Method for checking adulteration of a alocasia macrorrhiza in a alocasia odora

Abstract

This invention relates to the field of Acorus tatarinowii detection technology, specifically disclosing a method for detecting adulteration of Acorus tatarinowii with Acorus calamus. The method includes the following steps: preparation of test solution, preparation of reference solution, liquid chromatography-mass spectrometry (LC-MS) detection, and result determination. This invention uses 10% adulteration as the standard. If the peak area ratio is ≤3.0, it is determined that there is no significant risk of adulteration with Acorus tatarinowii; if the peak area ratio is >3.0, it is determined that there is a risk of adulteration with Acorus tatarinowii. Using this as the core indicator, accurate differentiation between Acorus tatarinowii and Acorus calamus can be achieved. This avoids the problem of difficulty in distinguishing by appearance and conventional chemical components from the perspective of component characteristics, ensuring the specificity of adulteration detection from the root. It achieves a quantitative assessment of the degree of adulteration with Acorus tatarinowii in Acorus tatarinowii, rather than a simple qualitative judgment, making the detection results more objective and referential.

Description

Technical Field

[0001] This invention belongs to the field of Acorus calamus detection technology, specifically relating to a method for detecting adulteration of Acorus calamus in Acorus calamus. Background Technology

[0002] Acorus tatarinowii is the dried rhizome of the plant Acorus tatarinowii Schott, belonging to the Araceae family. It is a commonly used traditional Chinese medicine and is widely used in the pharmaceutical field. Its quality is directly related to the safety and efficacy of related products. The 2025 edition of the Chinese Pharmacopoeia also clearly defines its origin.

[0003] In the circulation of Chinese medicinal materials, Acorus tatarinowii is often confused with Acorus calamus L., a closely related species. At the same time, there is a significant difference in market price between the two, which leads some merchants to mix Acorus calamus with Acorus tatarinowii for sale in order to seek improper profits. This has created a chaotic situation of adulteration, seriously disrupted the normal order of the Chinese medicinal materials market, and also brought great challenges to the quality control of medicines.

[0004] Currently, the quality control of Acorus tatarinowii mainly relies on the conventional quality control indicators specified in the Chinese Pharmacopoeia. However, the appearance of Acorus tatarinowii and Acorus calamus are not significantly different, and their chemical composition is also not significantly different. Conventional quality control indicators are difficult to effectively distinguish between the two, and cannot accurately identify and determine adulteration of Acorus calamus in Acorus tatarinowii.

[0005] In summary, no highly sensitive, specific, and quantifiable method for detecting adulteration in Acorus tatarinowii and Acorus calamus has been established in the current technology, which is not conducive to the existing regulations on the safety of Acorus tatarinowii drugs and the standardized development of the Chinese medicinal materials market. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting adulteration of Acorus calamus in Acorus gramineus, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for detecting adulteration of Acorus calamus in Acorus gramineus, using the peak area ratio of the cis / trans isomers of methyl isoeugenol as the criterion, is performed using liquid chromatography-mass spectrometry (LC-MS). The method includes the following steps:

[0009] S1. Preparation of the test solution:

[0010] Weigh 0.5-1g of Acorus tatarinowii powder, add 25-50mL of methanol, extract by sonication for 30 minutes, filter, and take the filtrate to obtain the test solution;

[0011] S2. Preparation of reference solution:

[0012] Using methanol as a solvent, cis-methylisoeugenol and trans-methylisoeugenol reference standards were prepared into reference solutions with concentrations of 1-10 μg / ml.

[0013] S3. Liquid chromatography-mass spectrometry (LC-MS) detection:

[0014] The test solution S1 and the reference solution S2 were injected into a liquid chromatography-mass spectrometry (LC-MS) instrument for detection. The detection parameters of the LC-MS instrument were as follows: the column was a C18-PFP column with a particle size of 1.7 μm, an inner diameter of 2.1 mm, and a length of 100-150 mm; the mobile phase A was 0.1% formic acid solution, and the mobile phase B was acetonitrile; the gradient elution program was as follows: 0-15 min, 50% A phase, 50% B phase; 15-16 min, linear transition from 50% A phase to 10% B phase, and linear transition from 50% B phase to 90% B phase; 16-20 min, 10% A phase, 90% B phase; the column temperature was 30-40℃; the injection volume was 1-10 μL; a triple quadrupole mass spectrometer was used, and multiple reaction monitoring (MRM) was performed in electrospray ionization (ESI) positive ion mode, with mass-to-charge ratios m / z 179.1→164.0 and 179.1→151.0 selected as the detection ion pairs;

[0015] S4. Result Determination:

[0016] Calculate the peak area ratio of the cis / trans isomers of methyl isoeugenol in the test solution. If the peak area ratio is ≤3.0, it is determined that there is no obvious risk of adulteration with Acorus tatarinowii. If the peak area ratio is >3.0, it is determined that there is a risk of adulteration with Acorus tatarinowii.

[0017] Preferably, the test solution in S1 is prepared as follows: Weigh about 0.5g of Acorus tatarinowii powder, place it in a stoppered conical flask, accurately add 25ml of methanol, seal tightly, weigh, sonicate for 45 minutes, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate.

[0018] Preferably, the concentration of the reference solution in S2 is 2.0 μg cis-methylisoeugenol and 2.0 μg trans-methylisoeugenol per 1 ml.

[0019] Preferably, the C18-PFP column is an octadecyl bonded silica-pentafluorophenyl mixed packing column.

[0020] Preferably, the parameters for the liquid chromatography-mass spectrometry (LC-MS) detection are: column temperature 40°C and injection volume 1 μL.

[0021] Preferably, the peak area ratio of the methyl isoeugenol cis / trans isomers of the pure Acorus tatarinowii is distributed in the range of 0.20 to 1, and the peak area ratio of the methyl isoeugenol cis / trans isomers of the pure Acorus tatarinowii is >50.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention accurately captures the inherent significant difference in the peak area ratio of methyl isoeugenol cis / trans isomers in *Acorus gramineus* and *Acorus calamus*. In pure *Acorus gramineus*, this ratio is stable between 0.20 and 1, while in pure *Acorus calamus*, it is greater than 50. The two ranges do not overlap. Using 10% adulteration as the standard, if the peak area ratio of the methyl isoeugenol cis / trans isomers is ≤3.0, then *Acorus gramineus* is determined to have no significant risk of adulteration with *Acorus calamus*; if the peak area ratio is >3.0, then *Acorus gramineus* is determined to have a risk of adulteration with *Acorus calamus*. Using this as the core indicator, accurate differentiation between *Acorus gramineus* and *Acorus calamus* can be achieved, avoiding the problem of difficulty in distinguishing by appearance and conventional chemical components at the component characteristic level, thus ensuring the specificity of adulteration detection from the root.

[0024] This invention provides clear parameter limits for the preparation processes of the test solution and reference solution. It also establishes standardized operating procedures for key parameters such as column specifications, mobile phase composition, gradient elution program, column temperature, injection volume, and mass spectrometry detection mode for liquid chromatography-mass spectrometry (LC-MS) detection. Furthermore, it offers optimized process parameters to avoid arbitrariness in the detection process. The detection steps are clear and the conditions are controllable, requiring no complex pretreatment procedures. It is easy for operators to master and can be carried out under routine laboratory conditions, making it suitable for batch sample screening. This invention achieves a quantitative assessment of the adulteration degree of *Acorus calamus* in *Acorus gramineus*, rather than a simple qualitative judgment, resulting in more objective and referential detection results.

[0025] This invention can be effectively applied to quality screening in the market circulation of Acorus tatarinowii and raw material acceptance in the production process, becoming an important technical support for the quality control of Acorus tatarinowii. It can effectively deter adulteration of Acorus tatarinowii in the Chinese medicinal materials market, curb the chaos of seeking improper profits by adulterating low-priced Acorus tatarinowii, and promote the standardized and healthy development of the Acorus tatarinowii Chinese medicinal materials market. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the chemical formulas of cis-methylisoeugenol and trans-methylisoeugenol of the present invention.

[0027] Figure 2 The structural diagram of trans-methylisoeugenol and the chromatogram of the cis-trans isomer reference standard MRM of methylisoeugenol are shown in the present invention.

[0028] Figure 3 This is the MRM chromatogram of the pure Acorus calamus sample of the present invention;

[0029] Figure 4 This is the MRM chromatogram of the pure water calamus sample of this invention;

[0030] Figure 5 MRM chromatogram of abnormal samples of Acorus calamus adulterated with water according to the present invention.

[0031] Figure 6 This is the MRM chromatogram of a normal sample of pure Acorus calamus. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1:

[0034] A method for detecting adulteration of Acorus calamus and Acorus gramineus in Acorus gramineus includes the following steps: I. Experimental materials

[0035] Test sample: Acorus tatarinowii powder (commercially available samples, including normal pure Acorus tatarinowii samples and abnormal samples suspected of being adulterated with water);

[0036] Reference standards: cis-methylisoeugenol reference standard (purity ≥98%), trans-methylisoeugenol reference standard (purity ≥98%);

[0037] Reagents: Methanol (chromatographic grade), acetonitrile (chromatographic grade), formic acid (chromatographic grade), ultrapure water;

[0038] Instruments: Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS), electronic analytical balance (accuracy 0.0001g), ultrasonic cleaner, stoppered conical flask (50mL), microporous filter membrane (0.22μm, organic phase), pipette, volumetric flask, etc.

[0039] II. Solution Preparation

[0040] (a) Preparation of test solution

[0041] Accurately weigh approximately 0.5g of Acorus tatarinowii powder and place it in a 50mL stoppered conical flask. Accurately add 25mL of methanol, seal tightly, and weigh. Place the conical flask in an ultrasonic cleaner and sonicate (300W power, 40kHz frequency) for 45 minutes. Remove and cool to room temperature, weigh again, and replenish the lost weight with methanol. Shake thoroughly and filter through a 0.22μm organic phase microporous membrane. Collect the filtrate to obtain the test solution, store in the dark, and use for later use.

[0042] (II) Preparation of reference solution

[0043] Accurately weigh appropriate amounts of cis-methylisoeugenol and trans-methylisoeugenol reference standards, place them in a 10 mL volumetric flask, dissolve them in methanol, and dilute to the mark. Shake well to prepare a mixed reference solution containing 2.0 μg of cis-methylisoeugenol and 2.0 μg of trans-methylisoeugenol per 1 mL. Filter the solution through a 0.22 μm organic phase microporous membrane. Store at 4℃ protected from light for later use.

[0044] III. Detection conditions for liquid chromatography-mass spectrometry (LC-MS)

[0045] Chromatographic conditions

[0046] Chromatographic column: C18-PFP column with octadecyl bonded silica gel and pentafluorophenyl mixed packing (particle size 1.7 μm, inner diameter 2.1 mm, length 100 mm);

[0047] Mobile phase: Phase A is 0.1% formic acid and ultrapure water, and Phase B is acetonitrile;

[0048] Gradient elution program: 0-15 min, Phase A 50%, Phase B 50%; 15-16 min, Phase A transitions from 50% to 10% linearly, Phase B transitions from 50% to 90% linearly; 16-20 min, Phase A 10%, Phase B 90%.

[0049] Column temperature: 40℃;

[0050] Injection volume: 1 μL;

[0051] Flow rate: 0.3 mL / min.

[0052] Mass spectrometry conditions: Ion source: Electrospray ionization (ESI), positive ion mode; Detection method: Multiple reaction monitoring (MRM); Characteristic detection ion pairs: For both cis and trans isomers of methyl isoeugenol, m / z 179.1→164.0 and m / z 179.1→151.0 were selected as quantitative and qualitative ion pairs; Mass spectrometry parameters: Ion source temperature 500℃, spray voltage 5500V, curtain gas, nebulizer gas, and auxiliary heating gas were all high-purity nitrogen, and the parameters were optimized to achieve symmetrical peak shape and the highest response value for the cis and trans isomers.

[0053] The characteristic peak areas of cis-methylisoeugenol and trans-methylisoeugenol in the chromatograms of each test sample solution were automatically calculated using a liquid chromatography-mass spectrometry (LC-MS) system with a matching workstation.

[0054] Accurately pipette 1 μL each of the mixed reference solution, pure Acorus calamus test solution, pure water Acorus calamus test solution, and suspected adulterated test solution, and inject them into the instrument under the above-described liquid chromatography-mass spectrometry (LC-MS) detection conditions. Acquire MRM chromatograms, identify the characteristic peaks of cis-methylisoeugenol and trans-methylisoeugenol in each chromatogram, and calculate the peak areas. The specific chromatographic analysis is as follows:

[0055] Reference standard chromatogram Figure 2 The spectrum clearly shows two characteristic peaks: cis-methylisoeugenol and trans-methylisoeugenol, with elution times of approximately 11.36 min and 11.37 min, respectively. The peak areas corresponding to the two ion pairs m / z 179.1→151.0 and m / z 179.1→164.0 are 1.379e+007 and 2.851e+006 (trans), respectively. The characteristic peaks are symmetrical and free from interference from other peaks, and can be used as a qualitative and quantitative reference for the identification of cis-trans isomers.

[0056] Spectrum of pure Acorus calamus (normal sample spectral). Figure 3 The elution times of the characteristic peaks of cis- and trans-methylisoeugenol in the chromatogram are consistent with those of the reference standard, with no additional impurities. The peak area ratio of cis- and trans-methylisoeugenol is stably distributed in the range of 0.20 to 1 (the ratio is 2.25 in this example), which is consistent with the component characteristics of pure Acorus tatarinowii.

[0057] Pure water calamus atlas, Figure 4 The chromatogram shows that the characteristic peak response values ​​of cis-methylisoeugenol are extremely high, while those of trans-methylisoeugenol are extremely low. The peak area ratio of cis / trans-methylisoeugenol is much greater than 50 (in this example, the ratio is 10.01 / 2.26), which is significantly different from that of pure Acorus tatarinowii. There is no overlap between the two peak areas.

[0058] Abnormal sample spectrum (Acorus calamus mixed with water) Figure 5The characteristic peak response value of cis-methyl isoeugenol in the spectrum was significantly higher than that of pure Acorus tatarinowii, while the characteristic peak response value of trans-methyl isoeugenol showed no significant change. The peak area ratio of cis / trans-methyl isoeugenol was greater than 3.0, exceeding the limit for determining no adulteration risk, indicating that Acorus tatarinowii was added to the sample.

[0059] If the peak area ratio of cis / trans-methylisoeugenol in the test sample is ≤3.0, then the Acorus calamus sample is determined to have no obvious risk of adulteration with Acorus calamus.

[0060] If the peak area ratio of cis / trans-methylisoeugenol in the test sample is >3.0, then the sample of Acorus calamus is considered to have a risk of adulteration with Acorus calamus.

[0061] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0062] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting adulteration of Acorus calamus in Acorus gramineus, characterized in that, Using the peak area ratio of the cis / trans isomers of methyl isoeugenol as the criterion, the method was analyzed using liquid chromatography-mass spectrometry (LC-MS), specifically including the following steps: S1. Preparation of the test solution: Weigh 0.5-1g of Acorus tatarinowii powder, add 25-50mL of methanol, extract by sonication for 30 minutes, filter, and take the filtrate to obtain the test solution; S2. Preparation of reference solution: Using methanol as a solvent, cis-methylisoeugenol and trans-methylisoeugenol reference standards were prepared into reference solutions with concentrations of 1-10 μg / ml. S3, Liquid chromatography-mass spectrometry (LC-MS) detection: The test solution S1 and the reference solution S2 were injected into a liquid chromatography-mass spectrometry (LC-MS) instrument for detection. The detection parameters of the LC-MS instrument were as follows: the column was a C18-PFP column with a particle size of 1.7 μm, an inner diameter of 2.1 mm, and a length of 100-150 mm; the mobile phase A was 0.1% formic acid solution, and the mobile phase B was acetonitrile; the gradient elution program was as follows: 0-15 min, 50% A phase, 50% B phase; 15-16 min, linear transition from 50% A phase to 10% B phase, and linear transition from 50% B phase to 90% B phase; 16-20 min, 10% A phase, 90% B phase; the column temperature was 30-40℃; the injection volume was 1-10 μL; a triple quadrupole mass spectrometer was used, and multiple reaction monitoring (MRM) was performed in electrospray ionization (ESI) positive ion mode, with mass-to-charge ratios m / z 179.1→164.0 and 179.1→151.0 selected as the detection ion pairs; S4. Result Determination: Calculate the peak area ratio of the cis / trans isomers of methyl isoeugenol in the test solution. With 10% adulteration as the standard, if the peak area ratio is ≤3.0, it is determined that there is no obvious risk of adulteration with Acorus tatarinowii. If the peak area ratio is >3.0, it is determined that there is a risk of adulteration with Acorus tatarinowii.

2. The method for detecting adulteration of Acorus calamus in Acorus gramineus according to claim 1, characterized in that: The test solution in S1 is prepared as follows: Weigh about 0.5g of Acorus tatarinowii powder, place it in a stoppered conical flask, accurately add 25ml of methanol, seal tightly and weigh, sonicate for 45 minutes, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate.

3. The method for detecting adulteration of Acorus calamus in Acorus gramineus according to claim 1, characterized in that: The concentration of the reference solution in S2 is 2.0 μg cis-methylisoeugenol and 2.0 μg trans-methylisoeugenol per 1 ml.

4. The method for detecting adulteration of Acorus calamus in Acorus gramineus according to claim 1, characterized in that: The C18-PFP column is an octadecyl-bonded silica gel-pentafluorophenyl mixed packing column.

5. The method for detecting adulteration of Acorus calamus in Acorus gramineus according to claim 1, characterized in that: The parameters for the liquid chromatography-mass spectrometry (LC-MS) detection are: column temperature 40℃, injection volume 1μL.

6. The method for detecting adulteration of Acorus calamus in Acorus gramineus according to claim 1, characterized in that: The peak area ratio of the cis / trans isomers of methyl isoeugenol in pure Acorus tatarinowii is distributed in the range of 0.20 to 1, and the peak area ratio of the cis / trans isomers of methyl isoeugenol in pure Acorus tatarinowii is >50.

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