Method for rapid separation and detection of berberine and epiberberine based on trapping ion mobility mass spectrometry

Abstract

This invention discloses a method for rapid separation and detection of berberine and epiberberine based on ion mobility mass spectrometry. The specific steps are as follows: berberine and epiberberine in the sample to be tested are mixed with cucurbituril [8] and magnesium ions to form a ternary complex; the obtained ternary complex is ionized using an electrospray ionization source; different complex ions are separated using nitrogen as the drift gas in the ion mobility mode; the separated ions are detected by tandem mass spectrometry; qualitative analysis of berberine and epiberberine is achieved based on the triple information of characteristic mass-to-charge ratio, mobility value and collision cross section; and quantitative analysis of berberine and epiberberine is achieved by external standard method or internal standard method. This invention uses cucurbituril [8] as a chiral auxiliary reagent and magnesium ions as a metal complexing agent, and uses ion mobility-mass spectrometry to distinguish berberine isomers, which can rapidly, with high resolution and high accuracy, separate and detect berberine and epiberberine.

Description

Technical Field

[0002] This invention belongs to the field of analytical detection technology of natural products berberine and epiberberine, specifically relating to a method for rapid separation and detection of berberine and epiberberine based on trapped ion mobility mass spectrometry. Background Technology

[0004] Berberine and epiberberine are important isoquinoline alkaloids widely found in traditional Chinese medicinal herbs such as Coptis chinensis and Phellodendron amurense. They are isomers with identical molecular weights, but subtle differences in substituents or hydrogenation levels lead to different spatial configurations. Modern pharmacological studies have shown significant differences in the anti-inflammatory, antibacterial, hypoglycemic, and antitumor bioactivities of berberine and epiberberine. Therefore, rapid and accurate separation and quantitative analysis of these two alkaloids are crucial for quality control of traditional Chinese medicinal herbs, research on their pharmacodynamic material basis, and new drug development. The molecular structures of berberine and epiberberine are as follows:

[0005]

[0006] Due to the high similarity in chemical structures between berberine and epiberberine, traditional separation and analysis methods face significant challenges. Although high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS) methods are widely used, they generally suffer from problems such as long analysis times (usually 30-60 minutes), insufficient resolution, difficulty in achieving baseline separation, and severe interference in complex matrices, affecting the accuracy of qualitative and quantitative analysis.

[0007] The existing patent document CN202410252464.5 discloses a method for determining the content of seven alkaloids in Huanglian Jiedu Decoction using a single measurement and multiple evaluation method. It uses readily available and inexpensive herbal root alkaloids as an internal standard and establishes a relative correction factor between herbal root alkaloids and six other alkaloids in Huanglian Jiedu Decoction: grandipine, tetrandrine, epiberberine, berberine, palmatine, and berberine. This allows for the determination of the content of the other six alkaloids in Huanglian Jiedu Decoction by measuring only herbal root alkaloids, and then using the correction factor to calculate the content of these alkaloids. This provides a simple, rapid, comprehensive, and accurate multi-index quality evaluation of Huanglian Jiedu Decoction, helping to ensure the product's quality control and stable efficacy, while saving detection costs and time. The chosen acetonitrile-ammonium formate (formic acid) mobile phase system ensures baseline separation of all analytes and is well-suited for mass spectrometry analysis, minimizing damage to the detection instrument and chromatographic column. Patent document CN202511358101.0 discloses a quality detection method for the Coptis chinensis-Gardenia jasminoides herbal pair. This method involves extensive experimentation to screen optimal mobile phase composition, elution program, mass spectrometry, flow rate, chromatographic column, and other analytical conditions. UPLC-Q-TOF / MS technology is used to analyze and identify the chemical components of the Coptis chinensis-Gardenia jasminoides herbal pair, identifying a total of 99 components: 41 from Coptis chinensis and 60 from Gardenia jasminoides. Two components are shared, primarily alkaloids, phenylpropanoids, and iridoids. This study primarily investigated the content of genipin 1-O-gentiopicroside, genipin glycoside, berberine, epiberberine, tetrandrine, purslane, berberine hydrochloride, and palmatine hydrochloride in the Coptis chinensis-Gardenia jasminoides herb pair using UPLC-PDA technology, focusing on organic acids and flavonoids. Systematic methodological investigation and content determination validated the method's good precision, stability, and repeatability, providing reliable technical support for the quality control and pharmacodynamic material basis research of the Coptis chinensis-Gardenia jasminoides herb pair. However, neither of the aforementioned patented technical solutions can achieve rapid, high-resolution differentiation and detection of berberine and epiberberine, and there is no record of rapid separation and qualitative and quantitative detection of berberine and epiberberine based on trapped ion mobility mass spectrometry.

[0008] Ion mobility mass spectrometry (IMS-MS) technology separates ions based on differences in their collision cross-section (CCS) in the gas phase, providing a new approach for distinguishing isomers. Among these, trapping ion mobility mass spectrometry (TIMS) offers advantages such as high resolution and fast analysis speed (milliseconds). However, berberine and epiberberine have relatively small spatial structural differences, and when directly analyzed using TIMS-MS, their ion mobility behaviors are extremely similar, making it difficult to achieve satisfactory separation. Therefore, there is an urgent need to develop a detection method that can effectively amplify the structural differences between berberine and epiberberine and achieve efficient separation. Summary of the Invention

[0010] To overcome the technical defects of low efficiency and poor separation of berberine and epiberberine in the prior art, this invention provides a rapid, high-resolution, and high-accuracy method for rapid separation and detection of berberine and epiberberine based on ion mobility mass spectrometry. This method uses cucurbituril [8] as a chiral auxiliary reagent and magnesium ions as a metal complexing agent, and uses ion mobility-mass spectrometry to distinguish berberine isomers, thereby realizing qualitative and quantitative analysis of berberine and epiberberine.

[0011] To solve the above-mentioned technical problems, this invention adopts the following technical solution: a method for rapid separation and detection of berberine and epiberberine based on trapping ion mobility mass spectrometry, the specific steps of which are as follows:

[0012] Step S1, Sample pretreatment: Berberine and epiberberine in the sample to be tested are mixed with cucurbituril[8] and magnesium ions to form a ternary complex;

[0013] Step S2, Ionization: The ternary complex obtained in step S1 is ionized using an electrospray ionization source;

[0014] Step S3, Trapping Ion Mobility Separation: Using the trapping ion mobility mode, with nitrogen as the drift gas, different complex ions generated in step S2 are separated.

[0015] Step S4, Mass spectrometry detection: Tandem mass spectrometry is used to detect the ions separated in step S3;

[0016] Step S5, Qualitative and quantitative analysis: Qualitative analysis of berberine and epiberberine is performed based on the triple information of characteristic mass-to-charge ratio, mobility value and collision cross section. Quantitative analysis of berberine and epiberberine is performed using external standard method or internal standard method.

[0017] Furthermore, the sample to be tested in step S1 includes Coptis chinensis extract, Phellodendron chinense extract, Coptis chinensis traditional Chinese medicine, Phellodendron chinense traditional Chinese medicine, health products, or biological fluids.

[0018] Further, in step S1, the molar ratio of cucurbituril [8] to berberine / epiberberine is 1:1 to 10:1, and the molar ratio of magnesium ions to berberine / epiberberine is 1:1 to 10:1.

[0019] Furthermore, the extraction solvent for sample pretreatment in step S1 is a methanol-water mixture with a volume ratio of 1:1 and containing 0.1% formic acid by volume.

[0020] Furthermore, the ionization mode described in step S2 is a positive ion mode.

[0021] Further, in step S3, the mobility scanning range is 0.50~1.80 V·s / cm², the ion aggregation time is 50.0 ms, the cycle time is 500.0 ms, the capillary voltage is 3600 V, the neutral gas pressure is 0.3 bar, the drying gas flow rate is 3.0 L / min, the drying temperature is 200 ℃, and the sample flow rate is 5.00 μL / min.

[0022] Furthermore, the specific steps of the method for rapid separation and detection of berberine and epiberberine based on trapped ion mobility mass spectrometry are as follows:

[0023] Step S1: Sample pretreatment

[0024] Standard samples: Weigh out berberine and epiberberine standards separately and dissolve them in the extraction solvent to prepare a concentration of 10. -4 The standard stock solution was prepared by dissolving 10 mol / L of cucurbituril[8] (CB[8]) in water to obtain an extraction solvent of 1:1 (v / v) and 0.1% (v / v) of formic acid. -4 A mol / L cucurbituril[8] solution was prepared by dissolving a magnesium-containing compound (such as MgCl2 or Mg(NO3)2) in water to a concentration of 10 mol / L. -4 A mol / L metal ion solution;

[0025] Before analysis, the standard stock solution, cucurbita urea[8] solution and magnesium ion solution were added to the extraction solvent at a volume ratio of 10:1:10 and diluted with the extraction solvent to a final concentration of 10. -5 mol / L, to form a stable ternary complex [M+CB[8]+Mg]²⁺, to be tested;

[0026] Sample to be tested: Weigh the sample to be tested containing berberine and epiberberine, add the extraction solvent and sonicate to dissolve, cool and filter, take the filtrate and dilute with the extraction solvent, take the diluted sample solution, add the sample solution, cucurbita[8] solution and magnesium ion solution to the extraction solvent at a volume ratio of 10:1:10, mix well and test;

[0027] Step S2: Ionization

[0028] An electrospray ionization (ESI) source is used to ionize the target analyte in positive ion mode, generating gaseous ions of the analyte.

[0029] Step S3: Ion Trapping Mobility Separation

[0030] The trapping ion mobility (TIMS) method was used for separation. The drift gas was high-purity nitrogen. The mobility scan range was set to 0.50–1.80 V·s / cm², the ion aggregation time was 50.0 ms, the cycle time was 500.0 ms, the capillary voltage was 3600 V, the neutral gas (nitrogen) pressure was 0.3 bar, the drying gas flow rate was 3.0 L / min, the drying temperature was 200 °C, and the sample flow rate was 5.00 μL / min.

[0031] Step S4: Tandem mass spectrometry detection

[0032] Tandem mass spectrometry (MS / MS) was used for detection. The first-stage mass spectrometry (MS1) was used to select the target complex ion ([M+CB[8]+Mg]²⁺), and the second-stage mass spectrometry (MS2) was used to obtain its fragment information or directly record the parent ion signal after ion mobility separation. The collision cross section (CCS) value of berberine and epiberberine complex was recorded simultaneously.

[0033] Step S5, Qualitative and Quantitative Analysis: Qualitative analysis is performed based on the characteristic mass-to-charge ratio (m / z), ion mobility (or migration time), and collision cross section (CCS) of the target complex. Since the spatial configuration difference between berberine and epiberberine is significantly amplified after the formation of the complex, they have different mobility behaviors in TIMS and can be distinguished according to the peak position, thus achieving qualitative analysis of berberine and epiberberine. Quantitative analysis of berberine and epiberberine is achieved using external standard method or internal standard method.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0035] (1) Fast separation speed: Based on the millisecond-level separation capability of TIMS, the single-needle analysis time is less than 1 minute, which is far superior to the traditional HPLC (30~60 minutes) and UPLC-MS (10~15 minutes) methods, and the analysis efficiency is improved by tens of times.

[0036] (2) Significantly enhanced isomer resolution: Cucurbituril[8] was innovatively introduced as a chiral auxiliary reagent and magnesium ions as a metal center to form a rigid ternary complex with berberine / epiberberine, effectively amplifying the small structural differences between the two and significantly increasing the difference in their collision cross section (CCS). Experiments showed that the separation degree (Rp-p) of the berberine-epiberberine complex could reach more than 0.72, achieving separation.

[0037] (3) High qualitative accuracy: The use of triple information of “characteristic m / z + mobility value + CCS value” for qualitative analysis greatly improves the ability to resist matrix interference. Even in complex Chinese medicine samples, the target analyte can be accurately identified with a qualitative accuracy of ≥99%.

[0038] (4) Excellent methodological performance: linear correlation coefficient (R²) > 0.99, low detection limit (up to ng / mL level), spiked recovery rate between 80% and 110%, relative standard deviation (RSD) < 5%, which fully meets the requirements for accurate detection of trace components in complex samples. Attached Figure Description

[0040] Figure 1 (a) Mass spectrum of berberine and [berberine / cucurbituril / metal complex] molecular ion peaks; (b) Mass spectrum of epiberberine and [epiberberine / cucurbituril / metal complex] molecular ion peaks.

[0041] Figure 2 This is a graph showing the ion mobility of berberine and epiberberine standards in Coptis chinensis extract.

[0042] Figure 3 The trapping ion mobility diagram of berberine and epiberberine in Coptis chinensis extract. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0045] Example 1

[0046] Analysis of standards and spiked samples

[0047] Sample preparation:

[0048] Standard solution: Accurately weigh berberine and epiberberine standards and dissolve them in the extraction solvent to prepare a solution with a concentration of 10. -4 A standard sample solution of mol / L was prepared, wherein the extraction solvent was a methanol-water mixture with a volume ratio of 1:1 and containing 0.1% formic acid. Cucurbita[8] was accurately weighed and dissolved in 1 mL of water to prepare a solution with a concentration of 10 mol / L. -4 A 10 mol / L cucurbituril [8] solution was prepared by dissolving MgCl2·6H2O in 1 mL of water. -4 A magnesium chloride solution of mol / L.

[0049] Complex solution I: Take 100 μL each of berberine and epiberberine stock solutions, mix them with 10 μL of cucurbituril[8] solution and 10 μL of magnesium chloride solution respectively, and make up to 1 mL with the above extraction solvent to make the final concentration of berberine / epiberberine 10⁻ 5mol / L. Let stand at room temperature for 15 minutes to allow for complete complexation.

[0050] Complex solution II: Take 100 μL each of berberine and epiberberine stock solutions, mix them with 100 μL of cucurbituril[8] solution and 100 μL of magnesium chloride solution respectively, and make up to 1 mL with the above extraction solvent to make the final concentration of berberine / epiberberine 10⁻ 5 mol / L. Let stand at room temperature for 15 minutes to allow for complete complexation.

[0051] TIMS-MS analysis conditions:

[0052] Instrument: Bruker TimsTOF Pro

[0053] Ion source: ESI, positive ion mode

[0054] Capillary voltage: 3600 V

[0055] Drying gas temperature: 200℃, drying gas flow rate: 3.0 L / min

[0056] Sample flow rate: 5 μL / min

[0057] Mobility parameters: scan range 0.60~1.60 V·s / cm², ion aggregation time 50 ms, cycle time 500 ms

[0058] Drift gas: High-purity nitrogen, 0.3 bar

[0059] Detection results: Clear complex ion peaks [Berberine+CB[8]+Mg]²⁺ and [Epiberberine+CB[8]+Mg]²⁺ are visible in the mass spectrum, with m / z values ​​of approximately 843. In the ion mobility diagram, the berberine complex and epiberberine complex show different migration times, with a resolution (Rp-p) of 1.88 and a collision cross section (CCS) difference greater than 10 Ų, achieving baseline separation. Figure 1 As shown.

[0060] Example 2

[0061] Isolation and detection of two alkaloids in Coptis chinensis

[0062] Sample pretreatment:

[0063] Weigh approximately 0.5 g of Coptis chinensis powder (passed through a 40-mesh sieve), accurately, and place it in a stoppered conical flask. Add 25 mL of a 70% (v / v) methanol-water solution containing 0.1% (v / v) formic acid, seal tightly, and weigh. Sonicate for 30 minutes, cool, and weigh again. Replenish the lost weight with the extraction solvent, shake well, and filter.

[0064] Take 100 μL of the above filtrate, add 10 μL of cucurbituril[8] solution and 100 μL of magnesium chloride solution, dilute to 1 mL with the above extraction solvent, filter through a 0.22 μm filter membrane, and analyze according to the TIMS-MS conditions in Example 1.

[0065] Detection results: Berberine and epiberberine were successfully detected in the Coptis chinensis sample, and the peak positions in their mobility plots perfectly matched those of the standard complex. Figures 2-3 As shown.

[0066] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for rapid separation and detection of berberine and epiberberine based on trapping ion mobility mass spectrometry, characterized in that... The specific steps are as follows: Step S1, Sample pretreatment: Berberine and epiberberine in the sample to be tested are mixed with cucurbituril[8] and magnesium ions to form a ternary complex; Step S2, Ionization: The ternary complex obtained in step S1 is ionized using an electrospray ionization source; Step S3, Trapping Ion Mobility Separation: Using the trapping ion mobility mode, with nitrogen as the drift gas, different complex ions generated in step S2 are separated. Step S4, Mass spectrometry detection: Tandem mass spectrometry is used to detect the ions separated in step S3; Step S5, Qualitative and quantitative analysis: Qualitative analysis of berberine and epiberberine is performed based on the triple information of characteristic mass-to-charge ratio, mobility value and collision cross section. Quantitative analysis of berberine and epiberberine is performed using external standard method or internal standard method.

2. The method for rapid separation and detection of berberine and epiberberine based on trapping ion mobility mass spectrometry according to claim 1, characterized in that: The sample to be tested in step S1 includes Coptis chinensis extract, Phellodendron chinense extract, Coptis chinensis traditional Chinese medicine, Phellodendron chinense traditional Chinese medicine, health products, or biological fluids.

3. The method for rapid separation and detection of berberine and epiberberine based on trapping ion mobility mass spectrometry according to claim 1, characterized in that: In step S1, the molar ratio of cucurbituril [8] to berberine / epiberberine is 1:1 to 10:1, and the molar ratio of magnesium ions to berberine / epiberberine is 1:1 to 10:

1.

4. The method for rapid separation and detection of berberine and epiberberine based on trapping ion mobility mass spectrometry according to claim 1, characterized in that: The extraction solvent for sample pretreatment in step S1 is a methanol-water mixture with a volume ratio of 1:1 and containing 0.1% formic acid by volume.

5. The method for rapid separation and detection of berberine and epiberberine based on trapping ion mobility mass spectrometry according to claim 1, characterized in that: The ionization mode described in step S2 is the positive ion mode.

6. The method for rapid separation and detection of berberine and epiberberine based on trapping ion mobility mass spectrometry according to claim 1, characterized in that: In step S3, the mobility scanning range is 0.50~1.80 V·s / cm², the ion aggregation time is 50.0 ms, the cycle time is 500.0 ms, the capillary voltage is 3600 V, the neutral gas pressure is 0.3 bar, the drying gas flow rate is 3.0 L / min, the drying temperature is 200 ℃, and the sample flow rate is 5.00 μL / min.

7. The method for rapid separation and detection of berberine and epiberberine based on trapping ion mobility mass spectrometry according to claim 1, characterized in that... The specific steps are as follows: Step S1: Sample pretreatment Standard samples: Weigh out berberine and epiberberine standards separately and dissolve them in the extraction solvent to prepare a concentration of 10. -4 The standard stock solution was prepared by dissolving 10 mol / L of cucurbituril[8] (CB[8]) in water to obtain an extraction solvent of 1:1 (v / v) and 0.1% (v / v) of formic acid. -4 A mol / L auxiliary reagent solution was prepared by dissolving a magnesium ion-containing compound in water to a concentration of 10 mol / L. -4 A mol / L metal ion solution; Before analysis, the standard stock solution, cucurbita urea[8] solution and magnesium ion solution were added to the extraction solvent at a volume ratio of 10:1:10 and diluted with the extraction solvent to a final concentration of 10. -5 mol / L, to form a stable ternary complex [M+CB[8]+Mg]²⁺, to be tested; Sample to be tested: Weigh the sample to be tested containing berberine and epiberberine, add the extraction solvent and sonicate to dissolve, cool and filter, take the filtrate and dilute with the extraction solvent, take the diluted sample solution, add the sample solution, cucurbita[8] solution and magnesium ion solution to the extraction solvent at a volume ratio of 10:1:10, mix well and test; Step S2: Ionization An electrospray ionization (ESI) source is used to ionize the target analyte in positive ion mode, generating gaseous ions of the analyte. Step S3: Trapped ion mobility separation was performed using Trapped Ion Mobility Separation (TIMS). The drift gas was high-purity nitrogen, the mobility scan range was set to 0.50~1.80 V·s / cm², the ion aggregation time was 50.0 ms, the cycle time was 500.0 ms, the capillary voltage was 3600 V, the neutral gas pressure was 0.3 bar, the drying gas flow rate was 3.0 L / min, the drying temperature was 200℃, and the sample flow rate was 5.00 μL / min. Step S4: Tandem mass spectrometry detection Tandem mass spectrometry (MS / MS) was used for detection. The first-stage mass spectrometry (MS1) was used to select the target complex ion ([M+CB[8]+Mg]²⁺), and the second-stage mass spectrometry (MS2) was used to obtain its fragment information or directly record the parent ion signal after ion mobility separation. The collision cross section (CCS) value of berberine and epiberberine complex was recorded simultaneously. Step S5, Qualitative and Quantitative Analysis: Qualitative analysis is performed based on the characteristic mass-to-charge ratio (m / z), ion mobility (or migration time), and collision cross section (CCS) of the target complex. Since the spatial configuration difference between berberine and epiberberine is significantly amplified after the formation of the complex, they have different mobility behaviors in TIMS and can be distinguished according to the peak position, thus achieving qualitative analysis of berberine and epiberberine. Quantitative analysis of berberine and epiberberine is achieved using external standard method or internal standard method.

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