Method for simultaneously determining contents of five traditional Chinese medicine components in rhizoma anemarrhenae based on UPLC-MS / MS method

Abstract

The invention discloses a method for simultaneously determining the contents of baohuoside, mangiferin, kaempferol, timosaponin A III and timosaponin B II in rhizoma anemarrhenae based on a UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method. The method is based on an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer, a methanol organic phase-0.1% formic acid water phase system is used as a mobile phase, a Waters Acquity UPLCBEH C18 column is selected, ESI is used as a mass spectrometry detection ion source, and the contents of the five traditional Chinese medicines in the rhizoma anemarrhenae powder are accurately quantified at the same time in a positive and negative ion synchronous scanning and multi-reaction monitoring mode. The method has the advantages of simple pretreatment, high detection sensitivity and good reproducibility, and can be used for identification or quality control of main components of corresponding traditional Chinese medicines.

Description

Technical Field

[0001] The present invention relates to the fields of traditional Chinese medicine and mass spectrometry analysis and detection, and particularly relates to an analytical method for simultaneously determining the contents of baohuoside, mangiferin, kaempferol, timosaponin AIII and timosaponin BII based on UPLC-MS / MS method. Background Art

[0002] Anemarrhena rhizome is the dried rhizome of Anemarrhena asphodeloides Bge. of the Liliaceae family, which has the effects of clearing heat and purging fire, nourishing yin and moistening dryness, and can be used for the treatment of diseases such as exogenous febrile diseases, high fever and restlessness, dry cough due to lung heat, hectic fever due to yin deficiency, internal heat and thirst, and constipation due to intestinal dryness. Modern pharmacological research has found that the main active ingredients of Anemarrhena rhizome are steroidal saponins and flavonoids, which have pharmacological effects such as hypoglycemic, antibacterial, anti-inflammatory, antipyretic, anti-Alzheimer's, antidepressant and anti-tumor effects.

[0003] "Chinese Pharmacopoeia" (2020 edition) has taken mangiferin (flavonoid) and timosaponin BII (steroidal saponin) as the quality control index components of Anemarrhena rhizome; in addition, there are also research reports that Anemarrhena rhizome contains other flavonoids (such as baohuoside and kaempferol) and steroidal saponins (such as timosaponin AIII) and other components. Therefore, the detection of the contents of the above-mentioned main active components of traditional Chinese medicine is of great significance for the quality control research of Anemarrhena rhizome.

[0004] At present, due to the different structural characteristics of flavonoid and saponin compounds, that is, their ultraviolet absorption abilities are different, most of the existing studies use liquid chromatography (HPLC) combined with ultraviolet detector (DAD) or evaporative light scattering detector (ELSD) to detect the above two types of substances separately; in addition, there are also studies based on HPLC-ELSD method to achieve the simultaneous quantification of the above two types of substances. However, the detection time of these HPLC analysis methods is often long, and the sensitivity is low, and the reproducibility is poor, which has certain limitations in the quantitative research of the above substances. In contrast, ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS) method combines the high-efficiency separation ability of ultra-high performance liquid chromatography and the high specificity and high sensitivity of mass spectrometry, can overcome the defects of the above analysis methods, can achieve the simultaneous detection and accurate quantification of flavonoid and saponin compounds, and the sample requires a shorter detection time.

[0005] However, there has been no research report on an analytical method for simultaneously and accurately determining the contents of baohuoside, mangiferin, kaempferol, timosaponin AIII and timosaponin BII based on UPLC-MS / MS method and applying it to the identification of main components and quality control research of Anemarrhena rhizome. Summary of the Invention

[0006] Objective of the Invention: The present invention aims to provide an analytical method for simultaneously determining the contents of baohuoside, mangiferin, kaempferol, timosaponin A Ⅲ, and timosaponin B Ⅱ based on UPLC-MS / MS method. By dissolving anemarrhena powder with 50% methanol solution, and then through pretreatment steps such as ultrasonic treatment, dilution, shaking, and high-speed centrifugation separation, finally, the supernatant is taken for detection. This method can accurately and reliably determine the contents of the above five traditional Chinese medicine components in anemarrhena powder, and is used for the identification of main components and quality control of traditional Chinese medicine.

[0007] The present invention provides the following solutions: First, accurately weigh appropriate amounts of reference substances of baohuoside, mangiferin, kaempferol, timosaponin A Ⅲ, and timosaponin B Ⅱ, and use DMSO or 50% methanol solution as a diluent to prepare a reference substance working stock solution with an appropriate concentration; Then, accurately weigh an appropriate amount of anemarrhena powder, dissolve it with 50% methanol solution, after ultrasonic treatment to fully dissolve it, then dilute the original solution, and obtain a test solution after high-speed centrifugation; Next, accurately transfer appropriate amounts of each reference substance working stock solution, use 50% methanol solution for gradient dilution to prepare a mixed calibration curve working solution, detect it on a UPLC-MS / MS instrument, and draw a linear fitting standard curve representing the relationship between the reference substance concentration and the peak area and calculate the corresponding linear fitting equation based on the peak areas of the obtained chromatograms; Finally, accurately transfer an appropriate amount of the test solution, detect it on a UPLC-MS / MS instrument, and calculate the contents of the above 5 traditional Chinese medicine components in the test solution by substituting the peak areas of the measured chromatograms into the linear fitting equation of the calibration curve.

[0008] The chromatographic conditions for detection by UPLC-MS / MS method in the present invention are as follows: Select a Waters Acquity UPLC ® BEH C18 column (100×2.1 mm, 1.7 μm) as the chromatographic column, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is methanol, and the gradient elution conditions are 0 - 3 min, 10% B; 3 - 17 min, 10 - 95% B; 17 - 17.5 min, 95 - 10% B; 17.5 - 20 min, 10% B. The total running time of liquid chromatography is 20 min, the flow rate is 0.3 ml / min; the column temperature is 40 °C, and the injection volume is 10 μl.

[0009] In the present invention, the mass spectrometry conditions for detection by UPLC-MS / MS method are as follows: The ESI ion source is selected, and positive and negative ion synchronous scanning is performed. Under the MRM acquisition mode, targeted detection of 5 target substances is completed. The MRM ion pairs of the target substances and the corresponding mass spectrometry parameters are listed in the following table. In addition, the parameters of each ion source in the ESI positive ion method are as follows: the curtain gas is 35.0 psi, the collision gas is Medium, the ionization voltage is 4500 V, the temperature is set at 450 °C, and the auxiliary heating gases are 50 and 50 psi respectively; in the ESI negative ion method, the ionization voltage is -4500 V, and the parameters such as curtain gas, collision gas, temperature, and auxiliary heating gas are set the same as those in the positive ion method.

[0010] * is used as the quantitative ion pair for calibration curve drawing and sample concentration quantification; the other is the qualitative ion pair to determine the chromatographic peak elution time.

[0011] In the present invention, the pretreatment process of the anemarrhena powder sample is as follows: Weigh an appropriate amount of anemarrhena powder and redissolve it with 50% methanol solution, sonicate for 30 min. After complete dissolution, the test solution stock solution is obtained. Then, accurately pipette an appropriate amount of the stock solution and dilute it again with 50% methanol solution. Then, mix it evenly at 2000 rpm for 5 min, then centrifuge at 12000 rpm for 5 min, and take the supernatant as the test solution for machine detection.

[0012] More preferably, the UPLC-MS / MS method described in the present invention uses a QTRAP 6500 plus triple quadrupole mass spectrometer.

[0013] Furthermore, the lowest quantitative limit of the 5 traditional Chinese medicine component substances under the UPLC-MS / MS method described in the present invention is only 0.2 ng / ml, and the method has high sensitivity.

[0014] Beneficial effects: A method for simultaneously determining the contents of baohuoside, mangiferin, kaempferol, timosaponin A Ⅲ and timosaponin B Ⅱ based on the UPLC-MS / MS method provided by the present invention has the following advantages compared with the prior art: (1) High sample detection efficiency: The method described in the present invention uses the ESI positive and negative ion synchronous scanning mode, which can realize the simultaneous detection of five substances, namely baohuoside, mangiferin, kaempferol, timosaponin A Ⅲ and timosaponin B Ⅱ, effectively improving the sample detection efficiency.

[0015] (2) High method sensitivity: The lowest quantitative limit of the 5 traditional Chinese medicine component substances under the method described in the present invention is only 0.2 ng / ml.

[0016] (3) Simple and convenient sample pretreatment: The present invention uses ultrasonic extraction for sample pretreatment. Compared with the previously reported heating reflux extraction method or impurity removal methods such as AB-8 macroporous adsorption resin, its operation is simpler, more convenient and faster, and can shorten the sample detection time. Description of the Drawings

[0017] Attached Figure 1 It is the detection standard curve graph of the mixed reference solution of 5 traditional Chinese medicine component substances Attached Figure 2 It is the detection chromatogram of the mixed reference solution of 5 traditional Chinese medicine component substances Attached Figure 3 It is the detection chromatogram of the test solution of 5 traditional Chinese medicine component substances Attached Figure 4 It is the chromatogram of the blank sample spiked solution of 5 traditional Chinese medicine component substances Specific Embodiments

[0018] The present invention will be further described in detail below in conjunction with specific implementation cases: 1. Experimental instruments: The instruments mainly include AB Sciex 6500 plus UPLC-MS / MS (composed of Sciex Exion LC AD ultra-high performance liquid chromatograph and QTRAP 6500 plus mass spectrometer); Eppendorf Peb-11 multi-well mixer and Eppendorf 5427R ultra-high speed constant temperature centrifuge.

[0019] 2. Liquid chromatography conditions: Waters Acquity UPLC is selected for liquid chromatography ® BEH C18 column (100×2.1 mm, 1.7 μm) is used as the chromatographic column. Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol. It is run with the mobile phase gradient shown in Table 1 below for 20 min, and the flow rate is 0.3 ml / min; the column temperature is 40 °C, and the injection volume is 10 μl.

[0020]

[0021] 3. Mass spectrometry conditions: ESI ion source was selected for mass spectrometry detection. Targeted detection of 5 analytes was completed under the synchronous positive and negative ion scanning and MRM acquisition mode. The MRM ion pairs of the analytes and the corresponding mass spectrometry parameters are listed in the following table. In addition, the parameters of each ion source in the ESI positive ion method were as follows: curtain gas was 35.0 psi, collision gas was Medium, ionization voltage was 4500 V, temperature was set at 450 °C, and auxiliary heating gas was 50 and 50 psi respectively; in the ESI negative ion method, the ionization voltage was -4500 V, and the parameters such as curtain gas, collision gas, temperature, and auxiliary heating gas were set the same as those in the positive ion method; the MRM ion pairs of the analytes and the corresponding mass spectrometry parameters are shown in Table 2 below.

[0022]

[0023] * was used as the quantitative ion pair for calibration curve plotting and sample concentration quantification; the other was the qualitative ion pair to determine the chromatographic peak elution time.

[0024] 4. Preparation of the mixed reference standard curve working solution: Preparation of the reference working stock solution: Weigh appropriate amounts of the reference standards of baohuoside, mangiferin, kaempferol, and timosaponin AIII precisely. First, use DMSO as the diluent to make the volume of each reference stock solution to a concentration of 1 mg / ml. Then, transfer appropriate amounts of each reference stock solution respectively, add 50% methanol solution and mix well for dilution to prepare 20 μg / ml of each reference working stock solution; in contrast, after weighing the reference standard of timosaponin BII precisely, directly use 50% methanol solution as the diluent and dilute it step by step to 20 μg / ml of the reference working stock solution for the preparation of the subsequent mixed calibration curve working solution.

[0025] Preparation of the mixed calibration curve working solution: Accurately transfer appropriate amounts of each reference working stock solution, and then use 50% methanol solution to prepare the mixed calibration curve working solution with a concentration range of 0.2 - 500 ng / ml by gradient dilution.

[0026]

[0027] 5. Sample treatment method: Weigh appropriate amounts of anemarrhena powder and redissolve it with 50% methanol solution, sonicate for 30 min to obtain the stock solution of the test sample. Then, precisely transfer appropriate amounts of the stock solution and dilute it again with 50% methanol solution. After that, mix well at 2000 rpm for 5 min, then centrifuge at 12000 rpm for 5 min, and take the supernatant as the sample for on-machine detection.

[0028] 6. Experimental results Standard curve: The standard curve results of each target were calculated based on the concentration gradient of the mixed standard curve working solution of the above five targets, and the peak area of the chromatogram of each target and the concentration of the corresponding standard curve solution were shown in Table 4 below. The results show that the linearity of the five targets is good under the method described in the present invention, and the correlation coefficient of the standard curve is r All of them were greater than 0.995, and the linear range also met the detection requirements.

[0029]

[0030] Specificity: Based on the chromatographic separation of the five target substances detected in the reference solution and the test solution, such as Figure 2-4 As shown, under the method of the present invention, the chromatographic peaks of the five targets are well separated, the retention times of the chromatographic peaks in different solutions are consistent, and there is no interference from impurity peaks near the target peaks. The method has good selectivity and specificity.

[0031] Limit of quantification: Based on the signal-to-noise ratio of the chromatographic peak response of the analyte (S / N=10), the limits of quantification of each Chinese medicinal ingredient, baohuoside, mangiferin, kaempferol, timosaponin B Ⅱ and timosaponin A Ⅲ, under this method were determined to be 0.2, 1, 1, 0.5 and 1 ng / ml, respectively.

[0032] Accuracy: A blank sample spike recovery experiment was conducted on the five target substances in a blank sample (i.e., 50% methanol solution). The ratio of the detected concentration value to the known added concentration (i.e., spike recovery) was calculated to represent the accuracy of the detection method. As shown in Table 5 below, the spike recovery rates of the five target substances were in the range of 89.1~106.0%, which met the detection requirements.

[0033]

[0034] In summary, the method of the present invention can realize the simultaneous determination of the contents of five traditional Chinese medicine ingredients, namely, baouside, mangiferin, kaempferol, timosaponin A Ⅲ and timosaponin B Ⅱ. The method has simple sample pretreatment, high detection sensitivity and good reproducibility, and can be applied to the identification and quality control of the main components of TCM Anemarrhena powder.

[0035] The above contents are preferred implementation cases of the present invention and are not intended to limit the scope of the present invention. For relevant detection personnel in this field, the contents and technical solutions of the present invention can be modified, or some characteristic parameters can be replaced by equivalents. Any improvements, equivalent replacements or modifications made within the ideas and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously determining the contents of baohuoside, mangiferin, kaempferol, timosaponin A Ⅲ and timosaponin B Ⅱ in Anemarrhenae Rhizoma by UPLC-MS / MS method, which is characterized in that, It includes the following steps: S1: Weigh an appropriate amount of baohuoside, mangiferin, kaempferol, timosaponin A Ⅲ, and timosaponin B Ⅱ reference substances accurately. Using DMSO or 50% methanol solution as a diluent, prepare working stock solutions of each reference substance at appropriate concentrations. S2: Weigh an appropriate amount of anemarrhena powder accurately, dissolve it in 50% methanol solution, and after ultrasonic dissolution, dilute and centrifuge to obtain a test solution. S3: Accurately pipette an appropriate amount of each working stock solution of the reference substances, prepare a mixed calibration curve working solution by gradient dilution with 50% methanol solution, detect it by UPLC-MS / MS method on the instrument. Based on the peak areas of the chromatograms of each reference substance of traditional Chinese medicine components obtained, draw a linear fitting standard curve showing the relationship between the concentration and peak area of each reference substance of traditional Chinese medicine components, and calculate the corresponding linear fitting equation. S4: Accurately pipette an appropriate amount of the test solution, detect it by UPLC-MS / MS method on the instrument. Based on the peak areas of the chromatograms of each traditional Chinese medicine component measured, substitute them into the linear fitting equation of the calibration curve to calculate the contents of the above 5 traditional Chinese medicine components in the test solution.

2. A method for simultaneously determining the contents of baohuoside, mangiferin, kaempferol, anemarsaponin A Ⅲ and anemarsaponin B Ⅱ based on UPLC-MS / MS method as described in claim 1, characterized in that: In step S1, for baohuoside, mangiferin, kaempferol, and timosaponin A Ⅲ reference substances, first use DMSO as a diluent to make the volume of each reference substance stock solution reach a concentration of 1 mg / ml. Then, respectively pipette an appropriate amount of each reference substance stock solution, add 50% methanol solution and mix well for dilution to prepare working stock solutions of each reference substance at 20 μg / ml for the preparation of the subsequent mixed calibration curve working solution. In contrast, timosaponin B Ⅱ reference substance directly uses 50% methanol solution as a diluent and is stepwise diluted into a working stock solution of 20 μg / ml reference substance.

3. A method for simultaneously determining the contents of baohuoside, mangiferin, kaempferol, anemarsaponin A Ⅲ and anemarsaponin B Ⅱ based on UPLC-MS / MS method as described in claim 1, characterized in that: In step S2, weigh an appropriate amount of anemarrhena powder, dissolve it in 50% methanol solution, sonicate for 30 min, and obtain the original test solution after complete dissolution. Then, accurately pipette an appropriate amount of the original solution and dilute it again with 50% methanol solution. Then mix well at 2000 rpm for 5 min, then centrifuge at 12000 rpm for 5 min, take the supernatant as the sample and detect it on the instrument.

4. A method for simultaneously determining the contents of baohuoside, mangiferin, kaempferol, anemarsaponin A Ⅲ and anemarsaponin B Ⅱ based on UPLC-MS / MS method as described in claim 1, characterized in that: In steps S3 and S4, the UPLC (Ultra Performance Liquid Chromatography) conditions are as follows: Waters Acquity UPLC ® BEH C18 column (100×2.1 mm, 1.7 μm) is used as the chromatographic column. Mobile phase A is an aqueous solution of 0.1% formic acid, and mobile phase B is methanol. The mobile phase gradient listed in the following table is run for 20 min at a flow rate of 0.3 ml / min; the column temperature is 40 °C, and the injection volume is 10 μl.

5. A method for simultaneously determining the contents of baohuoside, mangiferin, kaempferol, anemarsaponin A Ⅲ and anemarsaponin B Ⅱ based on UPLC-MS / MS method as described in claim 1, characterized in that: In steps S3 and S4, the mass spectrometry conditions are as follows: Select the ESI ion source, perform synchronous positive and negative ion scanning, and complete the targeted detection of 5 target substances in the MRM acquisition mode. The MRM ion pairs of the target substances and the corresponding mass spectrometry parameters are listed in the following table. In addition, for the ESI positive ion method, the parameters of each ion source are as follows: curtain gas is 35.0 psi, collision gas is Medium, ionization voltage is 4500 V, temperature is set at 450 °C, and the auxiliary heating gas is 50 and 50 psi respectively; for the ESI negative ion method, the ionization voltage is -4500 V, and the parameters such as curtain gas, collision gas, temperature, and auxiliary heating gas are set the same as those of the positive ion method. * is used as the quantitative ion pair for calibration curve drawing and sample concentration quantification; the other is the qualitative ion pair to determine the elution time of the chromatographic peak.

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