.0min, 80% A→5% A; 14.0-15.0min, 5% A; 15.0-15.5min, 5% A→10% A; 15.5-20.0min, 10% A); flow rate: 0.2mL / min; injection volume: 2μL; column temperature: 45℃. Fig.12 As shown, under this elution condition, some compounds are still not separated, and the elution ratio will be further adjusted and the elution time will be shortened.

[0080] Elution gradient ③:

[0081] Column: Thermo Accucore aQ column (2.1×100mm, 2.6μm); mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B), gradient elution (0-0.1min, 10% A; 0.1-3.0min, 10% A→13% A; 3.0-8.0min, 13% A; 8.0-8.5min, 13% A→17% A; 8.5-11.0min, 17% A; 11.0-13.0min, 17% A→70% A; 13.0-14.0min, 70% A→2% A; 14.0-15.0min, 2% A; 15.0-15.5min, 2% A→10% A; 15.5-17.0min, 10% A); flow rate: 0.2mL / min; injection volume: 2μL; column temperature: 45℃. Fig.13 As shown, under the elution conditions, each compound achieved baseline separation in its own channel, and the peak shape was good, which met the quantitative requirements.

Claims

1. A method for simultaneously determining the contents of multiple components in Huazhuo Sanjie Chubi prescription based on UHPLC-QqQ-MS / MS technology, characterized in that: The steps include: (1) Take the decoction pieces of each herbal medicine in the Huazhuo Sanjie Chubi prescription, add water to extract, add 0.1% formic acid water-acetonitrile solution to the extract and mix thoroughly, centrifuge, filter, and add the mixed internal standard solution to obtain the test solution; The Huazhuo Sanjie Chubi prescription is prepared from the following raw materials in weight ratio: 1-3 parts of Atractylodes lancea, 2-8 parts of Smilax glabra, 1-3 parts of Phellodendron chinense, 2-6 parts of Coix seeds, 2-4 parts of Sarcandra scutellariae, 1-3 parts of Radix Dioscoreae, 2-4 parts of Lysimachia chinensis, 2-4 parts of Cat's Claw, 1-3 parts of Verbena officinalis, and 1-3 parts of Cyathula officinalis. (2) Ultra-high performance liquid chromatography-tandem mass spectrometry was used to determine the content of multiple components in the test solution; The chromatographic conditions adopted by the ultra-high performance liquid chromatography are as follows: the chromatographic column is a Thermo Accucore aQ column; acetonitrile is used as mobile phase A, 0.1% formic acid aqueous solution is used as mobile phase B, and the gradient elution program is as follows: 0-0.1 min, 10%A; 0.1-3.0 min, 10%A→13%A; 3.0-8.0 min, 13%A; 8.0-8.5 min, 13%A→17%A; 8.5-11.0 min, 17%A; 11.0-13.0min, 17%A→70%A; 13.0-14.0 min, 70%A→2%A; 14.0-15.0 min, 2%A; 15.0-15.5 min, 2%A→10%A; 15.5-17.0 min, 10%A; The mass spectrometry conditions were as follows: HESI ion source, positive and negative ion switching mode: sheath gas: 40, auxiliary gas: 10; spray voltage: 3.8 (+) / 2.8 (-) kV; ion transfer tube temperature: 320 °C; nebulizer temperature: 350 °C; dwell time: 7.3 ms; carrier gas: N2; collision gas: Ar; scan mode: MRM, Q1 resolution: 7000; Q3 resolution: 7000; CID gas: 1.5 mTorr; The ingredients include trigonelline hydrochloride, citric acid, protocatechuic acid, catechin, caffeic acid, quercetin, phellodendron hydrochloride, verbena glycoside, isoschaftoside, taxifolin, ferulic acid, vitexin rhamnoside, isofraquinone, rutin, isoquercetin, astilbin, verbascoside, demethyleneberberine, kaempferol glycoside, isoschaftoside, quercetin, rosmarinic acid, amaranthone, berberine hydrochloride, jatrorrhizine hydrochloride, mulberry yellow, tectorius, and limonin.

2. The method according to claim 1, characterized in that In step (1), the volume ratio of 0.1% formic acid water to acetonitrile in the 0.1% formic acid water-acetonitrile solution is 9:

1.

3. The method according to claim 1, characterized in that In step (1), the mixed internal standard solution is a mixed solution of puerarin apiglycoside, strychnine and 2-hydroxycinnamic acid.

4. The method according to claim 1, characterized in that: In step (1), the extraction is performed by heating and reflux extraction, and the extraction is performed 1-3 times, each time for 1-2 hours.

5. The method according to claim 1, characterized in that In step (1), the volume ratio of the extract to the 0.1% formic acid water-acetonitrile solution is 1:

1.

6. The method according to claim 1, characterized in that In step (2), the chromatographic conditions adopted by the ultra-high performance liquid chromatography also include: a flow rate of 0.2 mL / min; an injection volume of 2 μL; and a column temperature of 45°C.

7. Use of the method according to any one of claims 1 to 6 in the quality control of Huazhuo Sanjie Chubi prescription.

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