Method for rapidly detecting whether cortex mori radicis is doped in cortex mori radicis medicinal material based on characteristic markers

Abstract

The invention discloses a method for rapidly detecting whether cortex mori radicis is doped in a cortex mori radicis medicinal material based on a characteristic marker, and belongs to the field of traditional Chinese medicine detection. According to the method disclosed by the invention, sanggenone C is determined as a characteristic chemical marker for distinguishing the cortex mori from the cortex mori radicis for the first time, and detection is carried out in an optimized positive ion multi-reaction monitoring mode by adopting a liquid chromatography-tandem mass spectrometry technology. Chromatographic behaviors and mass spectrum characteristics of a sample are investigated, and the content of sanggenone C is used as a judgment threshold value, so that whether the mulberry bark is doped in the white mulberry root bark or not is quickly and accurately identified. The method disclosed by the invention is high in specificity, high in sensitivity and simple and convenient to operate, can effectively solve the technical problem that the white mulberry root-bark and the Chinese mulberry bark are difficult to distinguish by a traditional method, and provides a reliable technical means for quality control and market supervision of the white mulberry root-bark medicinal material.

Description

Technical Field cathayana , Morus alba , Morus , Morus alba

[0001] The present invention relates to a method for quickly detecting whether mulberry bark药材 is adulterated with bark of Morus cathayana, belonging to the field of traditional Chinese medicine detection. Background Art

[0002] Mulberry bark is a commonly used traditional Chinese medicine. The Chinese Pharmacopoeia stipulates that its source is the dried root bark of the mulberry plant Morus alba ( Morus alba L.), so Morus alba ( Morus alba L.) is the legal source of mulberry bark药材. However, due to the fact that the root bark of Morus cathayana ( Morus cathayana Hemsl.) is also included in the local standards in many places such as Chongqing City, Hunan Province, and Zhejiang Province, and its medicinal material properties, especially after being cut into slices, are very similar to those of mulberry bark. It is extremely difficult to effectively distinguish by traditional methods such as morphological identification and microscopic identification, resulting in a large number of cases in the market where bark of Morus cathayana冒充 and adulterates mulberry bark.

[0003] Textual research on Chinese herbal medicines shows that the medicinal variety of mulberry bark does not record bark of Morus cathayana. Modern chemical composition research shows that there are significant differences between bark of Morus cathayana and mulberry bark, and its medicinal safety and effectiveness are still controversial. Adulterating mulberry bark with bark of Morus cathayana may seriously affect the quality, efficacy and medication safety of mulberry bark药材. Therefore, it is necessary to study a simple, rapid, accurate, stable and reliable detection method for adulterated bark of Morus cathayana in mulberry bark药材 and its slices to ensure the safety and effectiveness of clinical medication.

[0004] References [1] Zheng Tianbi. Research on the Quality Evaluation of Mulberry Bark Based on Chemical Constituents [D]. Jiangsu University, 2020. [2] Wei, Hua, et al. "Review of Bioactive Compounds from Root Barks of Morus Plants (Sang-Bai-Pi) and Their Pharmacological Effects [J]. Cogent Chemistry, Vol. 2, no. 1, 2016, p. 1212320, https: / / doi.org / 10.1080 / 23312009.2016.1212320. Summary of the Invention The purpose of the present invention is to overcome the deficiencies of the prior art and provide a detection method based on characteristic markers, which is simple to operate and accurate in results, for quickly discriminating whether mulberry bark药材 or its slices are adulterated with bark of Morus cathayana, so as to ensure the safety and effectiveness of clinical medication.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: A method for rapid detection of whether *Morus alba* root bark is adulterated with *Morus alba* bark based on characteristic markers includes: using liquid chromatography-tandem mass spectrometry (LC-MS) to detect whether the sample solution contains *Morus alba* root ketone C, a characteristic marker of *Morus alba* bark, through multiple reaction monitoring (MRM); wherein the MRM mode includes at least monitoring ion pairs m / z 709.2 → 641.2, and using a declustering voltage of 100 V-120 V and a collision voltage of 25 eV-35 eV.

[0006] Preferably, the multiple reaction monitoring mode further includes simultaneously monitoring at least one of the following ion pairs for qualitative confirmation of morula ketone C: m / z 709.2 → 339.1, declustering voltage is 90 V-110 V, collision voltage is 20 eV-30 eV; m / z 709.2 → 531.2, declustering voltage is 80 V-100 V, collision voltage is 30 eV-40 eV.

[0007] Preferably, the criterion for determining whether a sample is doped with mulberry bark is as follows: if the signal of the monitored ion pair m / z 709.2 → 641.2 is detected, and the quantitative result of mulberry root ketone C calculated based on the ion pair signal is higher than 0.006% (mass fraction), then the sample is determined to be doped with mulberry bark.

[0008] Preferably, the liquid chromatography conditions include: Column: C18 column packed with octadecylsilane-bonded silica gel; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution containing 0.01% to 0.1% (v / v) formic acid; Gradient elution was used.

[0009] More preferably, the gradient elution procedure is as follows: 0→25 minutes, mobile phase A 45%→50%; 25→26 minutes, mobile phase A 50%→45%; 26→30 minutes, mobile phase A 45%.

[0010] Preferably, the liquid chromatography conditions further include: a flow rate of 0.2-0.3 ml / min and a column temperature of 30-40℃.

[0011] Preferably, the mass spectrometry conditions include: Ion source: Electrospray ionization source; Ionization mode: Positive ion mode; Ion source temperature: 450-550℃; Air curtain airflow velocity: 35-45 psi / s; Spray airflow rate: 40-50 psi / s.

[0012] Preferably, the method for preparing the sample solution includes: taking sample powder, adding methanol for ultrasonic extraction for 30 minutes, filtering, diluting, filtering through a microporous membrane, and then injecting the sample.

[0013] More preferably, the methanol is pure methanol, the extraction material-to-liquid ratio is 1:200 (g / ml), and the extraction time is 30 minutes.

[0014] Compared with the prior art, the present invention has the following significant advantages: High specificity and accuracy: This study is the first to discover and confirm that mulberry root ketone C is a characteristic marker distinguishing Chinese mulberry bark from white mulberry bark. Employing a multiple reaction monitoring (MRM) mode, the study utilizes dual selective screening with both precursor and characteristic daughter ions to effectively eliminate interference from other components in the complex matrix of white mulberry bark, resulting in highly specific and accurate detection results.

[0015] High sensitivity: Based on optimized LC-MS / MS conditions, the method has a low detection limit, enabling reliable detection of trace adulteration (as low as 0.006%), meeting the needs of market supervision for identifying trace adulteration.

[0016] Fast and efficient: Simple sample pretreatment, short chromatographic analysis time (within 30 minutes), combined with the data acquisition speed of MRM mode, enables rapid screening of large batches of samples.

[0017] The results are objective and reliable: mass spectrometry is used for detection, and characteristic ion pairs and clear content thresholds are used as the basis for judgment, avoiding misjudgments caused by subjective experience in traditional methods. The data are objective and reproducible, providing solid technical support for quality control and standard setting.

[0018] With broad application prospects, this method can be used not only for the detection of adulteration in finished medicinal materials and decoction pieces, but also for quality supervision in all aspects of the production, circulation and use of Chinese medicinal materials. It has important practical value and social benefits for regulating the market and ensuring the safety and efficacy of Chinese medicine. Attached Figure Description

[0019] Figure 1 Total ion chromatograms of mulberry bark from three sources (chicken mulberry bark, Chinese mulberry bark, and mulberry bark).

[0020] Figure 2 OPLS-DA analysis plots of mulberry bark from three sources (left: principal component plot; right: model test plot).

[0021] Figure 3 OPLS-DA analysis plots of the mulberry bark group and the Chinese mulberry bark group (left: principal component plot; right: model test plot).

[0022] Figure 4: Primary and secondary mass spectrometry information of mulberry root ketone C (left: TIC image; right: MSMS image).

[0023] Figure 5 Specificity investigation chromatogram (monitoring ion pair: m / z 709.2 → 641.2).

[0024] Figure 6 Specificity investigation chromatogram (monitoring ion pair: m / z 709.2 → 339.1).

[0025] Figure 7 Specificity investigation chromatogram (monitoring ion pair: m / z 709.2 → 531.2).

[0026] Figure 8 Results of linearity study of mulberry root ketone C (standard curve).

[0027] Figure 9 Results of the analysis of significant differences between epidermal characteristics and mulberry root ketone C content. Detailed Implementation

[0028] The present invention will now be described in detail through specific embodiments.

[0029] Example 1: Screening of characteristic components of mulberry bark and Chinese mulberry bark based on UPLC-Q-TOF combined with PCA 1. Experimental Materials 1.1 Instruments and Reagents The equipment used was a Waters Acquity UPLC XEVO-G2 Q-TOF high-resolution time-of-flight mass spectrometer, a Mettler Toledo 0.01 g / 10,000 electronic balance, and an LC-350A ultrasonic herbal medicine processor (Shandong Jining Luchao Ultrasonic Equipment Co., Ltd.). Water was purified using a Millipore-Q Reference system; methanol and acetonitrile were of chromatographic grade; and formic acid was of mass spectrometric grade.

[0030] 1.2 Sample Source A total of 51 batches of mulberry bark samples were used for screening quality markers. All samples were collected by the research group. After morphological identification and DNA molecular identification, the original source was identified. For details, please refer to Table 1 Sample Information Table.

[0031] Table 1 Sample Information Table

[0032] 1.3 Source of reference standard A total of 23 reference standards were purchased. Detailed information is shown in Table 2.

[0033] Table 2. Reference Standard Information

[0034] 2. Experimental Methods 2.1 Liquid chromatography-mass spectrometry (LC-MS) conditions Liquid chromatography conditions: ACQUITYUPLCBEHC18 column (100 mm × 2.1 mm, 1.7 μm); mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile (B); gradient elution: 0–20 min, 3% → 10% B; 20–45 min, 10% → 35% B; 45–80 min, 35% → 75% B; 80–81 min, 75% → 95% B; 81–82 min, 95% B → 3% B; 82–85 min, 3% B. Detection wavelengths: 254 nm, 280 nm, 340 nm; column temperature: 35 °C; injection volume: 1 μl; flow rate: 0.3 ml / min.

[0035] Mass spectrometry conditions: Electrospray ionization (ESI+) was used, with a nebulizer gas (N2) flow rate of 600 L / h, a desolvation gas temperature of 350℃, an ion source temperature of 100℃, and a capillary voltage of 2.0 kV. The sampling cone voltage was 40 V. Detection was performed in MSE scan mode, with a collision energy of 20-40 V, a cone gas flow rate of 50 L / h, and a scan range of m / z 100-1500. Leucine-enkephalin (m / z: 556.2771(+)) was used as an external standard (Lock Spray™) for real-time mass correction, with a flow rate of 5 μl / min.

[0036] 2.2 Preparation of the test solution Take approximately 1.0 g of the above sample powder (passed through a No. 2 sieve), weigh it accurately, add 20 ml of 70% methanol accurately, sonicate for 30 min, filter, and the test solution is obtained.

[0037] 2.3 Preparation of reference solution Take appropriate amounts of the above reference standards into the volumetric flasks shown in the table below, dissolve them in methanol, and dilute to the mark to prepare the reference standard stock solution; measure appropriate amounts of the reference standard stock solution to prepare a mixed solution containing 10 μg of the above reference standard per 1 ml.

[0038] 2.4 Chemometric Analysis Mass spectrometry data were preprocessed with MarkerLynx to obtain the component distribution information of Marker retention time-mass-charge ratio (tR_m / z), and this information was used for differential Marker analysis using SIMCA13.0 software and SPSS26.0.

[0039] 3. Results 3.1 Identification of the main chemical components of mulberry bark The solutions were prepared according to steps “2.1~2.3” above, and mass spectra were collected. The total ion chromatograms of the three sources of mulberry bark are shown below. Figure 1 .

[0040] UPLC-Q-TOF-MS / MS data were processed using Masslynx 4.1. The major molecular ion peaks were assigned by combining Mass values ​​of standards, UV information, retention times from references, and molecular formula matching software. The results are shown in Table 3.

[0041] Table 3 Peak component attribution information

[0042] *: Comparison with reference standard.

[0043] 3.2 Differential Component Analysis Fifty-one batches of samples from three sources were divided into three groups based on identification results: Mulberry Bark (SBP), Chinese Mulberry Bark (HSP), and Chicken Mulberry Bark (JSP). Partial Least Squares-Discriminant Analysis (OPLS-DA) was used to extract the components of the independent variable X and the dependent variable Y, and then the correlation between the components was calculated. Figure 2 As a result, the mulberry bark group and the chicken mulberry bark group could not be completely separated. The model substitution test showed that the model prediction index Q2=0.442<0.5 and the dependent variable fit index R2Y=0.62, indicating that the model fit was poor and regrouping analysis was required.

[0044] Principal component plots and total mass chromatograms show that the chromatographic peaks of chicken mulberry bark and white mulberry bark are relatively similar. The chicken mulberry bark group was combined with the white mulberry bark group, and then compared with the Chinese mulberry bark group for OPLS-DA analysis. (See attached image). Figure 3 The results showed that the mulberry bark group and the Chinese mulberry bark group were completely separated. The model permutation test showed that the model prediction index Q2=0.604>0.5 and the dependent variable fit index R2Y=0.885, indicating that the model fit was good. Differential metabolite analysis was then performed using this group.

[0045] MetaboAnalystR 1.0.1 was used to analyze the differential metabolites in the SBP and HSP groups. Feature data points with parameter VIP>1 and ANOVA one-way variance P<0.05 were selected. The results are shown in Table 4.

[0046] Table 4. Information on differentially expressed metabolites between the Mulberry Bark Group and the Chinese Mulberry Bark Group

[0047] The violin plot further shows the distribution of each difference data point in the two groups. Eight pairs of ions were found to be significantly higher in the Huasangpi group than in the Sangbaipi group. These eight pairs of ions originated from three components: Cathayanon A, mulberry root ketone C, and mulberry root ketone A. Among them, Cathayanon A and mulberry root ketone C are epimers of each other and can be used as characteristic components that distinguish Huasangpi from Sangbaipi.

[0048]

[0049] The violin diagram further reveals 10 components, including morin A, morin G, morin T, morin B, morin H, morin G, cyclomorin, morin, 5-hydroxycoumarin-7-O-β-D-glucopyranoside, and oxidized resveratrol-3-O-glucopyranoside. These components are present in significantly higher amounts in the white mulberry bark group than in the Chinese mulberry bark group, and can be used as characteristic components to distinguish white mulberry bark from Chinese mulberry bark.

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] 3.3 Characteristic Component Analysis The characteristic components of mulberry bark and Chinese mulberry bark differ significantly. Chinese mulberry bark contains various mulberry root ketones, such as mulberry root ketone C and cathayanonon A, which can be used as indicators to detect adulteration of mulberry bark with Chinese mulberry bark. Mulberry bark mainly contains three categories of components: first, stilbene series components with oxidized resveratrol as the aglycone, such as morin A, morin F, and oxidized resveratrol-2-O-glucoside; second, flavonoid components, such as morin G, morinol, and cyclomorin; and third, DA-type adducts, such as morin H and morin T. All three categories of components can be used as indicators for the quality control of mulberry bark.

[0056] Example 2: Determination of Morus root ketone C content in Chinese mulberry bark by liquid chromatography-mass spectrometry 1. Instrument, reagent and sample information 1.1 Instruments LC30A-AB 4500 Q-trap LC-MS / MS (Shanghai Aibocaisi Analytical Instruments Trading Co., Ltd.); Waters G2 Q-TOF LC-MS / MS (Waters Corporation); LC-350A Ultrasonic Instrument (Luchao Instrument Factory, Shizhong District, Jining City); Mettler-Toledo XP205 Analytical Balance; METTLER TOLEDO XP204 Electronic Balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.).

[0057] 1.2 Reagents, consumables, and reference standards Chromatographic columns: Waters BEH C18 (2.1 mm × 100 mm, 1.7 μm); Phenomenex Kinetex XB-C18 (2.1 × 100 mm, 1.7 µm). Acetonitrile and formic acid were of mass spectrometry grade, water was primary purified water, and all other reagents were of analytical grade.

[0058] Sanggenone C (CAS: 80651-76-9, Purity: 99.3%, purchased from CATO).

[0059] 1.3 Sample Information Information on the experimental samples is shown in Tables 11 and 12.

[0060] 2. Establishment of detection methods 2.1 Selection of Detection Ion Pairs Based on the primary and secondary mass spectrometry information of mulberry root ketone C reference standard (see...) Figure 4 As can be seen, the molecular ion peak of mulberry root ketone C (C40H36O12) is m / z 709.2 [M+H]+. The molecule loses isoprene (-C5H8) to form a fragment at m / z 641.2. This m / z 641.2 fragment loses one molecule of H2O to form a fragment at m / z 623.2, and loses one molecule of resorcinol residue to form a fragment at m / z 531.2. Intramolecular fragmentation of the molecular ion peak forms fragments at m / z 339.1 and m / z 302.1. Based on the abundance of fragment ions, m / z 709.2→641.2, m / z 709.2→339.1, and m / z 709.2→531.2 were selected as detection ion pairs.

[0061] 2.2 Chromatographic and mass spectrometry conditions and system suitability tests Chromatographic conditions and system suitability test: Phenomenex Kinetex XB-C18 (2.1 × 100 mm, 1.7 µm) column; gradient elution with acetonitrile as mobile phase A and 0.01% formic acid solution as mobile phase B; flow rate 0.25 ml / min; column temperature 35℃.

[0062] Table 5 Gradient elution program

[0063] An electrospray ionization source was used in MRM(+) positive ion acquisition mode. Ionization voltage: 4500 V; ion source temperature: 500℃; curtain gas flow rate: 40 psi / s; spray gas flow rate: 45 psi / s; auxiliary heating gas flow rate: 35 psi / s. Detection parameters such as ion pair and collision energy in MRM mode are shown in Table 6. Injection volume: 1 µl.

[0064] Table 6. Detected ion pairs and detection parameters

[0065] 2.3 Solution Preparation Preparation of reference solution: Take an appropriate amount of mulberry root ketone C reference standard, accurately weigh it, place it in a volumetric flask, and add methanol to prepare a solution containing 0.03 µg of each per 1 ml.

[0066] Preparation of the test solution: Accurately weigh approximately 0.25 g of the test sample powder (passed through a No. 2 sieve), place it in a stoppered conical flask, accurately add 50 ml of methanol, weigh, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, cool, weigh again, replenish the lost weight with methanol, shake well, filter, accurately measure 1 ml of the filtrate, place it in a 10 ml volumetric flask, add methanol to the mark, shake well. Filter through a microporous membrane (0.22 μm), and collect the filtrate to obtain the test solution.

[0067] Assay: Accurately pipette 1 µl of the reference solution and the test solution into a liquid chromatograph-tandem mass spectrometer and measure accordingly.

[0068] 2.4 Selection of Extraction Method 2.4.1 Selection of Extraction Solvent Take the same sample powder (passed through a No. 2 sieve) and prepare the test sample according to the method in "2.3 Preparation of Test Sample Solution". The extraction efficiency of different solvents was investigated using the content of mulberry root ketone C as an indicator. The results showed that methanol had a higher extraction efficiency, therefore methanol was chosen as the extraction solvent.

[0069] Table 7. Investigation of Extraction Solvents

[0070] 2.4.2 Examination of extraction time Take the same batch of sample powder (passed through a No. 2 sieve) and prepare the test sample according to the method in "2.3 Preparation of Test Sample Solution". The extraction efficiency of different ultrasonic times was investigated using the content of mulberry root ketone C as an indicator. The results showed that the extraction efficiency did not increase with the extension of ultrasonic time; therefore, an extraction time of 30 minutes was selected.

[0071] Table 8. Examination of Extraction Time

[0072] 2.4.3 Investigation of the extraction material-to-liquid ratio Take the same batch of sample powder (passed through a No. 2 sieve) and prepare the test sample according to the method in "2.3 Preparation of Test Sample Solution". The extraction efficiency of different material-to-liquid ratios was investigated using the content of mulberry root ketone C as an indicator. The results showed that a material-to-liquid ratio of 1:200 was optimal.

[0073] Table 9. Investigation of the extraction material-liquid ratio

[0074] 2.5 Methodological Research 2.5.1 Specificity Research Take the following solutions: mulberry root ketone C reference solution, mulberry bark (SBP-3, negative sample, batch number: 20241119, Xinghua Township, Hong'an County, Hubei Province), and mulberry bark (SBP71, positive sample, batch number: 20250416, Lijia Village, Longba Town, Zhuxi County) test solution, and 1.00 ml of [mulberry bark (SBP-3, negative matrix sample) + mulberry root ketone C standard (concentration 111.8118 μg / ml)]. Determine the corresponding ion pairs according to the methods described in sections "2.1" and "2.2". Figure 5-7 It can be seen that the above characteristic ion pairs were not extracted from mulberry bark (negative sample), while the control, mulberry bark (positive sample), and mulberry bark matrix (negative) + standard solution could accurately detect three pairs of characteristic ions, indicating that the method has no interference from negative samples and has good specificity.

[0075] 2.5.2 Linear Relationship Accurately weigh 5.63 mg of mulberry root ketone C reference standard, place it in a 50 mL volumetric flask, dissolve it in methanol, and dilute to the mark. Shake well to obtain the stock solution (concentration 111.8118 μg / mL). Take an appropriate amount of the stock solution and dilute it sequentially to obtain concentrations of 4.4725, 1.7765, 0.8882, 0.3553, 0.1776, and 0.1421 μg / mL. Inject 1 μL of each solution into a high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. Prepare a standard curve with the peak area of ​​the reference standard as the ordinate and the concentration of the reference standard as the abscissa. Plot the standard curve with the concentration of the reference standard solution as the abscissa. The results show that mulberry root ketone C has a good linear relationship between 0.1421 and 4.4725 ng. The standard curve is shown in [Figure number missing]. Figure 8 .

[0076] 2.5.3 Accuracy (Recovery Rate) Approximately 0.25g of the same batch of sample powder (passed through a No. 2 sieve) was accurately weighed and added to the reference standard (i.e., accurately adding 0.5ml, 1.0ml, and 2.0ml of a 0.1118mg / ml mulberry root ketone C reference solution) at ratios of 1:0.5, 1:1, and 1:1.5 respectively before preparing the test solution. The test sample was then prepared according to the method in "2.3 Preparation of Test Solution". The sample was injected and the recovery rate and RSD of mulberry root ketone C were calculated. The average recovery rate was 105.63%, and the RSD was 4.27%.

[0077] 2.5.4 Repeatability Test Take 0.25 g of the same batch of mulberry bark powder (passed through a No. 2 sieve) and prepare 6 test samples according to the method in "2.3 Preparation of Test Sample Solution". Inject each sample separately for determination, and calculate the RSD of mulberry root ketone C content as 0.9%.

[0078] 2.5.5 Stability Test Take 0.25 g of the same batch of mulberry bark powder (passed through a No. 2 sieve) and prepare the test sample according to the method in "2.3 Preparation of Test Sample Solution". The test sample was measured at 0 h, 4 h, 8 h, 12 h, and 24 h. The RSD of the mulberry root ketone C content was calculated to be 1.3%, indicating that the test sample solution was stable within 24 h.

[0079] 2.5.6 Sensitivity Take the mulberry root ketone C reference standard and dilute it to an appropriate concentration so that the instrument's injection signal-to-noise ratio (S / N) is 3, which is the instrument's limit of detection. When the instrument's detection concentration is 0.0711 μg / ml, the signal-to-noise ratio (S / N) is 21.1, and the calculated limit of detection for mulberry root ketone C in the sample is 20 μg / g. When the signal-to-noise ratio (S / N) is 10:1, the limit of quantitation is 60 μg / g.

[0080] 2.5.7 Durability Test Take 0.25 g of Huasang bark powder (SBP71, batch number: 20250416, Lijia Village, Longba Town, Zhuxi County) (passed through a No. 2 sieve) and prepare the test sample according to the method in "2.3 Preparation of Test Sample Solution". Robustness tests were conducted using two different chromatographic columns on two different models of instruments. The results are shown in Table 10. The average content was 0.133%, and the SD was 1.89%, demonstrating the good robustness of this method.

[0081] Table 10 Durability Test Results

[0082] In summary, the methodological investigation was conducted in accordance with the "Guiding Principles for Validation of 9101 Analytical Methods" in Part IV of the 2025 edition of the Chinese Pharmacopoeia, and the method meets the relevant requirements.

[0083] Example 3: Application of the method for detecting adulteration in mulberry bark medicinal materials and processed slices 1. Detection of Morus root ketone C in primary samples The test solution was prepared according to the method described in section 2.3 of Example 2. The samples of 8 batches of Chinese mulberry bark (including the same batch of rough bark) and 31 batches of mulberry root bark (including the same batch of rough bark) were tested according to the conditions described in section 2.2. The results showed that morinone C was not detected in any of the 31 batches of mulberry root bark or rough bark, but was detected in all 8 batches of Chinese mulberry bark and rough bark. The morinone C content in Chinese mulberry bark ranged from 0.09% to 0.42%, with an average of 0.19%, while the morinone C content in rough bark ranged from 1.05% to 1.79%, with an average of 1.35%. The results are shown in Tables 11 and 12.

[0084] Table 11 Content of Morus root ketone C in the original sample, Morus bark

[0085] Table 12 Content of Morus root ketone C in mulberry bark samples

[0086] 2. Detection of Morus root ketone C in sampled specimens 257 batches of mulberry bark samples were prepared into test solutions using the method described in section 2.3 of Example 2, and tested under the conditions described in section 2.2. Mulberry root ketone C was detected in 213 batches of samples, indicating adulteration with mulberry bark. Table 13 lists some of the results from the 257 batches of samples.

[0087] Table 13 Detection of Morus root ketone C in sampled samples

[0088] Note: A: Fully peeled, with less than 10% of the outer skin remaining, and the outer skin is white / yellowish-white; B: Partially peeled, with approximately 10%-80% of the outer skin remaining, and residual brownish-yellow / orange-yellow outer skin is visible; C: Not peeled, with more than 80% of the outer skin remaining, and the outer skin is brownish-yellow or orange-yellow.

[0089] The results of the original sample analysis showed that the concentration of mulberry root ketone C was significantly higher in the outer bark of *Morus alba* root than in the outer bark of *Morus alba* bark. These 213 batches of samples containing mulberry root ketone C were statistically divided into three categories (A, B, and C) based on the degree of peeling (i.e., the extent of peeling). The statistical results are shown in Table 14. A statistical analysis of the significant differences among the three groups of samples with different degrees of peeling was performed, and the results are shown below. Figure 9 Within a p-value confidence interval of 0.05, the difference in mulberry root ketone C was significant among fully peeled (Group A), partially peeled (Group B), and unpeeled (Group C). The value of fully peeled mulberry bark was significantly lower than that of unpeeled and partially peeled processed pieces. The average value for fully peeled bark was 0.07%, nearly 10 times higher than the proposed limit of 0.006%. Therefore, this method and limit can accurately determine whether mulberry bark is adulterated with mulberry root ketone C.

[0090] Table 14. Statistical analysis of the content of mulberry bark and root ketone C in the sampled samples.

Claims

1. A method for rapid detection of whether *Morus alba* bark is adulterated with *Morus alba* bark based on characteristic markers, characterized in that: Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to detect whether the sample solution contained morin C, a characteristic marker of mulberry bark, using multiple reaction monitoring (MRM) mode. The MRM mode included monitoring ion pairs from m / z 709.2 to 641.2, and using a declustering voltage of 100 V-120 V and a collision voltage of 25 eV-35 eV.

2. The method according to claim 1, characterized in that, The multiple reaction monitoring mode also includes simultaneously monitoring at least one of the following ion pairs for qualitative confirmation of morula ketone C: m / z 709.2 → 339.1, declustering voltage is 90 V-110 V, collision voltage is 20 eV-30 eV; m / z 709.2 → 531.2, declustering voltage is 80 V-100 V, collision voltage is 30 eV-40 eV.

3. The method according to claim 1 or 2, characterized in that: If the signal of the monitored ion pair m / z 709.2 → 641.2 is detected, and the quantitative result of mulberry root ketone C calculated based on the ion pair signal is higher than 0.006%, it is determined to be doped with Chinese mulberry bark.

4. The method according to claim 1, characterized in that, The liquid chromatography conditions include: Column: C18 column packed with octadecylsilane-bonded silica gel; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution containing 0.01% to 0.1% formic acid; Gradient elution was used.

5. The method according to claim 4, characterized in that, The gradient elution procedure is as follows: 0→25 minutes, mobile phase A 45%→50%; 25→26 minutes, mobile phase A 50%→45%; 26→30 minutes, mobile phase A 45%.

6. The method according to claim 4, characterized in that, The liquid chromatography conditions also include: flow rate 0.2-0.3 ml / min; column temperature 30-40℃.

7. The method according to claim 1, characterized in that, The mass spectrometry conditions include: Ion source: Electrospray ionization source; Ionization mode: Positive ion mode; Ion source temperature: 450-550℃; Air curtain airflow velocity: 35-45 psi / s; Spray airflow rate: 40-50 psi / s.

8. The method according to claim 1, characterized in that, The method for preparing the sample solution includes: taking sample powder, adding methanol for ultrasonic extraction for 30 minutes, filtering, diluting, filtering through a microporous membrane, and then injecting the sample.

9. The method according to claim 8, characterized in that: The methanol is pure methanol, the extraction ratio is 1:200 (g / ml), and the extraction time is 30 minutes.

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