A method for determining magnolamine, argentin, and asarin in bamboo leaf pepper slices using high performance liquid chromatography.

Abstract

This invention provides a high-performance liquid chromatography (HPLC) method for the detection of magnolamine, lignans, and asaran in *Zanthoxylum bungeanum* slices. The chromatographic conditions are as follows: a CAPCELL PAK C18 column (250 mm × 4.6 mm, 5 μm); mobile phase A: 0.3% phosphoric acid solution; mobile phase B: acetonitrile; detection wavelengths: magnolamine 217 nm, lignans: 201 nm, asaran: 201 nm; column temperature: 25–35 °C; mobile phase flow rate: 0.5–0.8 mL / min; gradient elution. This method achieves complete separation of magnolamine, lignans, and asaran, enabling the detection of their contents in *Zanthoxylum bungeanum* slices. It features high sensitivity, strong specificity, simple experimental operation, and the ability to simultaneously determine both alkaloids and lignans, allowing for more comprehensive quality control of *Zanthoxylum bungeanum* slices.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting magnoflorine, phillyrin and asarinin in Zhuyejiao tablets by high performance liquid chromatography. BACKGROUND

[0002] Zhuyejiao tablets are oral tablets of traditional Chinese medicine developed by Lanzhou Foci Pharmaceutical Co., Ltd. for treating stomachache and abdominal pain of stasis type. The function is to clear heat and resolve toxins, activate blood and relieve pain, and is used for stomachache and abdominal pain of stasis type, fixed and obvious pain or rebound pain in the right lower abdomen caused by early acute simple appendicitis.

[0003] Zhuyejiao tablets are tablets made of Zhuyejiao extract, mainly containing alkaloids and lignans, and mainly playing the role of anti-inflammatory analgesia. However, according to the new drug standard: (number: WS3-04(Z-07)-99(Z)) using titration method only to determine the content of magnoflorine to represent the total alkaloid content, and the lignan content is not determined and the experimental process is complicated, the titration end point is fuzzy, the sensitivity is poor, the human operation is large, and the experimental results are easy to deviate. At present, there is no literature report on a more sensitive and more accurate method for determining the content of alkaloids and lignans in Zhuyejiao tablets. SUMMARY

[0004] The present application provides a method for detecting effective components in Zhuyejiao tablets by high performance liquid chromatography, determining the content of magnoflorine, phillyrin and asarinin, and realizing the determination of alkaloids and lignan compounds in Zhuyejiao tablets. The method has high sensitivity, strong specificity, simple experimental operation process, and can simultaneously determine two types of effective components of alkaloids and lignans, and can realize more comprehensive control of the quality of Zhuyejiao tablets.

[0005] The method for detecting magnoflorine, phillyrin and asarinin in Zhuyejiao tablets by high performance liquid chromatography provided by the present application comprises the following steps:

[0006] (1) Zhuyejiao tablets are added into methanol, ultrasonic treated for 40 min, taken out, cooled, supplemented with methanol to make up the weight loss, shaken, filtered, and the filtrate is taken, evaporated to dryness, the residue is dissolved in methanol and diluted to 25 ml, and filtered to obtain Zhuyejiao tablet test solution.

[0007] (2) The Zhuyejiao tablet test solution is injected into a high performance liquid chromatograph for detection, and the chromatographic conditions are as follows:

[0008] The chromatographic column was a CAPCELL PAK C18, with dimensions of 250 mm × 4.6 mm and a diameter of 5 μm. Mobile phase A consisted of 0.3% phosphoric acid solution (v / v) and acetonitrile (v / v). Detection wavelengths were: magnolol 217 nm, magnolol 201 nm, and asarone 201 nm. The column temperature was 25–35 °C. The mobile phase flow rate was 0.5–0.8 mL / min. Gradient elution was used, with the elution program being: 0.01 min → 25 min → 45 min → 45.01 min → 55 min. The volume percentage of mobile phase B was: 5% → 70% → 70% → 5% → 5%.

[0009] In the chromatogram of the test solution of *Zhuyejiao* slices, magnolamine, tylosin, and asarazin were completely separated. The peak areas in the chromatograms were substituted into the concentration-peak area standard curves of magnolamine, tylosin, and asarazin to calculate the content of magnolamine, tylosin, and asarazin in *Zhuyejiao* slices.

[0010] The ultrasonic processing power is 200W and the frequency is 40KHZ.

[0011] The concentration of bamboo leaf pepper slices in methanol is 0.01~0.02 g / mL.

[0012] The retention time of magnolol was 14.683 min, and the resolution was 2.237; the retention time of magnolol was 30.379 min, and the resolution was 3.281; the retention time of asarone was 32.388 min, and the resolution was 8.649.

[0013] The concentration-peak area standard curves for magnolol, tylosin, and asarone are as follows:

[0014]

[0015] Where x represents the concentrations of magnolol, magnolol, and asarol in μg / mL, and y represents the chromatographic peak area.

[0016] Resolution, used to evaluate the degree of separation between the analyte and the separated substances, is a key indicator of the separation efficiency of a chromatographic system. It is denoted by R, which is equal to the ratio of the difference in retention time between adjacent peaks to the average peak width of the two peaks. A larger R indicates better separation between adjacent components. Resolution is generally required to be ≥1.5.

[0017] The high-performance liquid chromatography (HPLC) method for detecting magnolamine, magnolamine, and asarone provided by this invention can achieve complete separation between the target peak and adjacent chromatographic peaks, with a resolution R≥1.6. This method enables the detection of the contents of magnolamine, magnolamine, and asarone in *Zanthoxylum bungeanum* slices. After six consecutive injections of the *Zanthoxylum bungeanum* slice solution, the RSD of magnolamine content was ≤1.2%, the RSD of magnolamine content was ≤1.2%, and the RSD of asarone content was ≤1.2%. The method exhibits good injection repeatability and robustness, and can be used for the quality control of *Zanthoxylum bungeanum* slices. Attached Figure Description

[0018] Figure 1 The spectrum of magnolamine;

[0019] Figure 2 The spectrum of magnolia biondii lipids:

[0020] Figure 3 The spectrum of asarone;

[0021] Figure 4 Chromatogram of the test solution of bamboo leaf pepper slices;

[0022] Figure 5 The chromatogram is of the reference solution;

[0023] Figure 6 The standard curve of magnolol;

[0024] Figure 7 Standard curve of magnolia biondii lipids:

[0025] Figure 8 This is the standard curve of asarone. Detailed Implementation

[0026] Example 1: Establishment of the detection method

[0027] 1. Screening of chromatographic conditions

[0028] Chromatographic conditions are determined by screening factors such as chromatographic column, mobile phase, wavelength, and flow rate.

[0029] Preparation of reference solution: Take appropriate amounts of magnolamine reference standard, magnolamine reference standard and asarone reference standard, accurately weigh them, add methanol to prepare reference solution. In the reference solution, the content of magnolamine is 0.4720072 mg / mL, the content of magnolamine is 0.0478 mg / mL, and the content of asarone is 0.0214 mg / mL.

[0030] Preparation of the test solution of bamboo leaf pepper slices: Take 10 bamboo leaf pepper slices, grind them into a fine powder, accurately weigh about 0.5g, place them in a stoppered conical flask, accurately add 50ml of methanol, seal tightly, weigh, sonicate for 40min, remove, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, evaporate to dryness, dissolve the residue in methanol and make up to 25ml, filter to obtain the solution.

[0031] (1) Chromatographic column: CAPCELL PAK C18 (250 mm × 4.6 mm, 5 μm)

[0032] Detection wavelengths: magnolol: 217 nm, magnolol: 201 nm, asarol: 201 nm; column temperature: 35℃; injection volume: 10 μL; flow rate: 0.8 mL / min;

[0033] Mobile phase A: Acetonitrile;

[0034] Mobile phase B: a phosphoric acid solution of sodium octane sulfonate (wherein, the mass fraction of sodium octane sulfonate is 0.4% and the volume fraction of phosphoric acid is 0.1%).

[0035] Elute isocratic according to mobile phase A: mobile phase B (24:76);

[0036] Conclusion: Using acetonitrile as mobile phase A and sodium octane sulfonate in phosphoric acid solution (sodium octane sulfonate mass fraction of 0.4% and phosphoric acid volume fraction of 0.1%) as mobile phase B, and eluting isocratically according to mobile phase A: mobile phase B (24:76), magnolol peaked at approximately 21.6 min, but magnolol and asarol did not peak.

[0037] (2) Mobile phase A: methanol; Mobile phase B: water;

[0038] Perform gradient elution under the following conditions;

[0039]

[0040] Conclusion: The methanol-water system used as reference standards oxyphylla and asarum oxyphylla resulted in excessively late peak elution and unstable baselines, leading to interference.

[0041] (3) Column: CAPCELL PAK C18 (250 mm × 4.6 mm, 5 μm)

[0042] Detection wavelengths: magnolol: 217 nm, magnolol: 201 nm, asarol: 201 nm; column temperature: 35℃; injection volume: 10 μL; flow rate: 0.8 mL / min;

[0043] Mobile phase A: Acetonitrile;

[0044] Mobile phase B: Water;

[0045] Using mobile phase A, gradient elution was performed under the following conditions;

[0046]

[0047] Conclusion: Using the acetonitrile-water system as reference standards for magnolamine, magnolol, and asarol, the peak elution time was moderate and the baseline was relatively stable. However, the peak shape of magnolamine was poor and the theoretical plate number was too low.

[0048] (4) Based on (3), try replacing the aqueous phase with a 0.3% phosphoric acid solution.

[0049] Column: CAPCELL PAK C18 (250 mm × 4.6 mm, 5 μm)

[0050] Detection wavelengths: magnolol: 217 nm, magnolol: 201 nm, asarol: 201 nm; column temperature: 35 ℃; injection volume: 10 μL; flow rate: 0.8 mL / min;

[0051] Mobile phase A: Acetonitrile

[0052] Mobile phase B: 0.3% phosphoric acid solution

[0053] Perform gradient elution under the following conditions;

[0054]

[0055] Conclusion: When using acetonitrile-0.3% phosphoric acid solution for gradient elution, the reference solution showed normal elution of magnolamine, oxyphylla, and asarazine, with theoretical plate number and resolution meeting the requirements. However, the resolution between the magnolamine peak and its adjacent peak in the test solution was 0.9, which did not meet the requirements.

[0056] (5) Try to change the gradient elution ratio of acetonitrile and 0.3% phosphoric acid solution based on (4);

[0057] Column: CAPCELL PAK C18 (250 mm × 4.6 mm, 5 μm)

[0058] Detection wavelengths: magnolol: 217 nm, magnolol: 201 nm, asarol: 201 nm; column temperature: 35℃; injection volume: 10 μL; flow rate: 0.8 mL / min;

[0059] Mobile phase A: Acetonitrile

[0060] Mobile phase B: 0.3% phosphoric acid solution

[0061] Perform gradient elution under the following conditions;

[0062]

[0063] Conclusion: After elution using the above gradient, the chromatographic peaks of magnolamine, magnolamine, and asarone in the reference solution and the test solution were completely separated, with a resolution greater than 1.7 and the theoretical plate number meeting the requirements. The chromatogram of the bamboo leaf pepper slices test solution is shown below. Figure 4 (Peak 1: Magnoliaine; Peak 2: Magnolia extract; Peak 3: Asarone). The chromatogram of the reference solution is shown below. Figure 5 (Peak 1: Magnolia alkaloid; Peak 2: Magnolia oleoresin; Peak 3: Asarone oleoresin).

[0064] 2. Screening of test sample solution preparation conditions

[0065] Extraction was performed using methanol, ethanol, and methanol hydrochloric acid, as well as two extraction methods: ultrasonic and reflux.

[0066] (1) Weigh approximately 0.5g of bamboo leaf pepper powder into three portions and place them in stoppered conical flasks. Add 50ml each of methanol, ethanol, and 5% hydrochloric acid methanol solution to each flask. Seal tightly, weigh, and sonicate (power 200W, frequency 40KHZ) for 40min. Remove, cool, and weigh again. Make up the weight loss with the corresponding reagents, shake well, filter, take the filtrate, evaporate to dryness, dissolve the residue in methanol, and make up to 25ml. Filter to obtain the bamboo leaf pepper sample solution. Calculate the content according to the chromatographic conditions in Example 1 (5). The results are shown in the table below:

[0067]

[0068] Conclusion: As shown in the table above, the extraction efficiency of ultrasonic extraction is: methanol > ethanol > 5% hydrochloric acid methanol. When using hydrochloric acid methanol solution, neither octocarcinol nor asarocorcinol was detected. Therefore, methanol is the most effective extraction solvent.

[0069] (2) Weigh approximately 0.5g of bamboo leaf pepper powder into three portions and place them in stoppered conical flasks. Add 50ml each of methanol, ethanol, and 5% hydrochloric acid methanol solution to each flask. Seal tightly, weigh, and heat under reflux for 40min. Remove, cool, and weigh again. Make up the weight loss with methanol, shake well, filter, take the filtrate, evaporate to dryness, dissolve the residue in methanol, and make up to 25ml. Filter. Calculate the content according to the chromatographic conditions in Example 1 (5). The results are shown in the table below:

[0070]

[0071] Conclusion: As shown in the table above, the extraction effect of heating and reflux extraction is: methanol > ethanol > 5% hydrochloric acid methanol. When using hydrochloric acid methanol solution, neither magnolol nor asarilol was detected. Therefore, methanol is the best extraction solvent. Combined with (1), it can be seen that using methanol as the extraction solvent and ultrasonic extraction results in more complete extraction and higher content.

[0072] (3) Weigh approximately 0.5g of bamboo leaf pepper powder into three portions and place them in stoppered conical flasks. Add 50ml of methanol to each flask, seal tightly, weigh, and extract using ultrasonic extraction (power 200W, frequency 40KHZ) for 30min, 40min, 50min, and 60min respectively. Remove, cool, weigh again, replenish the lost weight with methanol, shake well, filter, take the filtrate, evaporate to dryness, dissolve the residue in methanol, and dilute to 25ml. Filter to obtain the final product. Calculate the content according to the chromatographic conditions in Example 1 (5). The results are shown in the table below:

[0073]

[0074] Conclusion: As shown in the table above, the extraction efficiency of using methanol as the extraction solvent and ultrasonic extraction method is: 40 min > 50 min > 60 min > 30 min. Therefore, the optimal method for preparing the test solution is ultrasonic treatment with 50 ml of methanol for 40 min.

[0075] 3. Screening of detection wavelengths for magnolol, magnolol, and asarol

[0076] By examining the full-wavelength spectra of magnolol, magnolol, and asarol in the range of 190nm to 800nm ​​(spectral images of magnolol, magnolol, and asarol are shown in Figures 1-3), three wavelengths with absorption peaks were selected as candidate wavelengths. The optimal absorption wavelength was determined by comparing the peak area and resolution at each wavelength. The comparison results are shown in the table below:

[0077]

[0078] Conclusion: As shown in the table above, the optimal detection wavelengths are: magnolol: 217nm, magnolol: 201nm, and asarol: 201nm.

[0079] 4. Establishment of detection methods

[0080] (1) Take 10 slices of bamboo leaf pepper, grind them into a fine powder, accurately weigh about 0.5g, place them in a stoppered conical flask, accurately add 50ml of methanol, seal tightly, weigh, sonicate for 40min, take out, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, evaporate to dryness, dissolve the residue in methanol and make up to 25ml, filter to obtain the bamboo leaf pepper slice test solution;

[0081] (2) The test solution of bamboo leaf pepper slices was injected into a high-performance liquid chromatograph for detection. The chromatographic conditions were as follows:

[0082] Chromatographic column: CAPCELL PAK C18 (250 mm × 4.6 mm, 5 μm):

[0083] Detection wavelengths: magnolol: 217 nm, magnolol: 201 nm, asarol: 201 nm; column temperature: 35℃; injection volume: 10 μL; flow rate: 1.0 mL / min;

[0084] Mobile phase A: Acetonitrile

[0085] Mobile phase B: 0.3% phosphoric acid solution

[0086] Perform gradient elution according to the table below;

[0087]

[0088] Results: Magnoliin retention time: 14.683 min, resolution: 2.237; Magnolia oleoresin retention time: 30.379 min, resolution: 3.281; Asarone retention time: 32.388 min, resolution: 8.649. The chromatogram of the test solution of *Zanthoxylum bungeanum* slices is shown below. Figure 4 (Peak 1: Magnolia alkaloid; Peak 2: Magnolia oleoresin; Peak 3: Asarone oleoresin).

[0089] The method of this invention can achieve complete separation of magnolol, magnolol, and asarol.

[0090] Example 2: Validation of the method for determining the content of magnolol, magnolol, and asarol.

[0091] In accordance with the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 9101), we conducted methodological validation of the determination methods for the content of magnolol, magnolol, and asarol.

[0092] 1. Linear regression equation

[0093] Accurately weigh appropriate amounts of magnolamine reference standards, magnolamine reference standards, and asarone reference standards, and add methanol to prepare a mixed reference solution. The mixed reference solution contains 0.4720072 mg / mL of magnolamine, 0.0478 mg / mL of magnolamine, and 0.0214 mg / mL of asarone. Prepare a series of reference solutions with seven concentration gradients by diluting 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mL of the mixed reference solution to 10 mL with methanol. Inject the solutions under the chromatographic conditions determined in Example 1, and measure the peak area. The results are shown in Table 1. Plot a standard curve with the peak area integral value on the ordinate and the concentrations of magnolamine, magnolamine, and asarone on the abscissa. The concentration-peak area standard curves for magnolamine, magnolamine, and asarone are shown in Table 2.

[0094]

[0095] 2. Accuracy

[0096] A spiking recovery test was conducted. 0.25 g of *Zanthoxylum bungeanum* powder was accurately weighed and divided into three portions. 1.0 ml, 2.0 ml, and 3.0 ml of a mixed reference solution (concentrations: magnolol: 0.4720072 mg / ml, magnolol: 0.0478 mg / ml, asarol: 0.0214 mg / ml) were accurately added to each portion, respectively. After mixing, the solution was prepared according to the method for the test sample. The chromatographic conditions determined in Example 1 were followed, and the recovery rate was calculated using the following formula. The RSD was also calculated. The test results are shown in Tables 3, 4, and 5.

[0097] Recovery rate of fibroin % = [(Amount of fibroin measured - Amount of fibroin in the sample) / Amount of fibroin added] × 100%

[0098] Recovery rate of magnolia biondii esters % = [(Amount of magnolia biondii esters measured - Amount of magnolia biondii esters in the sample) / Amount of magnolia biondii esters added] × 100%

[0099] Recovery rate of asarone esters % = [(Amount of asarone esters measured - Amount of asarone esters in the sample) / Amount of asarone esters added] × 100%

[0100]

[0101] Experimental results show that this method has good accuracy.

[0102] 3. Repeatability test

[0103] Take 10 slices of Chinese pepper, grind them into a fine powder, accurately weigh about 0.5g, place them in a stoppered conical flask, accurately add 50ml of methanol, seal tightly, weigh, sonicate for 40min, remove, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, evaporate to dryness, dissolve the residue in methanol and make up to 25ml, filter to obtain the Chinese pepper sample solution.

[0104] The assay was repeated 6 times under the chromatographic conditions determined in Example 1, and the results are shown in Table 6.

[0105]

[0106] Experimental results show that this method has good repeatability.

[0107] 4. Precision test

[0108]

[0109] The experimental results show that the instrument has good precision.

[0110] 5. Content detection

[0111] The contents of magnolamine, tylosin, and asarone were determined in six different batches of bamboo leaf pepper slices:

[0112] Take 10 slices of bamboo leaf pepper from 6 different batches, grind them into a fine powder, accurately weigh about 0.5g, place them in a stoppered conical flask, accurately add 50ml of methanol, seal tightly, weigh, sonicate for 40min, remove, cool, weigh again, make up the weight loss with methanol, shake well, filter, take the filtrate, evaporate to dryness, dissolve the residue in methanol and make up to 25ml, filter to obtain the bamboo leaf pepper slice test solution;

[0113] (2) The test solution of bamboo leaf pepper slices was injected into a high-performance liquid chromatograph for detection. The chromatographic conditions were as follows:

[0114] Chromatographic column: CAPCELL PAK C18 (250 mm × 4.6 mm, 5 μm):

[0115] Detection wavelengths: magnolol: 217 nm, magnolol: 201 nm, asarol: 201 nm; column temperature: 35℃; injection volume: 10 μL; flow rate: 0.8 mL / min;

[0116] Mobile phase A: Acetonitrile

[0117] Mobile phase B: 0.3% phosphoric acid solution

[0118] Perform gradient elution according to the table below;

[0119]

[0120] The peak areas were substituted into the concentration-peak area standard curves of magnolamine, magnolamine, and asarone for calculation, and the contents of magnolamine, magnolamine, and asarone in the bamboo leaf pepper slices were obtained as shown in the table below:

[0121] .

Claims

1. A method for detecting magnoflorine, linarin and asarin by high performance liquid chromatography in Fagara zanthoxyloides, characterized in that, It comprises the following steps: (1) Put the leaf pepper pieces into methanol, and ultrasonically treat for 40 min, take out, cool, make up the weight loss with methanol, shake well, filter, take the filtrate, evaporate to dryness, dissolve the residue with methanol to 25 ml, filter to obtain the test solution of leaf pepper pieces; The preparation of the reference solution: take the magnolia base reference, the lutein reference and the asarone reference, add methanol to prepare the reference solution; (2) Inject the test solution of leaf pepper pieces into the high performance liquid chromatograph for detection, and the chromatographic conditions are as follows: The chromatographic column is CAPCELL PAK C18, the column specification is 250 mm x 4.6 mm, 5 μm, the mobile phase A is 0.3% phosphoric acid solution, and the mobile phase B is acetonitrile; the detection wavelength is 217 nm for magnolia base, 201 nm for lutein, and 201 nm for asarone; the column temperature is 25-35℃; the flow rate of the mobile phase is 0.5-0.8 mL / min; the gradient elution is performed, and the gradient elution program is: 0.01 min→25 min→45 min→45.01 min→55 min, the volume percentage of the mobile phase B is: 5%→70%→70%→5%→5%; The magnolia base, the lutein and the asarone in the test solution of leaf pepper pieces are completely separated; the peak areas of the magnolia base, the lutein and the asarone in the chromatogram are substituted into the concentration-peak area standard curve of the magnolia base, the lutein and the asarone to calculate the content of the magnolia base, the lutein and the asarone in the leaf pepper pieces.

2. The method for detecting magnoline, linarin and asarinin in lycopus lucidus tablet by high performance liquid chromatography according to claim 1, characterized in that, The ultrasonic treatment power is 200 W, and the frequency is 40 KHZ.

3. The method for detecting magnoline, linarin and asarinin in lycopus lucidus tablet by high performance liquid chromatography according to claim 1, characterized in that, The concentration of the leaf pepper pieces in methanol is 0.01-0.02 g / mL.

4. The method for detecting liriodendrin, magnolin and asarinin in psidium guajava L. leaves by high performance liquid chromatography according to claim 1, characterized in that, The retention time of the magnolia base is: 14.683 min, and the resolution is 2.237; the retention time of the lutein is 30.379 min, and the resolution is 3.281; the retention time of the asarone is 32.388 min, and the resolution is 8.

649.

5. The method for detecting magnoline, honkinolin and asarinin in the lycopus lucidus slice by high performance liquid chromatography according to claim 1, characterized in that, The concentration-peak area standard curve of the magnolia base, the lutein and the asarone is: Wherein, x is the concentration of the magnolia base, the lutein and the asarone, and the unit is μg / mL, and y is the chromatographic peak area.

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