Detection method of mangiferin and derivatives thereof

Abstract

The invention provides a method for detecting mangiferin and derivatives thereof. The method comprises the following steps: (1) sample pretreatment; (2) gradient elution separation; and (3) mass spectrum detection. By optimizing sample pretreatment, chromatographic separation and mass spectrometry detection conditions, the invention establishes a high-sensitivity and high-selectivity detection method for mangiferin and derivatives thereof.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to a method for detecting mangiferin and its derivatives. Background Technology

[0002] Mangiferin ( Mangiferin Mangiferin (Atractylodes macrocephala) is a plant-derived compound widely found in the roots, stems, and leaves of Anemarrhena asphodeloides (Liliaceae), the leaves and flowers of Belamcanda chinensis (Iridaceae), and the bark, leaves, and fruit of Mango (Anacardiaceae). It possesses rich pharmacological effects, such as antiviral, antitumor, hypoglycemic, antioxidant, and lipid-lowering properties. Mangiferin As a natural active ingredient, its detection is of great significance for the quality control of traditional Chinese medicine and the development of functional foods.

[0003] However, existing methods for detecting mangiferin still have many drawbacks, including: I. Traditional HPLC-UV methods have high detection limits (>50 ng / mL), which cannot meet the needs of trace analysis; Second, the national standard method involves complex pretreatment and has a large fluctuation in recovery rate (60-85%). Third, existing mass spectrometry methods mostly use single ion monitoring, which has weak resistance to matrix interference.

[0004] Therefore, it is necessary to propose a detection method for mangiferin and its derivatives to solve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting mangiferin and its derivatives. This invention establishes a highly sensitive and selective method for detecting mangiferin and its derivatives by optimizing sample pretreatment, chromatographic separation and mass spectrometry detection conditions.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for detecting mangiferin and its derivatives, the method comprising the following steps: (1) Sample pretreatment: After freeze-drying, the sample was extracted with supercritical CO2 fluid using a eutectic solvent; (2) Gradient elution separation: Gradient elution separation was performed using an HSS T3 column; (3) Mass spectrometry detection: MRM detection was performed using a triple quadrupole linear ion trap mass spectrometer in negative ion mode.

[0007] In this invention, further, in step (1), the eutectic solvent is composed of hydrogen bond acceptor and hydrogen bond donor in a molar ratio of 1:1.5-2.5; the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is lactic acid or oxalic acid.

[0008] In this invention, further, the eutectic solvent in step (1) is a mixture of choline chloride and lactic acid in a 1:2 molar ratio.

[0009] In this invention, the supercritical extraction conditions in step (1) are as follows: entrainer dosage: DES volume accounts for 10-15% of the total volume of supercritical CO2 fluid; temperature: 40-50℃; pressure: 20-30 MPa; dynamic extraction time: 30-50 min; CO2 flow rate: 1.5-2.5 L / min.

[0010] In this invention, further, step (1) specifically involves: pulverizing the freeze-dried sample to 100 mesh, injecting the eutectic solvent DES (choline chloride: lactic acid = 1:2 mol / mol) at a volume ratio of 12% into a supercritical extraction vessel, extracting for 40 min at 45°C, 25 MPa, and a CO2 flow rate of 2.0 L / min, and collecting the extract for purification through a 0.22 μm filter membrane.

[0011] In this invention, further, the gradient elution separation mobile phase in step (2) is: 0.1% formic acid water as phase A and 0.1% formic acid acetonitrile as phase B.

[0012] In this invention, further, the gradient elution separation mobile phase in step (2) is: 0.1% formic acid water as phase A and 0.1% formic acid acetonitrile as phase B.

[0013] In this invention, further, the gradient elution procedure of step (2) is as follows: 0-1 min maintain 20%B, 1-2 min increase to 50%B, 2-5 min increase to 90%B, 5-6 min maintain 90%B, 6-6.5 min decrease to 20%B and equilibrate to 9.5 min.

[0014] In this invention, the mass spectrometry detection in step (3) specifically uses three characteristic ion pairs: m / z 421.1→301.0, 421.1→385.0, and 421.1→259.0.

[0015] m / z 421.1 → 301.0: As a primary quantitative ion pair, it has a high response value and good stability, and is used for the accurate quantitative calculation of mangiferin; m / z 421.1 → 385.0: This serves as an auxiliary qualitative ion pair to verify the structural consistency of mangiferin molecules and ensure the reliability of the qualitative results. m / z 421.1 → 259.0: As a specific confirmatory ion pair, its fragmentation pathway is unique, which can effectively eliminate co-elution interference from complex matrices and further improve the specificity of detection results.

[0016] In this invention, the liquid chromatography conditions in step (2) further include: gradient elution flow rate of 0.20 mL / min; column temperature of 30 °C; and injection volume of 2.0 μL.

[0017] In summary, due to the adoption of the above technical solutions, the present invention has at least the following beneficial effects: This invention provides a method for detecting mangiferin and its derivatives. By systematically optimizing the pretreatment, chromatographic separation and mass spectrometry detection parameters, it effectively solves the technical problems of low detection sensitivity and poor specificity in existing technologies, and is particularly suitable for the accurate analysis of mangiferin components in complex matrices. Firstly, the triple ion-pair strategy used in the detection of mangiferin employs the following: 421.1 → 301.0 is the primary quantitative ion, which has high abundance and is used for quantitative calculation; 421.1 → 385.0 is the auxiliary qualitative ion, verifying the consistency of the compound; and 421.1 → 259.0 is the specificity confirmatory ion, which can eliminate matrix interference. This triple ion-pair strategy not only meets the requirements for high-sensitivity quantification but also ensures the reliability of the detection results through multi-dimensional verification, providing a reliable solution for trace component analysis in complex matrices. Furthermore, this application improves the detection specificity by establishing a triple characteristic ion-pair verification mechanism. Secondly, the use of an HSS T3 column improves the retention behavior of polar derivatives; Third, optimize the pretreatment method to improve the recovery rate of mangiferin. Attached Figure Description

[0018] Figure 1 Total ion chromatogram (TIC) of mangiferin extracted by DES-assisted supercritical CO2 extraction. Figure 2 The image shows the MRM chromatogram of mangiferin under gradient elution conditions. Detailed Implementation

[0019] The following embodiments can help those skilled in the art to more fully understand the present invention, but should not be construed as limiting the present invention in any way. Example

[0020] This embodiment provides a method for detecting mangiferin and its derivatives, which includes the following steps: (1) Sample pretreatment: ① DES preparation: Choline chloride and lactic acid were mixed at a molar ratio of 1:2 and stirred at 80°C until a homogeneous and transparent liquid was formed with a water content of <3% (measured value 2.1%). ② Supercritical extraction: Accurately weigh 500 mg of freeze-dried mango leaf powder (passed through a 100-mesh sieve) and place it in a supercritical extraction vessel. Inject the DES obtained in step ① into the system as an entrainer at a volume ratio of 12%, and perform dynamic extraction under the following conditions: Supercritical fluid: CO2 (purity ≥99.9%), temperature: 45℃, pressure: 25 MPa, CO2 flow rate: 2.0 L / min, extraction time: 40 min; ③ Post-processing: Collect the extract, concentrate it to near dryness by nitrogen blowing at 40℃, redissolve it with 1 mL of methanol, filter it through a 0.22 μm organic microporous membrane, and store it in a sample vial for testing.

[0021] The mangiferin extraction yields under different extraction methods are compared and grouped as shown in Table 1 below:

[0022] The control group consisted of freeze-dried sample powder. 500 mg of mango leaf sample was accurately weighed and placed in a centrifuge tube. 5 mL of 70% methanol extraction solution was added, and the mixture was vortexed for 2 min, followed by ultrasonic extraction for 30 min, and then centrifuged (12000 r / min, 4°C) for 10 min. The supernatant was filtered through a 0.22 μm microporous membrane and stored in a sample vial for later analysis.

[0023] Experimental groups 2-4 were the same as experimental group 1, differing only in the composition of the DES. All data were based on the same mango leaf sample (homogeneity verified by HPLC) and were completed by the same operator within 24 hours to eliminate sample / human error.

[0024] The test results are shown in Table 2:

[0025] Experimental group 1 vs. control group: directly demonstrates the effectiveness of the technology (extraction rate ↑37.1%); Experimental group 2: demonstrates that the oxalic acid donor effect is weaker than that of lactic acid (↓23.5% vs ↑37.1%), illustrating the necessity of lactic acid; Experimental groups 3-4: when the molar ratio exceeds 1:1.5-2.5, the extraction rate is lower than the optimal ratio, indicating that a better extraction amount can be obtained under the values ​​of this embodiment.

[0026] (2) Gradient elution separation: a. Column: Waters HSS T3 (1.8 μm, 2.1 × 100 mm); b. Mobile phase: 0.1% formic acid water (A) - 0.1% formic acid acetonitrile (B); c. Gradient elution procedure:

[0027] d, flow rate 0.20 mL / min; column temperature 30°C; injection volume 2 μL; e. The retention time of mangiferin was 2.21 min; Flow rate gradient screening test:

[0028] It was found that 0.20 mL / min achieved the optimal balance between column efficiency (N=15,000 / m), resolution (R=2.5), and column pressure (450 bar). High flow rate (0.30 mL / min): peak broadening (decreased theoretical plate number) may lead to insufficient resolution. Low flow rate (0.15 mL / min): analysis time increased (3.05 min), with limited improvement in column efficiency; the optimal flow rate was 0.20 mL / min. Using an HSS T3 column with a flow rate of 0.20 mL / min, high-resolution elution of mangiferin was achieved within 2.21 min (R > 2.0).

[0029] (3) Mass spectrometry detection: An AB SCIEX 5500+ Q TRAP triple quadrupole linear ion trap mass spectrometer equipped with an ESI ionization source was used in negative ion mode. The ion spray voltage (IS) was set to -4500V, and the temperature was set to 450°C. Ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 40, 40, and 20.0 psi, respectively, and the collision gas was set to 9 psi. The mass spectrometry parameters for mangiferin were as follows, using MRM scanning mode:

[0030] Mass spectrometry condition data: like Figure 1 As shown in the figure, the supercritical CO2 extraction efficiency of mangiferin in the corresponding embodiment was verified. The high-intensity peak of 1.7e8 proves that the DES solvent (choline chloride: lactic acid = 1:2) has excellent extraction efficiency, and the optimization of supercritical conditions (45℃ / 25MPa / 2L / min) is effective, with a peak shape symmetry factor of 1.05, which meets the USP requirements. In addition, the secondary peak appearing at 4.25-4.33 min in the figure is well separated from the main peak (resolution R=2.1>1.5), further verifying the excellent separation efficiency of the HSS T3 column under the conditions of this method.

[0031] like Figure 2 As shown, the main peak (2.21 min) has a high signal intensity, indicating that DES supercritical extraction has an excellent enrichment effect on the target analyte. The chromatographic peaks of the triple characteristic ion pairs completely overlap at the same retention time, with sharp and symmetrical peak shapes and no obvious tailing, meeting the system suitability requirements. Moreover, the signal-to-noise ratio is extremely high, fully demonstrating the excellent selectivity, sensitivity, and separation efficiency of this method.

[0032] The applicant further set up experimental groups with blank samples added with different concentrations of mangiferin standard, and measured them using the detection method of this invention and the national standard detection method, respectively.

[0033] The test results are compared in Table 3:

[0034] The data above clearly shows that the recovery rate of the detection method of this invention is consistently above 98%, with stable data approaching 100%, indicating that this method can accurately extract and detect mangiferin in samples with extremely high accuracy. In contrast, the recovery rate of the national standard detection method is generally below 85%, with some experimental groups even below 75%, and the detection results deviate significantly from the actual amount added. This fully demonstrates that, compared to the national standard detection method, the detection method of this invention has unexpected technical advantages, significantly improving accuracy and reliability, and enabling more precise detection of mangiferin.

[0035] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting mangiferin and its derivatives, characterized in that, The method includes the following steps: (1) Sample pretreatment: After freeze-drying, the sample was extracted with supercritical CO2 fluid using a eutectic solvent; (2) Gradient elution separation: Gradient elution separation was performed using an HSS T3 column; (3) Mass spectrometry detection: MRM detection was performed using a triple quadrupole linear ion trap mass spectrometer in negative ion mode.

2. The method according to claim 1, characterized in that, In step (1), the eutectic solvent is composed of hydrogen bond acceptors and hydrogen bond donors in a molar ratio of 1:1.5-2.5; the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is lactic acid or oxalic acid.

3. The method according to claim 1, characterized in that, The supercritical extraction conditions in step (1) are as follows: entrainer dosage: DES volume accounts for 10-15% of the total volume of supercritical CO2 fluid; Temperature: 40-50℃; Pressure: 20-30 MPa; Dynamic extraction time: 30-50 min; CO2 flow rate: 1.5-2.5 L / min.

4. The method according to claim 1, characterized in that, The specific steps (1) are as follows: freeze-dry the sample and pulverize it to 100 mesh, inject the eutectic solvent DES (choline chloride: lactic acid = 1:2 mol / mol) at a volume ratio of 12% into the supercritical extraction vessel, extract for 40 min at 45℃, 25 MPa and CO2 flow rate of 2.0 L / min, and collect the extract and purify it through a 0.22 μm filter membrane.

5. The method according to claim 1, characterized in that, The gradient elution mobile phase in step (2) is: 0.1% formic acid water as phase A and 0.1% formic acid acetonitrile as phase B.

6. The method according to claim 1, characterized in that, The gradient elution procedure in step (2) is as follows: 0-1 min maintain 20% B, 1-2 min increase to 50% B, 2-5 min increase to 90% B, 5-6 min maintain 90% B, 6-6.5 min decrease to 20% B and equilibrate to 9.5 min.

7. The method according to claim 1, characterized in that, The mass spectrometry detection in step (3) specifically uses three characteristic ion pairs: m / z 421.1→301.0, 421.1→385.0, and 421.1→259.

0.

8. The method according to claim 1, characterized in that, The liquid chromatography conditions in step (2) also include: gradient elution flow rate of 0.20 mL / min; column temperature of 30℃; and injection volume of 2.0 μL.

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