Method for determining alkaloid in Chinese mahonia stem

Abstract

The invention belongs to the technical field of Chinese mahonia stem alkaloid determination, and particularly relates to a method for determining alkaloid in Chinese mahonia stem. The method for determining the alkaloid in the Chinese mahonia stem comprises the following steps: performing microwave-assisted extraction on Chinese mahonia stem medicinal material powder in an extracting solution, and performing centrifugal filtration to obtain a solution to be tested; and carrying out UFLC-MS / MS analysis on the solution to be tested. According to the method, auxiliary extraction is performed on the alkaloid in the Chinese mahonia stems by adopting microwaves, and then the extracted alkaloid components are detected and analyzed by UFLC-MS / MS, so that the analysis and detection accuracy of the alkaloid in the Chinese mahonia stems can be greatly improved, and the analysis and detection efficiency is high; the determination method is simple, efficient and good in repeatability, and the quality of the Chinese mahonia stem medicinal material can be better evaluated.

Description

Technical Field

[0001] This invention relates to the field of alkaloid determination technology in Mahonia japonica, specifically to a method for determining alkaloids in Mahonia japonica. Background Technology

[0002] Mahonia bealei (Fort.) Carr. or Mahonia fortunei (Lindl.) Fedde., belonging to the Berberidaceae family, are dried stems. They are bitter and cold in nature, primarily entering the liver, stomach, and large intestine meridians. They possess the effects of clearing heat and drying dampness, purging fire and detoxifying. Mahonia bealei mainly contains alkaloids, flavonoids, volatile oils, and other chemical components, exhibiting pharmacological effects such as antioxidant, anti-inflammatory, analgesic, antibacterial, antiviral, and antitumor properties. Clinically, they are mainly used to treat red and swollen eyes, stomach heat and toothache, acute tonsillitis, bacterial dysentery, and tuberculosis, and are hailed as a "green medicine."

[0003] Currently, the quality control methods for Mahonia japonica mainly rely on high-performance liquid chromatography (HPLC) to detect and analyze its alkaloid components. Huang Yang et al., in their paper "Simultaneous Determination of Nine Chemical Components in Mahonia japonica by HPLC," disclosed a method for determining the chemical components in Mahonia japonica. Specifically, the sample was ultrasonically extracted (500W power, 40kHz frequency) in a hydrochloric acid-methanol solution (1:100, V / V) for 45 min, followed by HPLC analysis of the nine chemical components. Qiu Mingming et al., in their paper "Determination of Three Alkaloids in Mahonia japonica by HPLC," disclosed a method for determining alkaloids. This involved adding the sample to a hydrochloric acid-methanol solution (1:100, V / V), immersing it in cold water, followed by ultrasonic extraction, and then using HPLC to determine the contents of three components: berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride. However, these methods suffer from inaccurate analysis and long analysis times. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining alkaloids in Mahonia japonica, which solves the problems of poor accuracy and long analysis time in the existing technology of alkaloid analysis.

[0005] To solve the above problems, the technical solution of the method for determining alkaloids in Mahonia japonica of the present invention is as follows:

[0006] A method for determining alkaloids in Mahonia fortunei includes the following steps: microwave-assisted extraction of Mahonia fortunei powder in an extractant, followed by centrifugation and filtration to obtain a test solution; and then analysis of the test solution by UFLC-MS / MS.

[0007] This invention improves upon existing technologies and provides a method for determining alkaloids in Mahonia japonica. By employing microwave-assisted extraction of alkaloids from Mahonia japonica, and then using UFLC-MS / MS to detect and analyze the extracted alkaloid components, this method can greatly improve the accuracy of alkaloid analysis and detection in Mahonia japonica, while also being highly efficient and significantly reducing analysis time. This method is simple, efficient, and reproducible, enabling better evaluation of the quality of Mahonia japonica medicinal materials.

[0008] To further improve the solubility of alkaloids, preferably, the extract is a mixed solution of methanol and hydrochloric acid, wherein the volume fraction of hydrochloric acid is 0.5-2%.

[0009] To further reduce the surface tension and viscosity of the extract and increase the solubility and diffusivity of alkaloids in the extract, preferably, the extraction temperature is 60–100°C and the extraction time is 2–10 min.

[0010] To further improve the mass transfer effect of the extract, preferably, the liquid-to-solid ratio of the extract to the Mahonia japonica powder is (40-60) mL: 1 g.

[0011] To further improve the separation effect of alkaloids, preferably, the alkaloids include berberine, palmatine, purslane, magnoflorine, and schönthospermum erythrorhizonine.

[0012] To further improve detection efficiency, preferably, the mobile phase used in the UFLC-MS / MS analysis is 0.4% aqueous acetic acid (A)-acetonitrile (B), with a gradient elution mode of: 0.0-1.0 min, 90%-85% A; 1.0-7.0 min, 85% A; 7.0-8.0 min, 85%-70% A; 8.0-8.5 min, 70% A; 8.5-9.0 min, 70%-90% A.

[0013] To further improve the separation of analytes, preferably, the chromatographic column used in the UFLC-MS / MS analysis is an ACQUITY UPLC BEH C. 18 The column temperature of the chromatographic column is 30℃.

[0014] To further improve the accuracy of mass spectrometry analysis, the preferred mass spectrometry conditions for the UFLC-MS / MS analysis are as follows: ion source is an electrospray ionization (ESI) source with positive ionization; DL heating device temperature: 200℃; heating module temperature: 350℃; dryer flow rate: 13.0 L·min. -1 Atomizer flow rate: 2.0 L / min -1 The scanning method was multiple reaction monitoring (MRM). Attached Figure Description

[0015] Figure 1 Typical chromatograms of five alkaloids and internal standard compounds in Mahonia japonica medicinal materials;

[0016] Figure 2 A bar graph showing the extraction efficiency of five alkaloids in Mahonia japonica medicinal material by different types of extracts;

[0017] Figure 3 A bar graph showing the extraction efficiency of five alkaloids from Mahonia japonica with methanol of different volume fractions.

[0018] Figure 4 A bar graph showing the extraction efficiency of five alkaloids from Mahonia japonica with different volume fractions of hydrochloric acid.

[0019] Figure 5 A bar graph showing the extraction efficiency of five alkaloids in Mahonia japonica at different extraction temperatures.

[0020] Figure 6 A bar graph showing the extraction efficiency of five alkaloids in Mahonia japonica at different extraction times;

[0021] Figure 7 A bar graph showing the extraction efficiency of five alkaloids from Mahonia japonica medicinal material at different liquid ratios. Detailed Implementation

[0022] A method for determining alkaloids in Mahonia fortunei includes the following steps: microwave-assisted extraction of Mahonia fortunei powder in an extractant, followed by centrifugation and filtration to obtain a test solution; and then analysis of the test solution by UFLC-MS / MS.

[0023] In a specific embodiment, the determination method uses the internal standard method to test the alkaloid content. The test solution is obtained by mixing 50 μL of filtrate after membrane filtration with the internal standard solution at a volume ratio of 1:1. The internal standard solution is prepared by mixing chelidonine with methanol-water (50:50, v / v) at a concentration of 20 ng·mL. -1 Chelidonine solution.

[0024] In a specific embodiment, the chemical structural formulas of the alkaloids in the Mahonia wood and the internal standard in the internal standard solution are shown below:

[0025]

[0026] A, B, C, D, and E represent five alkaloids: berberine, palmatine, purslane, magnoflorine, and tebufenozide, respectively, while F represents the internal standard compound celandine.

[0027] In a specific embodiment, the Mahonia japonica medicinal material powder is obtained by drying Mahonia japonica medicinal material, pulverizing it, and passing it through a 50-80 mesh sieve.

[0028] In a specific embodiment, stirring is performed during the extraction process, and the stirring is magnetic stirring with an efficiency of 30% to 60%.

[0029] In a specific implementation, the power range of the microwave is 200 to 1000W.

[0030] In a specific embodiment, the centrifugal speed is 10,000 to 15,000 r / min. -1 The centrifugation time is 3 to 5 minutes.

[0031] In a specific embodiment, the filtration is performed using a filter membrane with a micropore size of 0.2 μm.

[0032] The embodiments of the present invention will be further described below with reference to specific examples. Unless otherwise specified, the chemical reagents involved in the following examples are all commercially available conventional products.

[0033] I. Specific Embodiments of the Method for Determining Alkaloids in Mahonia Tree of the Present Invention

[0034] Example 1

[0035] The method for determining alkaloids in Mahonia japonica provided in this embodiment includes the following steps:

[0036] 1) Preparation of internal standard solution: Accurately weigh a certain amount of chelidonine and add it to a methanol-water solution (50:50, v / v) to prepare a solution with a concentration of 100 μg·mL. -1 The chelidonine solution was then diluted with methanol-water solution (50:50, v / v) to a concentration of 20 ng / mL. -1 The chelidonine solution is the internal standard solution;

[0037] 2) Preparation of the test solution: After drying, the Mahonia japonica medicinal material was pulverized and passed through a 60-mesh sieve to obtain Mahonia japonica powder. 0.4 g of the Mahonia japonica powder was accurately weighed and placed in a microwave extraction vessel. 20 mL of extractant was added for microwave-assisted extraction (MAE). The microwave power was 500 W, and the extractant was methanol-hydrochloric acid (100:1, v / v). Stirring was performed during extraction, with the magnetic stirring efficiency set at 40%, the extraction time at 5 min, and the extraction temperature at 80 °C. After extraction, the mixture was allowed to stand and cool to room temperature. The weight loss was replenished with the extractant, and the mixture was shaken well. The mixture was then heated at 12000 r·min. -1 Centrifuge at a speed of 4 min, filter through a 0.2 μm microporous membrane, accurately transfer 50 μL of the filtrate, and mix it with the internal standard solution at a volume ratio of 1:1 to obtain the test solution;

[0038] 3) Chromatographic and mass spectrometric analysis: The contents of five alkaloids (berberine, palmatine, purslane, magnoflorine and tebufenozide) in the test solution obtained in step 2) were analyzed by high performance liquid chromatography-mass spectrometry (UFLC-MS / MS).

[0039] Chromatographic conditions: The chromatographic column was an ACQUITY UPLC BEH C. 18 The column (50 mm × 2.1 mm ID, 1.7 μm) used a mobile phase of 0.4% aqueous acetic acid (A)-acetonitrile (B) with gradient elution (0.0–1.0 min, 90%–85% A; 1.0–7.0 min, 85% A; 7.0–8.0 min, 85%–70% A; 8.0–8.5 min, 70% A; 8.5–9.0 min, 70%–90% A); the flow rate was 0.3 mL / min. -1 Column temperature: 30℃; Injection volume: 3μL;

[0040] Mass spectrometry conditions: Ion source was electrospray ionization (ESI) source, positive ionization mode; DL heating device: 200℃; heating module temperature: 350℃; dryer flow rate: 13.0 L·min -1 Atomizer flow rate: 2.0 L / min -1 The scanning method was multiple reaction monitoring (MRM). The optimized ion parameters for the analytes (five alkaloids and the internal standard compound chelidonine) are shown in Table 1. The final analysis time was 9 min.

[0041] Table 1. MRM parameters and retention times of five alkaloids and chebulicine.

[0042] Analytes m / z Collision energy (eV) Retention time (min) Magnolia alkaloids 342.2>297.3 20 1.9 Chelidonine (internal standard) 353.9>275.1 25 5.7 African tetrandrine 338.0>322.1 28 6.3 Root alkaloids 338.0>322.1 29 6.8 Berberine 335.9>322.1 31 8.9 Barmatine 352.2>336.2 29 8.8

[0043] In other embodiments, the extracts are ethanol, water, methanol with volume fractions of 100%, 90%, 70% and 50%, hydrochloric acid (0.5%)-methanol mixed solution, and hydrochloric acid (2.0%)-methanol mixed solution, respectively.

[0044] In other embodiments, the extraction temperatures are 60°C and 100°C, respectively.

[0045] In other embodiments, the extraction times are 2 min and 10 min, respectively.

[0046] In other embodiments, the liquid-to-solid ratio of the extract and the Mahonia japonica powder is 40 mL: 1 g and 60 mL: 1 g, respectively.

[0047] II. Comparative Example

[0048] Comparative Example 1

[0049] The method for determining alkaloids in Mahonia japonica provided in this comparative example differs from that in Example 1 in that the extraction is performed using the heated reflux method (HRE). Specifically, approximately 0.4 g of Mahonia japonica powder (dried, pulverized, and sieved through a 60-mesh sieve, the same raw material as in Example 1) is accurately weighed and placed in a 100 mL round-bottom flask. 20 mL of methanol-hydrochloric acid (100:1, v / v) is added, the weight is measured, and the mixture is heated under reflux for 2 hours. After cooling to room temperature, the weight is measured again, and the weight is replenished with the extraction solution. The mixture is shaken well and centrifuged for 4 minutes (12000 r·min). -1 The solution is filtered through a 0.2 μm microporous membrane, and 50 μL of the filtrate is precisely taken and mixed with the internal standard solution at a 1:1 ratio to obtain the test solution.

[0050] Comparative Example 2

[0051] The method for determining alkaloids in Mahonia japonica provided in this comparative example differs from that in Example 1 in that the extraction is performed using ultrasonic extraction (UAE). Specifically, approximately 0.4 g of Mahonia japonica powder (dried, pulverized, and sieved through a 60-mesh sieve, the same raw material as in Example 1) is accurately weighed and placed in a 50 mL Erlenmeyer flask. 20 mL of methanol-hydrochloric acid (100:1, v / v) is added, the weight is measured, and the mixture is soaked for 30 min. Ultrasonic treatment is then performed for 90 min (500 W, 40 kHz). After cooling to room temperature, the mixture is weighed, and the weight loss is compensated with the extraction solvent. The mixture is shaken well and centrifuged for 4 min (12000 r·min). -1 The solution is filtered through a 0.2 μm microporous membrane, and 50 μL of the filtrate is precisely taken and mixed with the internal standard solution at a 1:1 ratio to obtain the test solution.

[0052] Comparative Example 3

[0053] The method for determining alkaloids in Mahonia japonica provided in this comparative example differs from that in Example 1 in that the analytical method employs high-performance liquid chromatography (HPLC), and the chromatographic column, mobile phase gradient, and peak shape of the HPLC were optimized to obtain optimal test conditions. The optimal test conditions are as follows: the chromatographic column is a Platisil ODS column (150 mm × 4.6 mm, 5 μm), with a mobile phase gradient of 0.02 mol·L⁻¹. -1 KH₂PO₄ (containing 0.01% triethylamine, pH adjusted to 2.8 with H₃PO₄) was used as mobile phase A, and acetonitrile was used as mobile phase B. The elution method was gradient elution (0.0–10 min, 90%–80% A; 10–15 min, 80%–45% A; 15–20 min, 45% A; 20–25 min, 45%–90% A; 25–28 min, 90% A), with a flow rate of 1 mL / min. -1 The detection wavelength is 345nm.

[0054] Even under the optimal testing conditions described above, the isomers tetrandrine and purslane were not completely separated when analyzed by high-performance liquid chromatography (HPLC), and the analysis time was 28 min. Compared with HPLC, the UFLC-MS / MS method used in this invention can not only separate tetrandrine and purslane, but also significantly shorten the analysis time (9 min) and has a relatively simple mobile phase composition.

[0055] III. Experimental Examples

[0056] Experimental Example 1

[0057] 1. Examination of linearity, detection limit, and quantitation limit

[0058] 1) Preparation of a series of reference solutions: Accurately weigh appropriate amounts of berberine, palmatine, purslane, magnoflorine, and tetrandrine reference standards, and prepare solutions with methanol-water (50:50, v / v) to a concentration of 100.0 μg·mL⁻¹. -1 100.0 μg·mL -1 140.0 μg·mL -1 100.0 μg·mL -1 and 100.0 μg·mL -1 Prepare the reference stock solutions; then accurately pipette appropriate amounts of 5 reference stock solutions and mix them, dilute with methanol-water (50:50, v / v) to prepare solutions containing berberine, palmatine, purslane, magnoflorine, and tetrandrine at concentrations of 400.0 ng·mL. -1 400.0 ng·mL -1 800.0 ng·mL -1 200.0 ng·mL -1 80.0 ng·mL -1 A mixed reference solution was prepared; it was diluted with methanol-water (50:50, v / v) to prepare a series of reference solutions with different concentrations, ranging from 10.0 to 400.0 ng / mL. -1 Berberine, 10.0-400.0 ng / mL -1 (Barmartin), 20.0-800.0 ng / mL -1 (Phyllanthus alkaloids), 5.0-200.0 ng / mL -1 (Magnolia alkaloid) and 2.0-80.0 ng·mL -1 (African tetrandrine)

[0059] 2) Linearity calculation: A series of reference solutions were precisely pipetted and injected for analysis. A standard curve was established with the concentration of each analyte (x) as the abscissa and the peak area ratio of the analyte to the internal standard (y) as the ordinate. Linear regression was performed using the weighted least squares method, and the resulting linearity data are shown in Table 2. Table 2 shows that the five alkaloids exhibited good linearity within their respective linear ranges.

[0060] 3) Calculation of detection limit and quantitation limit: The mixed reference solution was gradually diluted and measured. The mass concentration of each reference solution at a signal-to-noise ratio (S / N) of 3 and a signal-to-noise ratio (S / N) of 10 were used as the detection limit and quantitation limit, respectively. The results are shown in Table 2.

[0061] Table 2. Linear relationships, limits of detection, and limits of quantitation for five alkaloids.

[0062]

[0063] 2. Precision test

[0064] A mixed reference solution with low, medium, and high concentrations was prepared, and six injections were administered at each concentration for three consecutive days. The peak areas of the five alkaloids were recorded. The intraday precision of berberine, palmatine, purslane, magnoflorine, and tetrandrine was calculated to be between 1.1% and 4.7%, and the interday precision was between 2.1% and 5.0%, indicating that the instrument precision was good.

[0065] 3. Repeatability test

[0066] Six test solutions were prepared from the same sample of Mahonia fortunei, and each solution was injected and analyzed. The RSDs of berberine, palmatine, purslane, magnoflorine, and tetrandrine were calculated to be 2.01%, 2.03%, 2.88%, 2.82%, and 3.75%, respectively, indicating that the determination method of the present invention has good repeatability.

[0067] 4. Stability test

[0068] One sample of the test solution from the repeatability test was placed at room temperature and analyzed at 0, 2, 4, 8, 12 and 24 hours. The peak areas of the five alkaloids were recorded, and the RSDs of berberine, palmatine, purslane, magnoflorine and tetrandrine were calculated to be 2.89%, 1.07%, 1.57%, 3.20% and 5.00%, respectively. The results indicate that the test solution is stable within 24 hours.

[0069] 5. Recovery test

[0070] Six portions of a sample of Mahonia japonica with known alkaloid content were accurately weighed. Each portion was further divided into six parts, and appropriate amounts of a mixed standard solution of five alkaloids were added to each. Microwave-assisted extraction was used to obtain the test solutions, which were then injected for analysis. The recovery rate was calculated based on the ratio of the measured alkaloid content to the actual added alkaloid content. The results are shown in Table 3. The original amount represents the alkaloid content in the Mahonia japonica with known alkaloid content, the added amount represents the alkaloid content in the mixed standard solution, and the measured amount represents the alkaloid content determined using the method of this invention. Table 3 shows that the recovery rate of the method provided by this invention is good and meets the analytical requirements.

[0071] Table 3 Extraction recovery rates of five alkaloids from Mahonia japonica (n=6)

[0072]

[0073]

[0074] 6. Determination of alkaloid content in Mahonia japonica samples

[0075] Eight batches of Mahonia fortunei powder from different origins were collected, with three samples from each batch. The powder was analyzed according to the method described in Example 1. The contents of berberine, palmatine, physalisine, magnoflorine, and tetrandrine in Mahonia fortunei were calculated using the internal standard method. The results of the five alkaloid content determination are shown in Table 4. Table 4 shows that among the Mahonia fortunei from different origins, physalisine had the highest content, tetrandrine had the lowest content, and the contents of berberine and palmatine were similar. Typical chromatograms of the five alkaloids and the internal standard compound in Mahonia fortunei are shown below. Figure 1 As shown, Figure 1 In the middle, 1 to 6 represent magnoflorine, celandine, stephania tetrandra, purslane, berberine, and palmatine, respectively.

[0076] Table 4. Results of the determination of the content of five alkaloids in eight batches of Mahonia japonica (n=3)

[0077]

[0078] Experiment Example 2

[0079] This experiment compares the contents of five alkaloids obtained by the determination methods of alkaloids in Mahonia japonica in Examples 1 and 2, and the comparison results are shown in Table 5.

[0080] Table 5 shows the content of five alkaloids obtained from the examples and comparative examples.

[0081]

[0082] As shown in Table 5, among the three extraction methods, the extraction method (MAE) used in this invention has the shortest extraction time, and the alkaloid content obtained after extraction is higher than that of the comparative example. The heating reflux method (HRE) of comparative example 1 has the longest extraction time and the lowest extraction efficiency. Although the alkaloid content obtained after extraction by ultrasonic extraction (UAE) in comparative example 2 is not much different from that of MAE, and the extraction rate of UAE is similar to that of MAE, the extraction time of UAE (90 min) is much higher than that of MAE used in this invention (5 min). It can be seen that the MAE method used in this invention has a shorter extraction time and higher extraction efficiency compared with HRE and UAE. This invention provides a simple and rapid method for the determination of alkaloids in Mahonia japonica.

[0083] Experimental Example 3

[0084] This experimental example optimizes the extraction parameters in the method for determining alkaloids in Mahonia japonica described in this invention.

[0085] 1. Optimal selection of extract type

[0086] Microwave-assisted extraction was performed using methanol, ethanol, and water as extraction solvents to investigate the effect of different extraction solvents on the extraction efficiency of alkaloids. The test results of the contents of the five alkaloids after extraction with different extraction solvents are shown below. Figure 2 As shown. From Figure 2 It can be seen that among the three extracts—methanol, ethanol, and water—methanol has the best extraction efficiency.

[0087] Microwave-assisted extraction with methanol of different volume fractions was performed to investigate the effect of methanol volume fraction on the extraction efficiency of alkaloids. The test results of the contents of five alkaloids after extraction with methanol of different volume fractions are as follows: Figure 3 As shown, from Figure 3 It can be seen that there is no significant difference in the extraction effect of the five alkaloids in methanol extracts with volume fractions of 100%, 90%, 70%, and 50%. Considering the ease of operation, methanol with a volume fraction of 100% is preferred as the extract.

[0088] Considering that the target compounds are all weakly basic, adding a small amount of acid to the extract can increase the solubility of the alkaloids in the extract. Therefore, the effect of adding different proportions of commercially available hydrochloric acid (0.5%, 1.0%, 2.0%, v / v) to pure methanol on the extraction efficiency of the alkaloids was also investigated. The test results of the contents of the five alkaloids are as follows: Figure 4 As shown. From Figure 4 It can be seen that adding 1.0% hydrochloric acid to methanol can significantly improve the extraction efficiency. However, further increasing the proportion of hydrochloric acid tends to decrease the extraction rate. Therefore, the preferred extraction solution is methanol-hydrochloric acid (100:1, v / v).

[0089] 2. Optimal extraction temperature

[0090] Microwave-assisted extraction was performed at different extraction temperatures to investigate the effect of different extraction temperatures on the extraction efficiency of alkaloids. The test results are as follows: Figure 5 As shown. From Figure 5 It can be seen that when the extraction temperature is increased from 60℃ to 80℃, the extraction efficiency of the five alkaloids is significantly improved. This is likely because increasing the temperature reduces the surface tension and viscosity of the extract, thereby increasing its solubility and diffusion capacity, thus increasing the extraction efficiency of the five alkaloids. However, when the temperature exceeds 80℃, the extraction efficiency of all alkaloids decreases, possibly because the excessively high temperature causes the degradation of some alkaloids. Therefore, the preferred extraction temperature is 80℃.

[0091] 3. Optimal extraction time

[0092] Microwave-assisted extraction was performed using different extraction times to investigate the effect of different extraction times on the extraction efficiency of alkaloids. The test results are as follows: Figure 6 As shown. From Figure 6 As can be seen, the extraction efficiency of the five alkaloids increased as the extraction time increased from 2 min to 5 min, but decreased when the extraction time exceeded 5 min. Therefore, the optimal extraction time is 5 min.

[0093] 4. Optimal liquid-to-solid ratio

[0094] Microwave-assisted extraction was performed using different liquid-to-solid ratios (40 mL:1 g, 50 mL:1 g, 60 mL:1 g) to investigate the effect of different liquid-to-solid ratios on the extraction efficiency of alkaloids. The test results are as follows: Figure 7 As shown. From Figure 7 As can be seen, the extraction efficiency of alkaloids tends to increase with the increase of the liquid-to-solid ratio. This may be due to the increased mass transfer driving force resulting from the increased liquid-to-solid ratio. When the liquid-to-solid ratio is 50 mL:1 g, the extraction efficiency of the five alkaloids reaches its maximum. With further increases in the liquid-to-solid ratio, the extraction efficiency does not significantly improve. Therefore, the preferred liquid-to-solid ratio is 50 mL:1 g.

[0095] Based on the optimization results, the optimal conditions for extracting five alkaloids from Mahonia japonica using MAE were determined to be: methanol-hydrochloric acid (100:1, v / v) as the extraction solution, liquid-to-solid ratio of 50 mL: 1 g, and extraction at 80 °C for 5 min.

[0096] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining alkaloids in Mahonia japonica, characterized in that, Includes the following steps: The powder of Mahonia fortunei was extracted with microwave-assisted extraction solution, and the solution was obtained by centrifugation and filtration. The solution was then analyzed by UFLC-MS / MS.

2. The method for determining alkaloids in Mahonia japonica as described in claim 1, characterized in that, The extract is a mixed solution of methanol and hydrochloric acid, wherein the volume fraction of hydrochloric acid is 0.5-2%.

3. The method for determining alkaloids in Mahonia japonica as described in claim 1, characterized in that, The extraction temperature is 60–100℃, and the extraction time is 2–10 min.

4. The method for determining alkaloids in Mahonia japonica as described in claim 1, characterized in that, The liquid-to-solid ratio of the extract to the Mahonia japonica powder is (40-60) mL: 1 g.

5. The method for determining alkaloids in Mahonia japonica as described in claim 1, characterized in that, The alkaloids include berberine, palmatine, purslane, magnoflorine, and schöntenine.

6. The method for determining alkaloids in Mahonia japonica as described in claim 1, characterized in that, The mobile phase used for the UFLC-MS / MS analysis was 0.4% acetic acid aqueous solution (A)-acetonitrile (B), with a gradient elution mode of 0.0-1.0 min, 90%-85% A; 1.0–7.0 min, 85% A; 7.0~8.0min, 85%~70%A; 8.0–8.5 min, 70% A; 8.5~9.0min, 70%~90%A.

7. The method for determining alkaloids in Mahonia japonica as described in claim 1, characterized in that, The chromatographic column used for the UFLC-MS / MS analysis was an ACQUITY UPLC BEH C. 18 The column temperature of the chromatographic column is 30℃.

8. The method for determining alkaloids in Mahonia japonica as described in claim 1, characterized in that, The mass spectrometry conditions for the UFLC-MS / MS analysis were as follows: ion source: electrospray ionization (ESI) source, positive ionization mode; DL heating device temperature: 200℃; heating module temperature: 350℃; dryer flow rate: 13.0 L·min. -1 Atomizer flow rate: 2.0 L / min -1 The scanning method was multiple reaction monitoring (MRM).

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