A method for determining the contents of 20 chemical components in Phyllanthus emblica by UHPLC-MS / MS

Abstract

The application belongs to the technical field of chemical analysis, and particularly relates to a method for determining the content of 20 chemical components in Phyllanthus emblica by UHPLC-MS / MS method. The Phyllanthus emblica medicinal material is dissolved in a methanol aqueous solution to obtain a test sample solution; the test sample solution is subjected to UHPLC-MS / MS detection; and the content of 20 chemical components in the Phyllanthus emblica medicinal material is obtained according to the peak area of the chromatographic peak in the obtained chromatogram and a linear regression equation. On the basis of UHPLC-MS / MS detection, the application applies online double collision energy, and can simultaneously and accurately, reliably and sensitively detect the content of 20 chemical components in the Phyllanthus emblica, thereby providing strong technical support for comprehensive quality evaluation and origin tracing of the Phyllanthus emblica medicinal material.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis technology, specifically relating to a method for determining the content of 20 chemical components in Phyllanthus emblica using UHPLC-MS / MS. Background Technology

[0002] Phyllanthus emblica ( Phyllanthi Fructus Phyllanthus emblica, belonging to the Euphorbiaceae family. Phyllanthus emblica The dried, ripe fruit of *Eupatorium fortunei* is mainly distributed in China and Thailand, with its main distribution areas in Yunnan, Fujian, Guangxi Zhuang Autonomous Region, and Sichuan Province in my country. It has wide applications in traditional Chinese medicine and food processing. Its chemical composition is complex, primarily containing tannins, phenolic acids, flavonoids, and organic acids, exhibiting various pharmacological activities such as antioxidant, anti-inflammatory, and hepatoprotective effects. Due to the influence of geographical conditions, climate, and environmental factors, the chemical composition and content of *Eupatorium fortunei* from different sources vary significantly, resulting in inconsistent quality. Therefore, establishing a comprehensive and accurate multi-component quantitative analytical method to reflect its intrinsic chemical composition is crucial for its quality control and evaluation.

[0003] Currently, existing technologies cannot accurately and reliably detect all 20 chemical components in Phyllanthus emblica simultaneously. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the content of 20 chemical components in Phyllanthus emblica using UHPLC-MS / MS. The method provided by this invention can simultaneously, accurately, reliably, and sensitively detect the content of 20 chemical components in Phyllanthus emblica, and can provide strong technical support for the comprehensive quality evaluation and origin traceability of Phyllanthus emblica medicinal materials.

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

[0006] This invention provides a method for determining the content of 20 chemical components in Phyllanthus emblica using UHPLC-MS / MS, comprising the following steps: dissolving Phyllanthus emblica in a methanol-water solution to obtain a test solution; performing ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS / MS) on the test solution; obtaining the content of the 20 chemical components in Phyllanthus emblica based on the peak areas of the chromatographic peaks and a linear regression equation, wherein the vertical axis of the linear regression equation represents the peak areas of the 20 chemical components, and the horizontal axis represents the concentrations of the 20 chemical components; the UHPLC conditions for the UHPLC-MS / MS detection include: mobile phases A and B, wherein mobile phase A is an aqueous formic acid solution, and mobile phase B is acetonitrile; the elution method is gradient elution, and the gradient elution program is: 0~3 min, with the volume percentage of mobile phase B changing uniformly from 3% to 25%, 3~4.5 For 4.5–5.5 min, the volume percentage of mobile phase B changes uniformly from 25% to 32.5%. For 5.5–10 min, the volume percentage of mobile phase B remains at 32.5%. For 10–11 min, the volume percentage of mobile phase B changes uniformly from 32.5% to 55%. The volume percentage of mobile phase B was uniformly changed from 55% to 80% at a constant rate. The mass spectrometry conditions for the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection included: an electrospray ion source, a negative ion scanning mode, and a multiple reaction monitoring (MRM) detection mode. The 20 chemical components included gallic acid, ellagic acid, chebulic acid, chebulic biphenyl acid, corilagin, geranyl oleoresin, L-malic acid, methyl gallate, ethyl gallate, protocatechuic acid, eugenol, naringenin, sennaol, epicatechin, catechin, quercetin, piperidin, quercetin, isoquercitrin, and rutin. This invention establishes an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS / MS) method to achieve simultaneous and accurate quantification of 20 chemical components in Phyllanthus emblica. The method provided by this invention is accurate, reliable, and sensitive, and can provide strong technical support for the comprehensive quality evaluation and origin traceability of Phyllanthus emblica.

[0007] Furthermore, in this invention, the linear regression equations for 20 chemical components are shown in Table 8. Currently, traditional mass spectrometry quantitative methods often fail to reach the upper limit of quantitation (ULOQ) for high-concentration components when faced with samples with extremely wide concentration ranges due to detector saturation. This invention, based on ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS / MS) detection, applies online double collision energy (ODCE) to achieve simultaneous and accurate quantification of 20 chemical components in Phyllanthus emblica. This invention targets six components with high content—gallic acid, ellagic acid, chebulic acid, chebulic biphenyl acid, corilagin, and geranyl oleoresin—using ODCE to broaden their upper limit of quantitation by constructing a dual calibration curve (DCC). The linear regression equations for the 20 chemical components provided by this invention show good linearity within their respective detection ranges (r>0.999), with an average recovery rate of 93.25%~104.77%. This invention solves the problem of insufficient quantitative range of high-content components by introducing the ODCE strategy, and can provide strong technical support for the comprehensive quality evaluation and origin traceability of Phyllanthus emblica. Attached Figure Description

[0008] Figure 1 MRM chart for standard reference ( Figure 1 (A) and sample MRM map ( Figure 1 (B in the middle)

[0009] Figure 2 The response intensity-collision energy curves for gallic acid, ellagic acid, chebulic acid, chebulic biphenyl acid, corilagin, and geraniol are shown. Detailed Implementation

[0010] This invention provides a method for determining the content of 20 chemical components in Phyllanthus emblica using UHPLC-MS / MS, comprising the following steps:

[0011] The Phyllanthus emblica herb was dissolved in an aqueous methanol solution to obtain the test solution;

[0012] The test solution was subjected to ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry. The contents of 20 chemical components in Phyllanthus emblica were obtained based on the peak areas of the chromatographic peaks in the obtained chromatograms and the linear regression equation. The vertical axis of the linear regression equation was the peak area of ​​the 20 chemical components, and the horizontal axis was the concentration of the 20 chemical components.

[0013] The ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS / MS) conditions for detection include: mobile phases A and B, where mobile phase A is an aqueous formic acid solution and mobile phase B is acetonitrile; the elution method is gradient elution, and the gradient elution program is as follows: 0–3 min, the volume percentage of mobile phase B changes uniformly from 3% to 25%; 3–4.5 min, the volume percentage of mobile phase B changes uniformly from 25% to 32.5%; 4.5–5.5 min, the volume percentage of mobile phase B remains at 32.5%; 5.5–10 min, the volume percentage of mobile phase B changes uniformly from 32.5% to 55%; 10–11 min, the volume percentage of mobile phase B changes uniformly from 55% to 80%.

[0014] The mass spectrometry conditions for the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection include: the ion source is an electrospray ion source, the scanning mode is a negative ion scanning mode, and the detection mode is multiple reaction monitoring.

[0015] The 20 chemical components include gallic acid, ellagic acid, chebulic acid, chebulic biphenyl acid, corilagin, geraniol, L-malic acid, methyl gallate, ethyl gallate, protocatechuic acid, eugenol, naringenin, sennaol, epicatechin, catechin, quercetin, piperidin, quercetin, isoquercetin, and rutin.

[0016] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0017] This invention dissolves Phyllanthus emblica in an aqueous methanol solution to obtain a test solution. Preferably, the Phyllanthus emblica is in powder form. The methanol content in the aqueous methanol solution is preferably 50% by mass. Before dissolution, the Phyllanthus emblica powder is preferably sieved through a No. 3 sieve. The dissolution is performed under ultrasonic conditions, with a preferred ultrasonic power of 250-350 W, and 300 W in some examples. The preferred ultrasonic frequency is 30-50 Hz, and 40 Hz in some examples. The initial solution obtained from the dissolution is further preferably subjected to membrane filtration to obtain the test solution. The membrane filtration preferably uses a microporous membrane, with a preferred pore size of 0.22 μm.

[0018] In this invention, the volume percentage of formic acid in the formic acid aqueous solution is 0.05~0.15%, most preferably 0.1%.

[0019] In this invention, the flow rate of the mobile phase is 0.2~0.4 mL / min, and most preferably 0.3 mL / min.

[0020] In this invention, the column temperature of the chromatographic column is preferably 30~35 °C. The injection volume is preferably 1.5~2 μL.

[0021] In this invention, the chromatographic column is preferably ACQUITY UPLC® HSS T3. The column specifications are preferably 2.1 × 100 mm and 1.8 μm.

[0022] In this invention, the preferred mass spectrometry conditions further include: a capillary voltage of 1.5 kV; an ion source temperature of 150°C; a desolvation gas temperature of 400°C; a cone-shaped gas flow rate of 150 L / h; a desolvation gas flow rate of 800 L / h; a cone-shaped backflush gas flow rate of 30 L / h; and argon as the collision gas, preferably with a purity of 99.99%. The flow rate of the collision gas is preferably 0.15 mL / min.

[0023] In this invention, the multiple reaction ion monitoring parameters detected by mass spectrometry include ion mode, parent ion (m / z), daughter ion (m / z), fragmentation voltage (V) and collision energy (V).

[0024] In this invention, the collision energy of gallic acid, ellagic acid, chebulic acid, chebulic biphenyl acid, corilagin and geraniol among the 20 chemical components is determined using a double collision energy method.

[0025] The preferred method for determining the double collision energy of gallic acid, ellagic acid, chebulin, chebulin biphenyl acid, corilagin, and geraniol includes: changing the collision energy within the range of 1 to 99 eV, with the collision energy change increment being 2 eV, to obtain the response intensity of each analyte. Then, using the response intensity as an indicator, a nonlinear regression is performed on the obtained response intensity data to obtain the response intensity-collision energy curves of gallic acid, ellagic acid, chebulin, chebulin biphenyl acid, corilagin, and geraniol; based on the response intensity-collision energy curves, the two collision energies (CE) corresponding to the maximum response intensity (MRI) and 10% of the MRI are selected as the optimal double collision energy values ​​(DCE). The final determined double collision energies (high CE / low CE) are: gallic acid (14 / 6 eV), ellagic acid (36 / 24 eV), chebulic acid (12 / 4 eV), chebulic biphenyl acid (56 / 24 eV), corilagin (38 / 21 eV) and geraniol (58 / 29 eV).

[0026] In this invention, the parameters for multi-reaction ion monitoring of the 20 chemical components are shown in Table 1.

[0027] In this invention, the linear regression equations for the 20 chemical components are shown in Table 8.

[0028] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] 1. Instruments and Materials

[0031] 1.1 Instruments

[0032] ACQUITY UHPLC I-Class ultra-high performance liquid chromatograph (Waters Corporation, USA); Xevo TQ-S triple quadrupole tandem mass spectrometer (Waters Corporation, USA); MassLynx analytical software (Waters Corporation, USA); Milli-Q IQ7005 ultrapure water preparation system (Millipore); 5424 R high-speed centrifuge (Eppendorf GmbH, Germany); AS 60 / 220. R2 0.0001 ppm balance (Radiwag GmbH, Poland); G3KT 18273 vortex mixer (Thermo Fisher Scientific, USA).

[0033] 1.2 Materials

[0034] Standard reference standards: Gallic acid (batch number: MUST-16022801), Ellagic acid (batch number: DSTDR000401), Terminalia chebulic acid (batch number: DST231116-244), Terminalia biphenyl acid (batch number: DST200927-064), Corilagin (batch number: DST210311-012), Geraniol (batch number: DST230619-008), L-malic acid (batch number: DST201124-038), Methyl gallate (batch number: DST200424-077), Ethyl gallate (batch number: DSTDM006301), Protocatechuic acid (batch number: DST191102-081). Eugenol (batch number: DSTDD030901), naringenin (batch number: DST211116-100), sennaol (batch number: DSTDS004201), epicatechin (batch number: DST200711-035), catechin (batch number: DE0147-0020), quercetin (batch number: DST180521-028), piperidin (batch number: DSTDH004102), quercetin (batch number: DST190413-006), isoquercetin (batch number: DSTTY000601), and rutin (batch number: DST210520-017) were purchased from Chengdu Dest Biotechnology Co., Ltd., China, and all had a purity greater than 98%. Methanol and acetonitrile (chromatographic grade) were purchased from Fisher Scientific, USA; formic acid was purchased from ROE, USA; and ultrapure water was prepared using a Milli-Q ultrapure water preparation system. Phyllanthus emblica samples were collected from different producing areas in China and Thailand. Specifically: batches S1, S2, and S3 were from Sichuan Province, China; batches S4, S5, and S6 were from Fujian Province, China; batches S7, S8, and S9 were from Guangxi Zhuang Autonomous Region, China; and batch S10 was from Thailand.

[0035] 2. Methods and Results

[0036] 2.1 Chromatographic conditions

[0037] Chromatographic column: ACQUITY UPLC® HSS T3 (2.1 × 100 mm, 1.8 μm; Waters); Mobile phase: Phase A was 0.1% formic acid aqueous solution, and Phase B was acetonitrile. Gradient elution program: 0–3 min, 3%–25% B; 3–4.5 min, 25%–32.5% B; 4.5–5.5 min, 32.5%–32.5% B; 5.5–10 min, 32.5%–55% B; 10–11 min, 55%–80% B. Flow rate: 0.3 mL / min; Column temperature: 35 °C; Injection volume: 2.0 μL.

[0038] 2.2 Mass Spectrometry Conditions

[0039] Ion source: Electrospray ionization (ESI); Scanning mode: Negative ion scanning mode; Detection mode: Multiple reaction monitoring (MRM); Capillary voltage: 1.5 kV; Ion source temperature: 150℃; Desolvent gas temperature: 400℃; Conical orifice gas flow rate: 150 L / h; Desolvent gas flow rate: 800 L / h; Conical orifice backflush gas flow rate: 30 L / h; Collision gas: Argon (purity 99.99%, flow rate 0.15 mL / min). Detailed MRM parameters for 20 chemical components are shown in Table 1.

[0040] Table 1 Mass Spectrometry Parameters of 20 Chemical Components

[0041]

[0042] 2.3 Solution Preparation

[0043] 2.3.1 Preparation of the reference solution

[0044] Accurately weigh appropriate amounts of gallic acid, ellagic acid, chebulic acid, chebulic biphenyl acid, corilagin, geraniol, L-malic acid, methyl gallate, ethyl gallate, protocatechuic acid, eugenol, naringenin, sennaol, epicatechin, catechin, quercetin, piperidin, quercetin, isoquercitrin, and rutin standard references, dissolve them in methanol to prepare a standard reference solution with a concentration of 1 mg / mL, and store it in a refrigerator at 4℃ for later use.

[0045] 2.3.2 Preparation of the test solution

[0046] Accurately weigh 50.0 mg of Phyllanthus emblica powder (passed through a No. 3 sieve), place it in a stoppered conical flask, accurately add 10 mL of 50% methanol, seal tightly, weigh, and sonicate (300 W power, 40 kHz frequency) for 30 min. After cooling, make up the weight loss, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0047] 2.4 Methodological Examination

[0048] 2.4.1 Establishment of Online Double Collision Energy Strategy (ODCE) and Double Correction Curve (DCC)

[0049] The experiments of this invention revealed that the concentrations of gallic acid, ellagic acid, chebulic acid, chebulic biphenyl acid, corilagin, and geraniol in Phyllanthus emblica samples far exceeded the upper limit of quantification for conventional mass spectrometry methods. To address this issue, this invention employs an online dual-collision energy strategy.

[0050] First, this invention obtains the response intensity of each analyte by varying the collision energy within the range of 1–99 eV (in 2 eV steps). Then, using the response intensity as an indicator, nonlinear regression is performed on the obtained response intensity data to derive the response intensity-collision energy curves for gallic acid, ellagic acid, chebulic acid, chebulic biphenyl acid, corilagin, and geraniol. This invention plots the response intensity-collision energy curves for these six chemical components (gallic acid, ellagic acid, chebulic acid, chebulic biphenyl acid, corilagin, and geraniol). Figure 2 As shown, Figure 2 In this figure, A represents the response intensity-collision energy curve of gallic acid. Figure 2 In the figure, B represents the response intensity-collision energy curve of ellagic acid. Figure 2 In the figure, C represents the response intensity-collision energy curve of chebulic hypoglycine. Figure 2 In the figure, D represents the response intensity-collision energy curve of chebulic biphenyl acid. Figure 2 In this context, E represents the Corilagin response intensity-collision energy curve. Figure 2 In the equation, F represents the response intensity-collision energy curve of geranium. The fitting equations for the response intensity-collision energy curves of the six chemical components are as follows:

[0051] Gallic acid: Y = 0.00536 + 0.9576 × exp(-0.5 × ((X - 13.90544) / 3.867985) 2 Ellagic acid: Y = 0.000947 + 2.533 × exp(0.25 - (X - 31.87524) / 6.723) × [erf((X - 31.87524) / 6.723 - 0.5) + 1] / 2; Terminalia chebula acid: Y = 0.01603 + 0.4909 × exp(-2 × ((X - 11.7562) / 6.67626) 2 ); Terminalia biphenyl acid: Y = -0.01016 + 0.9336 × exp(-0.5 × ((X - 56.04315) / 15.59837) 2 ); Corilagin: Y=-0.02852+2.1055×exp(-(X-30.87985) / 15.04267)×Φ((X-30.87985) / 6.8147-0.45300); Geraniol: Y=0.005+0.887×exp(-0.5×((X-58.16) / 13.76) 2 );

[0052] The different response intensities under different parameter settings indicate that mass spectrometry exhibits different sensitivities to detection under these conditions.

[0053] This invention selects two collision energies (CE) corresponding to the maximum response intensity (MRI) and 10% of the MRI as the optimal dual collision energy values ​​(DCE). The finally determined dual collision energies (high CE / low CE) are: gallic acid (14 / 6 eV), ellagic acid (36 / 24 eV), chebulic acid (12 / 4 eV), chebulic biphenyl acid (56 / 24 eV), corilagin (38 / 21 eV), and geraniol (58 / 29 eV).

[0054] This invention is based on Figure 2 The curves used in the study determined the double collision energy (DCE) of the six components, and the optimal concentration monitoring range (CMC) was determined based on the collision energy to facilitate concentration monitoring over a wider range. Based on the selected double collision energy, double calibration curves (DCC) were established for these six high-content components. Taking gallic acid as an example, at 14 eV, the linear equation was Y = 626.774X + 10124.6, applicable to a concentration range of 75–6000 ng / mL; at 6 eV, the linear equation was Y = 0.15047X + 1187.41, applicable to a concentration range of 6000–300000 ng / mL. The results show that the ODCE strategy successfully expanded the quantitative range of these six chemical components, and the accuracy of the calculated concentration to the actual concentration was between 85.70% and 107.86% (Tables 2, 3, 4, 5, 6, and 7).

[0055] Table 2. Accuracy of Calculated Gallic Acid Concentration at Two-Collision Energy

[0056]

[0057] Table 3. Accuracy of Calculated Ellagic Acid Concentration under Two-Collision Energy

[0058]

[0059] Table 4. Accuracy of Calculated Concentration of Terminalia chebula under Two-Collision Energy

[0060]

[0061] Table 5. Accuracy of Calculated Concentration of Terminalia chebula Biphenyl Acid under Two-Collision Energy

[0062]

[0063] Table 6. Accuracy of Corilagin Concentration Calculation at Two-Collision Energy

[0064]

[0065] Table 7. Accuracy of Calculated Geraniol Concentration under Two-Collision Energy

[0066]

[0067] 2.4.2 Standard Curve Regression Equation

[0068] Accurately measure an appropriate amount of the reference stock solution and dilute it with methanol to prepare a mixed reference solution with the following concentrations: gallic acid 300 μg / mL, ellagic acid 60 μg / mL, chebulic acid 30 μg / mL, chebulic biphenyl acid 100 μg / mL, corilagin and geraniol 150 μg / mL, L-malic acid 8 μg / mL, methyl gallate 10 μg / mL, quercetin 6 μg / mL, isoquercitrin, quercetin, ethyl gallate, rutin and naringenin 2 μg / mL, and protocatechuic acid, sennaol, catechin, epicatechin, taurine and syringaldehyde 0.4 μg / mL. Different concentrations of mixed reference solutions were obtained by sequentially diluting the solutions with methanol by 2, 2.5, 2, 2, 2.5, 2, 2, 2, 2.5, and 2 times. Each concentration of these solutions was then injected for analysis. Weighted least squares regression was performed, with the x-axis representing the analyte concentration and the y-axis representing the analyte peak area, and a weighting coefficient of 1 / X. Linear regression equations for 20 components, including gallic acid, ellagic acid, methyl gallate, and quercetin, were obtained. The reference solution concentrations calculated at signal-to-noise ratios (S / N) of 3 and 10 were used as the limits of detection (LLOD) and quantitation (LLOQ), respectively. The results are shown in Table 8. The MRM plot (… Figure 1 As can be seen, the peaks of each chemical component have good shapes and do not interfere with each other.

[0069] Table 8. Standard curve regression equations for 20 chemical components

[0070]

[0071]

[0072] Figure 1 In the figure, A represents the MRM diagram of the standard control. Figure 1 B in the image represents the MRM map of the sample. Figure 1 In Chinese: 1 represents L-malic acid, 2 represents chebulic acid, 3 represents gallic acid, 4 represents protocatechuic acid, 5 represents methyl gallate, 6 represents corilagin, 7 represents geraniol, 8 represents catechin, 9 represents epicatechin, 10 represents chebulic biphenyl acid, 11 represents rutin, 12 represents ethyl gallate, 13 represents isoquercitrin, 14 represents ellagic acid, 15 represents syringaldehyde, 16 represents taurine, 17 represents quercetin, 18 represents sennaol, 19 represents quercetin, and 20 represents naringenin.

[0073] 2.4.2 Precision Experiment

[0074] Precision (within day): 50.0 mg of coarse powder of Phyllanthus emblica (batch S1) was accurately weighed and prepared into a test solution using the preparation method described in section "2.3.2". Six consecutive injections were performed, and the RSD values ​​of the peak areas of each chemical component were calculated. The results are shown in Table 9, indicating good intra-day precision of the instrument.

[0075] Precision (daytime): 50.0 mg of crude powder of Phyllanthus emblica (batch S1) was accurately weighed and prepared into a test solution using the preparation method described in section "2.3.2". The sample was injected twice consecutively over three days. The RSD values ​​of the peak areas of each chemical component were calculated. The results are shown in Table 10, indicating good daytime precision of the instrument.

[0076] Table 9. Intra-day precision results for 20 chemical components (n=6)

[0077]

[0078] Table 10. Daytime precision results for 20 chemical components (n=6)

[0079]

[0080] 2.4.3 Repeatability Test

[0081] Take 50.0 mg of crude powder of Phyllanthus emblica (batch S1) and accurately weigh it. Prepare 6 test solutions in parallel using the preparation method under section "2.3.2". Inject the solutions separately and calculate the RSD values ​​of the concentrations of each chemical component. The results are shown in Table 11, indicating that the method has good repeatability.

[0082] Table 11. Repeatability results for 20 chemical components (n = 6, ng / mL)

[0083]

[0084] 2.4.4 Stability Test

[0085] Take 50.0 mg of crude powder of Phyllanthus emblica (batch S1), accurately weigh it, and use the preparation method under "2.3.2" to inject the prepared test solution at 0, 2, 4, 8, 12 and 24 h respectively. Calculate the RSD value of the peak area of ​​each chemical component. The results are shown in Table 12, indicating that each chemical component has good stability.

[0086] Table 12. Stability results of 20 chemical components (n=6)

[0087]

[0088] 2.4.5 Recovery Test

[0089] Take 25.0 mg of crude powder of Phyllanthus emblica (batch S1), add a certain amount of mixed reference solution, prepare the sample using the preparation method under "2.3.2", inject the sample separately, and calculate the recovery rate of each chemical component. The results are shown in Table 13.

[0090] Table 13 Recovery results of 20 chemical components (n = 6)

[0091]

[0092] 2.5 Content Determination

[0093] Accurately weigh each batch of Phyllanthus emblica crude powder, prepare the test solution according to the method in section “2.3.2”, and determine the concentration of 20 chemical components according to the established chromatographic and mass spectrometric conditions. Record the concentration and calculate the content. The results are shown in Tables 14 and 15.

[0094] Table 14 Content of 20 chemical components (μg / g) in different batches of Phyllanthus emblica (n = 3)

[0095]

[0096] Table 15 Content of 20 chemical components (μg / g) in different batches of Phyllanthus emblica (n = 3)

[0097]

[0098] Note: "-" in Table 15 indicates below the limit of quantitation (less than 0.04 ng / mL).

[0099] In Tables 14 and 15: C1: Gallic acid; C2: Ellagic acid; C3: Terminaliacetic acid; C4: Terminalia biphenyl acid; C5: Corilagin; C6: Geraniol; C7: L-malic acid; C8: Methyl gallate; C9: Ethyl gallate; C10: Protocatechuic acid; C11: Syringaldehyde; C12: Naringenin; C13: Sennaol; C14: Epicatechin; C15: Catechin; C16: Quercetin; C17: Taxodiacetin; C18: Quercetin; C19: Isoquercetin; C20: Rutin.

[0100] As demonstrated by the above embodiments, this invention, based on UHPLC-MS / MS combined with online double collision energy (ODCE), successfully established an analytical method capable of simultaneously and accurately determining 20 chemical components in Phyllanthus emblica. This method is not only sensitive, accurate, and reliable, but also effectively solves the problem of insufficient quantitative range in high-content component analysis using traditional mass spectrometry by constructing dual calibration curves. Application of this method to the analysis of Phyllanthus emblica from different origins revealed the differences in its chemical composition, providing a scientific basis and technical reference for the quality standardization and origin traceability of Phyllanthus emblica medicinal materials.

[0101] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for determining the contents of 20 chemical components in Phyllanthus emblica by UHPLC-MS / MS, characterized in that, The method comprises the following steps: dissolving the phyllanthus niruri medicinal material in a methanol aqueous solution to obtain a test sample solution; detecting the test sample solution by using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, obtaining the contents of 20 chemical components in the phyllanthus niruri medicinal material according to the peak areas of the chromatographic peaks in the obtained chromatogram and a linear regression equation, wherein the ordinate of the linear regression equation is the peak area of the 20 chemical components, and the abscissa is the concentration of the 20 chemical components; the ultra-high performance liquid chromatography conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection comprise that the mobile phase is mobile phase A and mobile phase B, the mobile phase A is a formic acid aqueous solution, and the mobile phase B is acetonitrile; the elution mode is gradient elution, and the gradient elution program is as follows: 0-3 min, the volume percentage of the mobile phase B changes uniformly from 3% to 25%, 3-4.5 min, the volume percentage of the mobile phase B changes uniformly from 25% to 32.5%, 4.5-5.5 min, the volume percentage of the mobile phase B remains 32.5%, 5.5-10 min, the volume percentage of the mobile phase B changes uniformly from 32.5% to 55%, 10-11 min, the volume percentage of the mobile phase B changes uniformly from 55% to 80%; and the chromatographic column is an ACQUITY UPLC HSS T3; the mass spectrometry conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry detection comprise that the ion source is an electrospray ion source, the scanning mode is a negative ion scanning mode, and the detection mode is a multiple reaction ion monitoring mode; the 20 chemical components comprise gallic acid, ellagic acid, chebulagic acid, chebulinic acid, corilagin, delphinine, L-malic acid, methyl gallate, ethyl gallate, protocatechuic acid, syringaldehyde, naringenin, eriodictyol, epicatechin, catechin, quercetin, taxifolin, quercitrin, isoquercitrin and rutin; the parameters of the multiple reaction ion monitoring of the 20 chemical components are as follows: 。 2. The method of claim 1, wherein, the volume percentage of formic acid in the formic acid aqueous solution is 0.05-0.15%.

3. The method of claim 1, wherein, the flow rate of the mobile phase is 0.2-0.4 mL / min.

4. The method of claim 1, wherein, the column temperature of the chromatographic column is 30-35 ℃, and the injection amount is 1.5-2 μL.

5. The method according to claim 1 or 4, characterized in that, the specification of the chromatographic column is 2.1×100 mm, 1.8 μm.

6. The method of claim 1, wherein, the mass spectrometry conditions further comprise that the capillary voltage is 1.5 kV, the ion source temperature is 150 ℃, the desolvation gas temperature is 400 ℃, the cone gas flow rate is 150 L / h, the desolvation gas flow rate is 800 L / h, the cone gas backflush flow rate is 30 L / h, the collision gas is argon, the purity of the argon is 99.99%, and the flow rate of the collision gas is 0.15 mL / min.

7. The method of claim 1, wherein, the mass percentage of methanol in the methanol aqueous solution is 50%; the dissolving is performed under ultrasonic conditions, the power of the ultrasonic is 250-350 W, and the frequency is 30-50 Hz.

8. The method according to claim 1 or 7, characterized in that, the initial solution obtained by the dissolving further comprises that the initial solution is subjected to membrane filtration to obtain the test sample solution, the membrane filtration uses a microporous filter membrane, and the pore size of the microporous filter membrane is 0.22 μm.

9. The method of claim 1, wherein, The linear regression equation of 20 chemical components was: 。

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