UHPLC-QE-MS method for separating and determining fingerprint components in traditional Chinese medicine composition

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a UHPLC-QE-MS method for separating and determining fingerprint components in a traditional Chinese medicine composition. The traditional Chinese medicine composition consists of the following components in parts by mass: 1-5 parts of leech and 0.5-2.5 parts of ligusticum wallichii. The fingerprint component is prepared from any one or more of saikoside D3, jatrorrhizine, palmatine, (+)-zearalene, oroxylin A, epiberberine, acacetin, genistein, timosaponin B II and tangeretin. According to the method, octadecyl silane bonded silica gel is used as a chromatographic column filler, a 0.05-0.5% formic acid aqueous solution is used as a mobile phase A, a 0.05-0.5% formic acid acetonitrile solution is used as a mobile phase B, fingerprint components in the traditional Chinese medicine composition are separated through linear elution gradient, and mass spectrometry is adopted for detection. Based on the method, the quality consistency and stability of the preparation can be effectively monitored, and support is provided for quality control of the preparation.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a UHPLC-QE-MS method for separating and determining fingerprint components in traditional Chinese medicine compositions. Background Technology

[0002] Chronic kidney disease (CKD) is a major global health concern characterized by progressive loss of kidney function, with high prevalence, low awareness, and poor prognosis. There are many causes of CKD, including glomerulonephritis, tubulointerstitial kidney disease, renal vascular disease, metabolic diseases, connective tissue diseases causing kidney damage, infectious kidney damage, and congenital and hereditary kidney diseases. In recent years, the prevalence of CKD has been increasing annually, but to date, there is still no targeted and effective treatment for non-dialysis CKD patients.

[0003] Traditional Chinese medicine (TCM) therapies have been proven to safely and effectively slow the progression of chronic kidney disease (CKD) through the synergistic effects of multiple targets and pathways, and can serve as an important component of CKD prevention and treatment programs. Chronic kidney disease falls under the TCM categories of "chronic renal failure," "edema," and "guan ge" (a TCM term referring to a specific condition or condition). Master Zheng Xin, a renowned TCM physician, proposed that the main pathogenesis of CKD lies in "blood stasis throughout the entire course of the disease," and treatment should focus on "removing blood stasis and unblocking the meridians," emphasizing the importance of blood-activating and stasis-removing methods. Based on this, Professor Zheng Xin developed a TCM capsule preparation (Hirudo and Ligusticum striatum capsules, medical institution preparation approval number: Yu Yao Zhi Zi Z20170001) using leeches and Ligusticum striatum as the main ingredients. Leeches have strong blood-breaking and mass-dissolving effects, while Ligusticum striatum has the functions of dispelling wind and dampness, activating blood, and relieving pain. The combined use of these two herbs primarily utilizes the "blood-activating and stasis-removing" method to achieve the effect of "removing blood stasis and unblocking the meridians." However, existing traditional Chinese medicine capsule formulations composed of leeches and Ligusticum chuanxiong still have two problems that need to be addressed: 1) the source of raw materials is uncontrollable, which has a certain impact on drug quality; 2) non-standard operation during the soaking and preparation of soft materials of traditional Chinese medicine can also affect drug quality. Therefore, it is necessary to establish a quality model to ensure the safety, efficacy and uniformity of the formulation products. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a method for separating fingerprint components in traditional Chinese medicine compositions based on UHPLC-QE-MS.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for separating fingerprint components in a traditional Chinese medicine composition based on UHPLC-QE-MS, wherein the traditional Chinese medicine composition comprises the following components in parts by weight: 1-5 parts of leech and 0.5-2.5 parts of Ligusticum chuanxiong; the fingerprint components include one or more of the following: buperidin D3, purslane, berberine, (+)-gibberellin, berberine A, epiberberine, farnesin, genistein, anemarrhenasaponin B II, and hesperidin; in the UHPLC-QE-MS method, octadecylsilane-bonded silica gel is used as the column packing, 0.05-0.5% formic acid aqueous solution is used as mobile phase A, and 0.05-0.5% formic acid acetonitrile solution is used as mobile phase B, and the fingerprint components in the traditional Chinese medicine composition are separated by a linear elution gradient.

[0007] Preferably, the traditional Chinese medicine composition consists of the following components in parts by weight: 2 parts leech and 1 part chuanxiong.

[0008] Preferably, the traditional Chinese medicine composition is prepared by the following method:

[0009] 1) Weigh out the prescribed amounts of leeches and chuanxiong, and grind them into the finest powder;

[0010] 2) Using 20-40% ethanol as a wetting agent, the fine powder of the drug obtained in step 1) is made into a soft mass, granulated by shaking, and dried at 50-70℃ to obtain the final product.

[0011] As a preferred option, the wetting agent is 30% ethanol.

[0012] As a more preferred method, in step 2), the drying temperature is 60°C.

[0013] As a more preferred method, the powder obtained in step 2) is granulated, filled into capsule No. 1, and finally filled into a high-density polyethylene bottle for oral solid medicine to obtain a capsule.

[0014] Preferably, the powder obtained in step 2) is used as an extractant in methanol and water to prepare a sample solution.

[0015] More preferably, the volume ratio of methanol to water in the extract is 3-10:1, more preferably 4:1.

[0016] As a more preferred embodiment, the concentration of the internal standard in the extract is 10 μg / mL.

[0017] As a preferred option, the sample preparation method is as follows:

[0018] Dissolve the powder obtained in step 2) in water, centrifuge at 12000 rpm for 15 min at 4 ℃; take the supernatant and add the extraction solution; vortex for 30 s, sonicate in an ice-water bath for 5 min; let stand at -40 ℃ for 1 h; centrifuge the sample at 12000 rpm for 15 min at 4 ℃; take the supernatant, filter, and obtain the final product.

[0019] As a more preferred method, the centrifugation conditions include a centrifugal force of 13800 × g and a radius of 8.6 cm.

[0020] As a better option, a 0.22 μm microporous membrane is used for filtration.

[0021] Preferably, the gradient elution program is set as follows:

[0022] Within 0-11 minutes, mobile phase A decreased from 75-95% to 15-35%, while mobile phase B increased from 5-25% to 65-85%.

[0023] Within 11-12 minutes, mobile phase A decreased from 15-35% to 1-5%, while mobile phase B increased from 65-85% to 95-99%.

[0024] For 12-14 minutes, mobile phase A is maintained at 1-5%, and mobile phase B is maintained at 95-99%.

[0025] Within 14-14.1 minutes, mobile phase A increases from 1-5% to 75-95%, while mobile phase B decreases from 95-99% to 5-25%.

[0026] 14.1-15 minutes, mobile phase A is maintained at 75-95%, and mobile phase B is maintained at 5-25%;

[0027] For 15-16 minutes, mobile phase A is maintained at 75-95%, and mobile phase B is maintained at 5-25%.

[0028] Preferably, the flow rate is 0.3-1.0 mL / min and the column temperature is 35-40℃.

[0029] Preferably, the flow rate is 0.5 mL / min and the column temperature is 40℃.

[0030] Preferably, mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is a 0.1% formic acid acetonitrile solution; the gradient elution program is set as follows:

[0031] Within 0-11 minutes, mobile phase A decreased from 85% to 25%, while mobile phase B increased from 15% to 75%.

[0032] In 11-12 minutes, mobile phase A decreased from 25% to 2%, while mobile phase B increased from 75% to 98%.

[0033] For 12-14 minutes, mobile phase A is maintained at 2%, and mobile phase B is maintained at 98%.

[0034] In 14-14.1 minutes, mobile phase A increased from 2% to 85%, while mobile phase B decreased from 98% to 15%.

[0035] 14.1-15 minutes, mobile phase A is maintained at 85%, and mobile phase B is maintained at 15%;

[0036] For 15-16 minutes, mobile phase A is maintained at 85% and mobile phase B is maintained at 15%.

[0037] The second objective of this invention is to provide a method for identifying fingerprint components in traditional Chinese medicine compositions and determining their content.

[0038] To achieve the above objectives, the present invention adopts the following technical solution:

[0039] A method for identifying fingerprint components in a traditional Chinese medicine composition and determining their content involves separating the fingerprint components in the traditional Chinese medicine composition using the aforementioned method and detecting them using mass spectrometry to obtain a chromatogram.

[0040] As a preferred method, mass spectrometry detection was performed using a Q Exactive Focus mass spectrometer, combined with Xcalibur software, to acquire MS and MS / MS data in IDA acquisition mode, with the mass spectrometry range of 100 to 1500 for each acquisition cycle.

[0041] Preferred mass spectrometry conditions include: spray gas flow rate: 40-50 Arb; auxiliary gas flow rate: 10-20 Arb; capillary temperature: 350-450 ℃; full mass spectrum resolution: 60000-80000; MS / MS resolution: 15000-20000; collision energy: 15 / 30 / 45 in NCE mode; spray voltage in positive ion mode: 3.0-5.0 kV, and / or spray voltage in negative ion mode: -3.0-5.0 kV.

[0042] More preferably, the mass spectrometry conditions include: spray gas flow rate: 45 Arb; auxiliary gas flow rate: 15 Arb; capillary temperature: 400 °C; full mass spectrum resolution: 70000; MS / MS resolution: 17500; collision energy: 15 / 30 / 45 in NCE mode; spray voltage in positive ion mode: 4.0 kV, and / or spray voltage in negative ion mode: -4.0 kV.

[0043] Preferably, the precursor ion of saiko saponin D3 is [M+H]. + m / z 903.491 / 903.516, daughter ions m / z 85.027, 255.208, 273.219; the parent ion of purslane is [M]. + m / z 338.137, daughter ions m / z 323.112, 322.106, 294.110, 308.089; the parent ion of berberine is [M]. +m / z 352.152, daughter ions are m / z 336.123, 337.131, 308.125, 322.106; the parent ion of (+)-gibberellin is [M+H]. + m / z 319.147, daughter ions m / z 298.141, 283.119, 255.124, 254.118, 191.094; the parent ion of pectin A is [MH]. - m / z 283.060, daughter ions m / z 268.037, 240.043, 239.034, 211.039; the parent ion of berberine is [M]. + m / z 336.123, daughter ions m / z 320.092, 321.099, 292.097, 306.074; the parent ion of farnesin is [MH]. - m / z 283.060, daughter ions m / z 268.037, 240.044, 211.040; parent ion of genistein is [M+H]. + m / z 271.060, daughter ions m / z 253.049, 169.012, 123.006; the parent ion of Anemarrhena asphodeloides BII is [MH]. - m / z 919.489 / 919.516, daughter ions m / z 757.430, 101.025, 71.014; parent ion of hesperidin is [M+H] + m / z 373.126, daughter ions m / z 358.105, 343.080, 297.075, 325.071.

[0044] As preferred, the following were identified: 236.42±0.5 min for bufotaside D3; 165.86±0.5 min for bufotaside; 205.25±0.5 min for berberine; 46.73±0.5 min for (+)-gibberelline; 147.72±0.5 min for phloroglucinol A; 162.66±0.5 min for epiberberine; 212.62±0.5 min for farnesin; 188.29±0.5 min for genistein; 235.98±0.5 min for anemarrhenasaponin BII; and 414.21±0.5 min for hesperidin.

[0045] The beneficial effects of this invention are as follows:

[0046] This invention utilizes a self-developed UHPLC-QE-MS method to accurately separate and identify various chemical components from traditional Chinese medicine compositions, including bufotaside D3, purslane, berberine, (+)-gibberellin, berberine A, epiberberine, farnesin, genistein, anemarrhenasaponin BII, and hesperidin. Through qualitative and quantitative analysis of these components, a comprehensive quality model can be established to effectively monitor the consistency and stability of the preparation's quality. This invention provides reliable data support for the quality control of Hirudo medicinalis capsules. Attached Figure Description

[0047] Figure 1 Chromatogram (positive ion mode) of the traditional Chinese medicine capsule preparation prepared in Example 1, detected by UHPLC-QE-MS.

[0048] Figure 2 Chromatogram of the traditional Chinese medicine capsule preparation prepared in Example 1 (negative ion mode) detected by UHPLC-QE-MS. Detailed Implementation

[0049] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0050] Example 1. Traditional Chinese Medicine Capsule Preparation and its Preparation Method

[0051] 1. Prescription

[0052] A traditional Chinese medicine composition comprising the following components in parts by weight: 2 parts leech and 1 part chuanxiong.

[0053] 2. Preparation method of capsule formulation

[0054] 1) Weigh out the prescribed amounts of leeches and chuanxiong, and grind them into 100 mesh;

[0055] 2) Using 30% ethanol as a wetting agent, the fine powder of the drug obtained in step 1) is made into a soft mass, granulated by shaking (20 mesh sieve), and dried at 60°C;

[0056] 3) Granulate the powder obtained in step 2), fill it into No. 1 capsules, and finally fill it into oral solid medicine high-density polyethylene bottles (30 capsules / bottle) to obtain the product.

[0057] Example 2. Clinical Case

[0058] A total of 1283 non-dialysis patients diagnosed with CKD from August 2018 to December 2023 in the applicant's unit were included. The propensity score matching (PSM) method was used to balance the treatment group and the control group, with 360 patients in each group. The changes in indicators such as blood routine, liver and kidney function, fecal occult blood and 24-hour urine protein quantification were used as the main detection indicators to evaluate the therapeutic safety and efficacy of the traditional Chinese medicine capsule preparation of Example 1.

[0059] The results of follow-up on white blood cell count, hemoglobin, blood glucose, urine protein, urine occult blood, fecal occult blood, aspartate aminotransferase, alanine aminotransferase, albumin, prealbumin, serum uric acid, serum creatinine, serum potassium, bicarbonate, and 24-hour urine protein quantification in the intervention group (using Hirudo Tongluo Capsules) and the control group (not using Hirudo Tongluo Capsules) are shown in Table 1. The median concentration of serum creatinine (Cre) in the treatment group was 192 μmol / L (IQR: 145-258), which was significantly lower than that in the control group (212 μmol / L (IQR: 168-293)) (p=0.026), suggesting that the traditional Chinese medicine capsule preparation of the present invention has a renal function protective effect.

[0060] Table 1

[0061]

[0062] Note: "No" corresponds to "0", and "Yes" corresponds to "1"; 1 Pearson chi-square test; Wilcoxon rank-sum test.

[0063] Example 3. Liquid chromatography-mass spectrometry analysis of compound traditional Chinese medicine combination

[0064] 1. Sample preparation method

[0065] Dissolve the powdered herbal medicine in water in the traditional Chinese medicine capsule preparation prepared in Example 1, and centrifuge at 12,000 rpm (centrifugal force 13,800 (×g), radius 8.6 cm) for 15 min at 4 ℃; take 300 μL of supernatant into an EP tube, add 1000 μL of extraction solution (methanol:water = 4:1, internal standard concentration 10 μg / mL); vortex for 30 s, sonicate in an ice-water bath for 5 min; after standing at -40 ℃ for 1 h, centrifuge the sample at 12,000 rpm (centrifugal force 13,800 (×g), radius 8.6 cm) for 15 min at 4 ℃; carefully take the supernatant and filter it through a 0.22 μm microporous membrane into a vial for instrumental analysis.

[0066] 2. Chromatographic conditions

[0067] UHPLC-QE-MS was used for detection. Specifically, a Thermo Fisher Scientific Vanquish+Q Exactive Focus mass spectrometer was used; the column was packed with octadecylsilane-bonded silica gel, preferably an ACQUITY UPLC BEH C18 column (2.1*100 mm, 1.7 μm); the injection volume was 5 μL, the flow rate was 0.5 mL / min, and the column temperature was 40°C; the mobile phase consisted of mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (0.1% formic acid acetonitrile solution); the multi-step linear elution gradient program was as follows:

[0068] Within 0-11 minutes, mobile phase A decreased from 85% to 25%, while mobile phase B increased from 15% to 75%.

[0069] In 11-12 minutes, mobile phase A decreased from 25% to 2%, while mobile phase B increased from 75% to 98%.

[0070] For 12-14 minutes, mobile phase A is maintained at 2%, and mobile phase B is maintained at 98%.

[0071] In 14-14.1 minutes, mobile phase A increased from 2% to 85%, while mobile phase B decreased from 98% to 15%.

[0072] 14.1-15 minutes, mobile phase A is maintained at 85%, and mobile phase B is maintained at 15%;

[0073] For 15-16 minutes, mobile phase A is maintained at 85% and mobile phase B is maintained at 15%.

[0074] 3. Mass spectrometry conditions

[0075] Mass spectrometry analysis was performed using a Q Exactive Focus mass spectrometer in conjunction with Xcalibur software, acquiring MS and MS / MS data in IDA acquisition mode. In each acquisition cycle, the mass spectrometry range was 100 to 1500 MHz. The top three ions in each cycle were screened, and the corresponding MS / MS data were further acquired. Spray gas flow rate: 45 Arb; auxiliary gas flow rate: 15 Arb; capillary temperature: 400 °C; full mass spectrum resolution: 70000 ohms; MS / MS resolution: 17500 ohms; collision energy: 15 / 30 / 45 ohms in NCE mode; spray voltage: 4.0 kV (positive ion mode) or -4.0 kV (negative ion mode).

[0076] The parent ion of saiko saponin D3 is [M+H]. +m / z 903.491 / 903.516, daughter ions m / z 85.027, 255.208, 273.219; the parent ion of purslane is [M]. + m / z 338.137, daughter ions m / z 323.112, 322.106, 294.110, 308.089; the parent ion of berberine is [M]. + m / z 352.152, daughter ions are m / z 336.123, 337.131, 308.125, 322.106; the parent ion of (+)-gibberellin is [M+H]. + m / z 319.147, daughter ions m / z 298.141, 283.119, 255.124, 254.118, 191.094; the parent ion of pectin A is [MH]. - m / z 283.060, daughter ions m / z 268.037, 240.043, 239.034, 211.039; the parent ion of berberine is [M]. + m / z 336.123, daughter ions m / z 320.092, 321.099, 292.097, 306.074; the parent ion of farnesin is [MH]. - m / z 283.060, daughter ions m / z 268.037, 240.044, 211.040; parent ion of genistein is [M+H]. + m / z 271.060, daughter ions m / z 253.049, 169.012, 123.006; the parent ion of Anemarrhenasaponin B II is [MH]. - m / z 919.489 / 919.516, daughter ions m / z 757.430, 101.025, 71.014; parent ion of hesperidin is [M+H] + m / z 373.126, daughter ions m / z 358.105, 343.080, 297.075, 325.071.

[0077] Liquid chromatography-mass spectrometry (LC-MS) results are as follows Figure 1 and Figure 2 As shown, based on existing database information and known literature data, a total of 124 chemical components can be observed, including 44 terpenoids, 36 flavonoids, 27 phenylpropanoids, 2 quinones, and 17 alkaloids. The top ten peak chemical components are shown in Table 2 below.

[0078] Table 2

[0079]

[0080] Based on existing literature, saponins (saikosaponin D3, anemarrhenasaponin B II) in animals and in vitro models have shown that they alleviate renal interstitial fibrosis and proteinuria by inhibiting pathways such as TGF-β / Smad and NF-κB. Alkaloids (berberine, epiberberine, purslane [usually referring to matrine / oxymatrine]) have also shown preclinical protective signals against diabetic nephropathy and renal fibrosis (involving AMPK, TGF-β, EMT, etc.). Flavonoids / isoflavones (farnesin, genistein, hesperidin) have been shown to inhibit inflammatory and fibrotic pathways such as NF-κB, NLRP3, and TGF-β in in vitro and in vivo studies, and improve oxidative stress and metabolic abnormalities. Therefore, the common mechanism of the main chemical components of Hirudo medicinalis capsules is to reduce renal interstitial fibrosis through anti-inflammatory, antioxidant, and TGF-β / Smad / NF-κB inhibition.

Claims

1. A method for separating fingerprint components in traditional Chinese medicine compositions based on UHPLC-QE-MS, characterized in that, The traditional Chinese medicine composition comprises the following components in parts by weight: 1-5 parts of leech and 0.5-2.5 parts of Ligusticum chuanxiong; the fingerprint components include one or more of the following: buperidin D3, purslane, berberine, (+)-gibberellin, berberine A, epiberberine, farnesin, genistein, anemarrhenasaponin B II, and hesperidin; in the UHPLC-QE-MS method, octadecylsilane-bonded silica gel is used as the column packing, 0.05-0.5% formic acid aqueous solution is used as mobile phase A, and 0.05-0.5% formic acid acetonitrile solution is used as mobile phase B, and the fingerprint components in the traditional Chinese medicine composition are separated by a linear elution gradient.

2. The method according to claim 1, characterized in that, The traditional Chinese medicine composition is prepared by the following method: 1) Weigh out the prescribed amounts of leeches and chuanxiong, and grind them into 80-120 mesh; 2) Using 20-40% ethanol as a wetting agent, the fine powder of the drug obtained in step 1) is made into a soft mass, granulated by shaking, and dried at 50-70℃ to obtain the final product.

3. The method according to claim 2, characterized in that, The powder obtained in step 2) is used as an extraction solvent in methanol and water to prepare a sample solution.

4. The method according to claim 1, characterized in that, The gradient elution program is set as follows: Within 0-11 minutes, mobile phase A decreased from 75-95% to 15-35%, while mobile phase B increased from 5-25% to 65-85%. Within 11-12 minutes, mobile phase A decreased from 15-35% to 1-5%, while mobile phase B increased from 65-85% to 95-99%. For 12-14 minutes, mobile phase A is maintained at 1-5%, and mobile phase B is maintained at 95-99%. Within 14-14.1 minutes, mobile phase A increases from 1-5% to 75-95%, while mobile phase B decreases from 95-99% to 5-25%. 14.1-15 minutes, mobile phase A is maintained at 75-95%, and mobile phase B is maintained at 5-25%; For 15-16 minutes, mobile phase A is maintained at 75-95%, and mobile phase B is maintained at 5-25%.

5. The method according to claim 1, characterized in that, The flow rate was 0.3-1.0 mL / min, and the column temperature was 35-40℃.

6. The method according to claim 1, characterized in that, The mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid acetonitrile solution; the gradient elution program is set as follows: Within 0-11 minutes, mobile phase A decreased from 85% to 25%, while mobile phase B increased from 15% to 75%. In 11-12 minutes, mobile phase A decreased from 25% to 2%, while mobile phase B increased from 75% to 98%. For 12-14 minutes, mobile phase A is maintained at 2%, and mobile phase B is maintained at 98%. In 14-14.1 minutes, mobile phase A increased from 2% to 85%, while mobile phase B decreased from 98% to 15%. 14.1-15 minutes, mobile phase A is maintained at 85%, and mobile phase B is maintained at 15%; For 15-16 minutes, mobile phase A is maintained at 85% and mobile phase B is maintained at 15%.

7. A method for identifying fingerprint components in traditional Chinese medicine compositions and determining their content, characterized in that, The fingerprint component in the traditional Chinese medicine composition is separated by the method according to any one of claims 1-6, and detected by mass spectrometry to obtain a chromatogram.

8. The method according to claim 7, characterized in that, The parent ion of saiko saponin D3 is [M+H]. + m / z 903.491 / 903.516, daughter ions m / z 85.027, 255.208, 273.219; the parent ion of purslane is [M]. + m / z 338.137, daughter ions m / z 323.112, 322.106, 294.110, 308.089; the parent ion of berberine is [M]. + m / z 352.152, daughter ions are m / z 336.123, 337.131, 308.125, 322.106; the parent ion of (+)-gibberellin is [M+H]. + m / z 319.147, daughter ions m / z 298.141, 283.119, 255.124, 254.118, 191.094; the parent ion of pectin A is [MH]. - m / z 283.060, daughter ions m / z 268.037, 240.043, 239.034, 211.039; the parent ion of berberine is [M]. + m / z 336.123, daughter ions m / z 320.092, 321.099, 292.097, 306.074; the parent ion of farnesin is [MH]. - m / z 283.060, daughter ions m / z 268.037, 240.044, 211.040; parent ion of genistein is [M+H]. + m / z 271.060, daughter ions m / z 253.049, 169.012, 123.006; the parent ion of Anemarrhenasaponin B II is [MH]. - m / z 919.489 / 919.516, daughter ions m / z 757.430, 101.025, 71.014; parent ion of hesperidin is [M+H] + m / z 373.126, daughter ions m / z 358.105, 343.080, 297.075, 325.

071.

9. The method according to claim 7, characterized in that, Mass spectrometry conditions included: spray gas flow rate: 40-50 Arb; auxiliary gas flow rate: 10-20 Arb; capillary temperature: 350-450 ℃; full mass spectrum resolution: 60000-80000; MS / MS resolution: 15000-20000; collision energy: 15 / 30 / 45 in NCE mode; spray voltage in positive ion mode: 3.0-5.0 kV, and / or spray voltage in negative ion mode: -3.0-5.0 kV.

10. The method according to claim 7, characterized in that, Retention time of 236.42±0.5 min, identified as saikosaponin D3; retention time of 165.86±0.5 min, identified as purslane; retention time of 205.25±0.5 min, identified as berberine; retention time of 46.73±0.5 min, identified as (+)-gibberelline; retention time of 147.72±0.5 min, identified as phloroglucinol A; retention time of 162.66±0.5 min, identified as epiberberine; retention time of 212.62±0.5 min, identified as farnesin; retention time of 188.29±0.5 min, identified as genistein; retention time of 235.98±0.5 min, identified as anemarrhenasaponin B II; retention time of 414.21±0.5 min, identified as hesperidin.

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