Construction method and application of aspongopus characteristic peptide spectrum

Abstract

The invention discloses a construction method and application of an aspongopus characteristic peptide spectrum, and the method comprises the following steps: preparing an aspongopus test sample solution and an aspongopus control medicinal material reference substance solution, analyzing by an ultra-high performance liquid chromatography-mass spectrometer, and determining the following six characteristic ion peaks with double-charge parent ions from a peptide spectrogram of the test sample, the characteristic peptide spectrum of the aspongopus is as follows: m / z is 469.263 to 522.312, 617.811 to 731.402, 559.780 to 760.400, 649.800 to 785.350, 623.840 to 430.260, and 789.926 to 1167.596. The characteristic peptide spectrum of the aspongopus can be used for preparing the aspongopus. According to the method, true and false identification of the aspongopus medicinal material, the standard decoction and the formula granules can be simultaneously realized, and adulterants such as jatropha curcas, lysimachia plicata and amblycoris nuda can be effectively distinguished. The method is high in specificity, good in reproducibility, easy and convenient to operate and high in detection efficiency, the technical problem that a traditional identification method is not suitable for a water extraction preparation is solved, and reliable guarantee is provided for quality control of the aspongopus formula granules.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for constructing a characteristic peptide spectrum of *Stachys chinensis* and its application. Background Technology

[0002] Nine-scented insects are insects belonging to the family Stink bug (Vibrio vulgaris). Coridius chinensis The dried form of *Dallas*. It has the effects of regulating qi and relieving pain, warming the middle jiao and tonifying yang, and is often used for stomach cold and distending pain, liver and stomach qi pain, kidney deficiency and impotence, and lower back and knee pain. *Nine-fragrant insect* formula granules are made from *Nine-fragrant insect* slices through modern technology of extraction, concentration, drying, and granulation, preserving the clinical effects of *Nine-fragrant insect* while also considering portability. The 2025 edition of the *Pharmacopoeia of the People's Republic of China* mainly identifies *Nine-fragrant insect* based on its morphological characteristics and thin-layer chromatography, but this identification method has significant limitations for formula granules that have undergone water extraction and processing.

[0003] Nine-scented bugs belong to the family Stink bugs. This group of insects has many varieties with similar characteristics and is often used interchangeably in traditional Chinese medicine. Currently, there are few reports on the identification of adulterants and adulterants of nine-scented bugs. Tan Siyin et al. established a multi-site specific polymerase chain reaction (PCR) method for simultaneously identifying nine-scented bugs and their adulterants. By analyzing the cytochrome C oxidase subunit I (COI) gene sequence of nine-scented bugs and their adulterants, screening for single nucleotide polymorphism (SNP) sites, designing specific identification primers, and optimizing the PCR reaction system, they were able to accurately identify nine-scented bugs, *Hemiberlesia lataniae*, *Tetracentrum brevicornum*, *Trichoderma brevicornum*, and *Trichoderma gracilis* simultaneously. This provides a reference for research on adulteration and adulteration of nine-scented bug medicinal materials, as well as for the quality of medicinal materials and the safety of medication use (Tan Siyin, Pan Liye, Song Ye, et al. Simultaneous identification of nine-scented bugs and their adulterants by multi-site specific PCR [J]. Journal of Guangdong Pharmaceutical University, 2025, 41(04):119-128.). Xiong Xiran et al. identified *Amanita muscaria* medicinal materials based on DNA barcoding technology, analyzed the classification relationship between species of *Amanita muscaria* medicinal materials at the molecular level, obtained the COI gene sequence using PCR amplification and sequencing methods, and used MEGA11.0 software to analyze and process the data, calculate the base information and genetic distance, and construct a neighbor-joining (NJ) phylogenetic tree. The results showed that, except for *Amanita muscaria*, other adulterants such as *Amanita muscaria* could cluster separately in the NJ phylogenetic tree. The average genetic distance between *Amanita muscaria* and other adulterants was 0.207. The COI gene had a significant DNA barcoding gap and could be used as an effective DNA barcoding gene in *Amanita muscaria* medicinal materials, providing a new basis for the classification and species identification of *Amanita muscaria* medicinal materials (Xiong Xiran, Li Sha, Jin Fansen, et al. Identification of *Amanita muscaria* medicinal materials based on DNA barcoding technology [J]. Chinese Medicinal Herbs, 2024, 47(12):2996-3000.).

[0004] Based on existing literature, current research on the quality control of *Corydalis yanhusuo* mainly focuses on medicinal materials and processed medicinal slices, with limited research on the quality control of its water extract. Patent CN119331982A discloses a primer combination and its application, as well as an identification method, for multiplex PCR identification of *Corydalis yanhusuo*, *Hemiptera chinensis*, *Tetracentron sinense*, *Trichophyton septemlobus*, and *Trichophyton davidii*. This multiplex PCR method establishes a specific identification method for *Corydalis yanhusuo* raw materials. Under the same amplification conditions, it can simultaneously identify the original *Corydalis yanhusuo* and its adulterants, including *Hemiptera chinensis*, *Tetracentron sinense*, *Trichophyton septemlobus*, and *Trichophyton davidii*. It can also simultaneously identify *Corydalis yanhusuo* medicinal materials and their adulterants, including *Hemiptera chinensis*, *Tetracentron sinense*, *Trichophyton septemlobus*, and *Trichophyton davidii*, as well as processed *Corydalis yanhusuo* processed medicinal slices and their adulterants. Patent CN109580893B discloses a method for identifying *Stachys affinis* medicinal materials, including morphological identification, microscopic identification, and efficacy identification. The efficacy identification includes a pain-induced tail-flick test, a writhing test, and an antibacterial effect test. This method combines visual observation with internal component efficacy analysis to identify *Stachys affinis* medicinal materials, improving the accuracy of identification. However, the methods in the above-mentioned patents have long experimental cycles and complex operating procedures. None of them have conducted research on the identification of *Stachys affinis* water extracts or different species, and the quality evaluation research is not comprehensive enough, failing to effectively control the quality level of *Stachys affinis* formula granules. Summary of the Invention

[0005] Objectives of the invention: The first objective of this invention is to provide a method for constructing a characteristic peptide spectrum of *Corydalis yanhusuo* that is highly specific, reproducible, stable, low in detection cost, and highly efficient. The second objective of this invention is to provide the application of the characteristic peptide spectrum in identifying *Corydalis yanhusuo* and its adulterants.

[0006] Technical solution: The method for constructing a characteristic peptide profile of the stink bug according to the present invention includes the following steps:

[0007] (1) Prepare the test sample solution and the reference solution of the nine-fragrant insect reference material respectively;

[0008] (2) The test solution and reference solution prepared in step (1) were analyzed by ultra-high performance liquid chromatography-mass spectrometry to obtain the corresponding peptide spectra;

[0009] (3) Using the peptide spectrum of the reference medicinal material as a reference, the following 6 characteristic ion peaks with double-charged precursor ions were determined from the peptide spectrum of the test sample to constitute the characteristic peptide spectrum of *Corydalis yanhusuo*:

[0010] Peak 1: Mother ion m / z 469.263 (double charge), daughter ion m / z 522.312;

[0011] Peak 2: Mother ion m / z 617.811 (double charge), daughter ion m / z 731.402;

[0012] Peak 3: Mother ion m / z 559.780 (double charge), daughter ion m / z 760.400;

[0013] Peak 4: Mother ion m / z 649.800 (double charge), daughter ion m / z 785.350;

[0014] Peak 5: Mother ion m / z 623.840 (double charge), daughter ion m / z 430.260;

[0015] Peak 6: Mother ion m / z 789.926 (double charge), daughter ion m / z 1167.596.

[0016] Furthermore, the analytical conditions of the ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) are as follows: Stationary phase: The chromatographic column is packed with octadecylsilane-bonded silica gel; Mobile phase: Acetonitrile or 0.05-0.15% formic acid acetonitrile as mobile phase A, and 0.05%-0.15% formic acid as mobile phase B, using gradient elution: 0-10 min, 10-25% A, 10-11 min, 25-10% A; Mass spectrometry detection: Electrospray ionization (ESI) positive ion mode, multiple reaction monitoring (MRM).

[0017] Furthermore, the flow rate is 0.25-0.35 mL per minute; the column temperature is 25-35℃.

[0018] Preferably, the mobile phase A is 0.1% formic acid acetonitrile, the mobile phase B is 0.1% formic acid, the flow rate is 0.3 ml per minute, and the column temperature is 30°C.

[0019] Preferably, the chromatographic column has a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.9 μm.

[0020] Preferably, the injection volume of the test solution and the reference solution is 1 μl-3 μl.

[0021] Preferably, the chromatographic column is an EC-C18, HSS T3, or EP-C18 column.

[0022] Preferably, the test sample in step (1) is selected from *Corydalis yanhusuo* medicinal material, *Corydalis yanhusuo* standard decoction, *Corydalis yanhusuo* preparation intermediate or *Corydalis yanhusuo* preparation finished product, or *Corydalis yanhusuo* adulterant.

[0023] Furthermore, the preparation of the test solution and / or reference solution in step (1) includes extraction and enzymatic hydrolysis steps.

[0024] Furthermore, the extraction is performed using ultrasonic extraction or reflux extraction, with an extraction time of 15-60 minutes; the enzymatic hydrolysis uses trypsin, with a trypsin dosage of 100-300 μL and a hydrolysis time of 2-8 hours.

[0025] Furthermore, the trypsin solution is prepared fresh for immediate use, and is prepared by adding 1 wt% ammonium bicarbonate solution to trypsin for sequence analysis to a solution with a concentration of 1 mg / mL.

[0026] Furthermore, the extractant in the extraction step is a 1 wt% ammonium bicarbonate solution.

[0027] A further preferred method for preparing the test solution is as follows: Take 0.2-1g of the standard decoction of *Corydalis yanhusuo*, place it in a stoppered conical flask, add 1wt% ammonium bicarbonate solution, sonicate (power 250W, frequency 40kHz) for 15-60min, cool, shake well, filter, take the filtrate, add trypsin solution, enzymatically hydrolyze at 37℃ for 2-8 hours, centrifuge at 12000 rpm / min for 10 minutes, and take the supernatant as the test solution.

[0028] A further preferred method for preparing the reference solution of the control medicinal material is as follows: Take 1-3g of *Corydalis yanhusuo* (a type of insect) as a reference medicinal material, place it in a stoppered conical flask, soak it in water for 30 minutes, heat and reflux to extract for 30-60 minutes, cool, shake well, filter, collect the filtrate and evaporate to dryness; dissolve the residue in 1wt% ammonium bicarbonate solution, transfer it to a centrifuge tube, add the above-mentioned trypsin solution, and enzymatically hydrolyze at 37℃ for 2-8 hours; centrifuge at 12000 rpm / min for 10 minutes, collect the supernatant, and obtain the reference solution of the control medicinal material.

[0029] The application of the characteristic peptide spectrum described in this invention in identifying *Symplocos chinensis* and distinguishing its adulterants.

[0030] Preferably, the characteristic peptide profile is applicable to both the medicinal material *Corydalis yanhusuo* and the standard decoction of *Corydalis yanhusuo*.

[0031] Furthermore, the presence or absence of characteristic ion peaks in the characteristic peptide spectrum can distinguish between adulterants such as *Stachys chinensis*. If all six characteristic ion peaks are detected, the sample is *Stachys chinensis*. Only peak 3 is detected in *Sterculia serratifolia*. Only peaks 4-6 are detected in *Sterculia scabra*. No characteristic ion peaks are detected in *Sterculia scabra*.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The characteristic peptide spectrum method established by the present invention is based on liquid chromatography-mass spectrometry and uses characteristic peptides as identification means, providing a new and reliable analytical method for the identification of adulterants of *Corydalis yanhusuo*, and the results are relatively objective and accurate; (2) The characteristic peptide spectrum established by the method of the present invention screens out 6 groups of detection ion pairs to establish the characteristic peptide spectrum of *Corydalis yanhusuo*, which can effectively distinguish different kinds of animal drugs, and can simultaneously control the quality of *Corydalis yanhusuo* medicinal materials and its water extracts, providing an important guarantee for the quality of *Corydalis yanhusuo* formula granules; (3) The method is simple to operate, has high detection efficiency, good sensitivity and reproducibility, and low detection cost. It can simultaneously realize the confirmation of the authenticity of *Corydalis yanhusuo* medicinal materials and its preparations and the identification of adulterants, providing a strong guarantee for the quality control of *Corydalis yanhusuo* raw materials and the industrialization of formula granules. Attached Figure Description

[0033] Figure 1 Characteristic peptide spectrum of Nine-fragrant Insect Formula Granules;

[0034] Figure 2 Characteristic peptide spectrum of the standard decoction of *Corydalis yanhusuo* (a type of insect);

[0035] Figure 3 Characteristic peptide spectrum of the medicinal material *Corydalis yanhusuo*;

[0036] Figure 4 A comparison diagram of characteristic peptide spectra of the standard decoction of *Nine-Fragrance Insect* and the control medicinal materials;

[0037] Figure 5 To examine the characteristic peptide spectra of different extraction methods;

[0038] Figure 6 To examine characteristic peptide profiles at different extraction times;

[0039] Figure 7 Characteristic peptide profiles for investigating trypsin dosage;

[0040] Figure 8 To examine the characteristic peptide profiles based on the enzymatic hydrolysis time;

[0041] Figure 9 This is a diagram illustrating the specificity test of the standard decoction of *Nine-Fragrance Insect*.

[0042] Figure 10 Chromatographic images of characteristic peptides from the standard decoction of *Nine-Fragrance Insect* on different chromatographic columns;

[0043] Figure 11 Figure 1 shows the characteristic peptide spectrum of the standard decoction of *Corydalis yanhusuo* at different column temperatures.

[0044] Figure 12 A graph showing the characteristic peptide profiles of the standard decoction of *Nine-Fragrance Insect* at different flow rates;

[0045] Figure 13Comparative peptide profiles of genuine and counterfeit *Nine-fragrant insect* standard decoctions;

[0046] Figure 14 This is a diagram illustrating the specificity of characteristic peptide spectra of different types of animal drugs. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0048] Unless otherwise specified, all instruments, materials and reagents used in the following examples are commercially available.

[0049] Main instruments and equipment:

[0050] (1) Main reagents:

[0051] Acetonitrile, methanol (chromatographic grade, Thermo Fisher Scientific); formic acid (mass spectrometry grade, Aladdin); trypsin (sequence analysis grade, Sigma, catalog number 0000310680); ammonium bicarbonate (analytical grade); the experimental water was Watson's distilled water.

[0052] (2) Sample source:

[0053] The reference material of *Symplocos edulis* was purchased from the National Institutes for Food and Drug Control (batch number: 121651-202103).

[0054] The standard decoctions for nine-fragrant insects, the standard decoctions for adulterants (sawtooth bug, small wrinkled bug, and hemp-skin bug), as well as the standard decoctions for mantis egg case, scorpion, male silkworm moth, centipede, and roasted gecko, were all provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0055] Example 1: Method for constructing characteristic peptide profiles of *Corydalis yanhusuo* (a type of insect).

[0056] This embodiment provides a method for constructing the characteristic peptide profile of *Corydalis yanhusuo* as described in claim 1. The test sample includes *Corydalis yanhusuo* medicinal material, standard decoction, pharmaceutical intermediate, formulation granules, and adulterants (such as *Sterculia serrulata*, *Sterculia scabra*, and *Sterculia fuciformis*). The following description uses the standard decoction of *Corydalis yanhusuo* as an example; the specific steps are as follows:

[0057] 1. Prepare the test sample solution and the reference solution of the nine-fragrant insect medicinal material separately;

[0058] (1) Preparation of reference solution: Take 1g of the reference herb *Corydalis yanhusuo*, place it in a stoppered conical flask, add 50ml of water, soak for 30 minutes, heat under reflux for 30 minutes, cool, shake well, filter, take 25ml of the filtrate, evaporate to dryness, add 1wt% ammonium bicarbonate solution to the residue to 1ml, add 100μl of trypsin solution (take trypsin for sequence analysis, add 1wt% ammonium bicarbonate solution to prepare a solution containing 1mg per 1ml, prepare immediately before use), shake well, enzymatically hydrolyze at 37℃ for 4 hours, centrifuge at 12000 rpm / min for 10 minutes, take the supernatant as the reference solution for the reference herb.

[0059] (2) Preparation of test solution: Take 0.5g of the standard decoction powder of *Corydalis yanhusuo*, place it in a stoppered conical flask, add 20ml of 1wt% ammonium bicarbonate solution, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, take 1ml of the filtrate into a centrifuge tube, add 100μl of trypsin solution (take trypsin for sequence analysis, add 1wt% ammonium bicarbonate solution to prepare a solution containing 1mg per ml, prepare immediately before use), shake well, enzymatically hydrolyze at 37℃ for 4 hours, centrifuge at 12000 rpm / min for 10 minutes, take the supernatant as the test solution.

[0060] 2. The test solution and reference solution prepared in step (1) were analyzed by ultra-high performance liquid chromatography-mass spectrometry to obtain the corresponding peptide spectra;

[0061] Chromatographic conditions: The column was packed with octadecylsilane-bonded silica gel (column length 100 mm, inner diameter 2.1 mm, particle size 1.9 μm); 0.1% formic acid acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B. Gradient elution was performed as shown in the table below. The flow rate was 0.3 mL per minute, the column temperature was 30 °C, and the injection volume was 2 μL.

[0062] Mass spectrometry conditions: triple quadrupole mass spectrometry, electrospray ionization (ESI) positive ion mode, multiple reaction monitoring (MRM).

[0063] Table 1 Gradient Elution Table

[0064]

[0065] 3. Using the peptide spectrum of the *Corydalis yanhusuo* reference material as a reference, the following six characteristic ion peaks with double-charged precursor ions were determined from the peptide spectrum of the test sample to constitute the characteristic peptide spectrum of *Corydalis yanhusuo*.

[0066] Using *Nine-fragrant insect* formula granules, standard decoction, and medicinal materials as samples, test samples were prepared and determined according to the above method. The characteristic peptide spectra obtained are as follows: Figure 1 , Figure 2 and Figure 3As shown, all three types of samples exhibit consistent characteristic ion peaks.

[0067] Furthermore, by systematically comparing the peptide spectra of multiple batches of genuine *Nine-Fragrance Insects* (including control material, raw material, standard decoction, and formula granules) with common adulterants (saw-toothed bug, small wrinkled bug, and hemp-skin bug) (see...), the analysis was further conducted. Figure 13 Six characteristic ion peaks were identified that were consistently present in all genuine products but absent or significantly different in adulterated products. The specific characteristic peptide spectrum of *Corydalis yanhusuo* was finally determined, as shown in the table below:

[0068] Table 2. Ion pairs detected by characteristic peptide spectra of *Corydalis yanhusuo*.

[0069]

[0070] like Figure 4 As shown, the characteristic peptide spectrum of the standard decoction of *Corydalis yanhusuo* contains all six ion peaks mentioned above, and the retention times of each peak are highly consistent with those of the reference medicinal material, indicating that the characteristic peptide spectrum has good reproducibility and representativeness, and can be used as a specific basis for the identification of *Corydalis yanhusuo*.

[0071] Example 2: Validation of the applicability of characteristic peptide profiles in multiple batches of samples

[0072] To verify the reproducibility and representativeness of the characteristic peptide profile of *Corydalis yanhusuo* constructed in Example 1, this example systematically determined the peptide profiles from multiple sources.

[0073] (1) Sample information

[0074] Three batches of Nine-Fragrance Insect Formula Granules, 17 batches of Nine-Fragrance Insect Medicinal Materials, and 17 batches of Nine-Fragrance Insect Standard Decoctions;

[0075] All samples were prepared as test solutions according to the method described in Example 1 and then measured.

[0076] (2) Test results

[0077] The test results of the test solution are shown in Tables 3-5.

[0078] Table 3. Determination results (peak area) of Nine-Fragrance Insect Formula Granules

[0079]

[0080] Table 4. Determination results (peak area) of the standard decoction sample of *Corydalis yanhusuo*.

[0081]

[0082] Table 5. Results of the determination of the peak area of ​​the *Nine-Fragrance Insect* medicinal material sample.

[0083]

[0084] Qualitative analysis showed that the six characteristic ion peaks in the characteristic peptide spectra of 3 batches of formula granules, 17 batches of standard decoctions and 17 batches of medicinal materials could be detected stably, with good peak shape and stable response.

[0085] The above results confirm that the characteristic peptide spectrum has good reproducibility and representativeness in genuine samples of *Corydalis yanhusuo* (a type of medicinal herb) from different origins and its water extracts, and can be used as a reliable basis for the identification of *Corydalis yanhusuo*.

[0086] Example 3: Establishment and Methodological Study of Characteristic Peptide Spectroscopy Method for Nine-Fragrance Insect Standard Decoction

[0087] Based on the characteristic peptide spectrum of *Corydalis yanhusuo* (containing 6 characteristic ion pairs) established in Example 1, this embodiment systematically optimizes the preparation method of the test sample solution and conducts comprehensive methodological validation of the established ultra-high performance liquid chromatography-mass spectrometry analysis method, mainly including the examination of specificity, precision, intermediate precision, stability, repeatability and robustness.

[0088] The preparation of the reference solution, chromatographic conditions, and mass spectrometry conditions were the same as in Example 1, and will not be repeated here.

[0089] 1. Optimization of the preparation method of the test solution

[0090] Using the standard decoction of *Corydalis yanhusuo* as a sample, and the ratio of total peak area to sample weight as an evaluation index, the key parameters of the pretreatment of the test sample were systematically investigated.

[0091] (1) Examination of different extraction methods

[0092] Take 0.5 g of sample, in duplicate, and treat each sample separately by ultrasonication (250 W, 40 kHz, 30 minutes) or reflux (30 minutes). Perform the remaining procedures according to the sample solution preparation process in Example 1. Inject the sample for analysis, record the peak area, and calculate "total peak area / sample weight". The results are shown in Table 6. Figure 5 .

[0093] Table 6. Results of characteristic peptide spectra determination using different extraction methods (total peak area / sample weight)

[0094]

[0095] The results showed that the total peak area / sample weight obtained by ultrasonic extraction was 2.75 × 10⁻⁶. 6 The value was significantly higher than that of the reflux method (6.83 × 10⁻⁶). 5 Therefore, ultrasonic extraction was chosen as the extraction method.

[0096] (2) Examination of different extraction times

[0097] Take 0.5 g of sample, make three parallel aliquots, and sonicate for 15 minutes, 30 minutes, and 60 minutes respectively. Perform the remaining operations according to the sample solution preparation procedure in Example 1. Inject the sample for analysis, record the peak area, and calculate "total peak area / sample weight". The results are shown in Table 7. Figure 6 .

[0098] Table 7. Results of peptide profile determination at different extraction times (total peak area / sample weight)

[0099]

[0100] The results showed that the response values ​​obtained at 30 minutes and 60 minutes were similar (both were 2.75 × 10⁻⁶). 6 ), 15 minutes slightly lower (2.55×10) 6 Taking into account both efficiency and effectiveness, the ultrasound time was determined to be 30 minutes.

[0101] (3) Investigation of trypsin dosage

[0102] Take 0.5 g of sample, in triplicate, and sonicate for 30 minutes. Then add 50 μL, 100 μL, and 200 μL of trypsin solution (prepare a solution containing 1 mg per ml by adding 1 wt% ammonium bicarbonate solution to trypsin for sequence analysis, to be prepared immediately before use). Perform the remaining operations according to the sample solution preparation procedure in Example 1. Inject the sample for analysis, record the peak area, and calculate "total peak area / sample weight". The results are shown in Table 8. Figure 7 .

[0103] Table 8. Results of characteristic peptide profile determination at different trypsin dosages (total peak area / sample weight)

[0104]

[0105] The results showed that the total response value was highest at 100 μL (2.75 × 10⁻⁶). 6 Since the response of each characteristic peak is balanced, the amount of trypsin used is determined to be 100 μL.

[0106] (4) Investigation of enzymatic hydrolysis time

[0107] Take 0.5 g of sample, divide into three parallel portions, sonicate for 30 minutes, add 100 μL of trypsin solution, and incubate at 37 ℃ for 2 hours, 4 hours, and 8 hours respectively. Perform the remaining operations according to the sample solution preparation procedure in Example 1. Inject the sample for analysis, record the peak area, and calculate "total peak area / sample weight". The results are shown in Table 9. Figure 8 .

[0108] Table 9. Results of peptide profile determination at different enzymatic digestion times (total peak area / sample weight)

[0109]

[0110] The results showed that the highest total peak area / sample weight ratio was obtained after 4 hours of enzymatic hydrolysis (2.75 × 10⁻⁶). 6 Since extending the time to 8 hours did not improve the enzyme digestion, the enzyme digestion time was determined to be 4 hours.

[0111] (5) Determination of the preparation method of the test solution

[0112] Based on the above research results, the method for preparing the test solution for the characteristic peptide profile of the Nine-Fragrance Insect Standard Decoction is determined as follows:

[0113] Take 0.5g of the standard decoction of *Corydalis yanhusuo*, place it in a stoppered conical flask, add 20ml of 1wt% ammonium bicarbonate solution, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, take 1ml of the filtrate into a centrifuge tube, add 100μl of trypsin solution (prepare a solution containing 1mg per ml by adding 1wt% ammonium bicarbonate solution to trypsin for sequence analysis, and prepare it immediately before use), shake well, incubate at 37℃ for 4 hours, centrifuge (12000 rpm) for 10 minutes, and take the supernatant as the test solution.

[0114] 2. Validation of the characteristic peptide profiling methodology

[0115] (1) Specificity examination

[0116] Take a negative sample lacking *Corydalis jiunifolia* and prepare a negative sample solution according to the test sample preparation method. Accurately pipette 2 μl each of the negative sample solution, the reference medicinal material solution, and the test sample solution (*Corydalis jiunifolia* standard decoction), and inject them into a liquid chromatography-tandem mass spectrometry (LC-MS / MS) instrument. Determine the results according to the chromatographic and mass spectrometric conditions of Example 1. See [Figure showing the results]. Figure 9 .

[0117] The results showed that no characteristic ion peaks were detected at the corresponding retention times in the chromatograms of the test solution and the reference medicinal material, indicating that the negative sample did not interfere with the detection of characteristic ion pairs in the method, and that the method is specific.

[0118] (2) Precision test

[0119] Take samples from the same batch (Standard decoction of Nine-Fragrance Insects), prepare test solutions according to the test solution preparation method, inject 2 μl of the sample 6 times consecutively, record the peak area of ​​each ion pair, and calculate the peak area RSD value. The results are shown in Table 10.

[0120] Table 10 Precision Experiment Results (Peak Area)

[0121]

[0122] The results showed that the RSD values ​​of the peak areas of each ion pair were all less than 2%, indicating good precision.

[0123] (3) Intermediate precision test

[0124] Take samples from the same batch (Standard decoction of Nine-Fragrance Insects), and have two experimenters, A and B, prepare three portions of each sample according to the preparation method of the test solution. Measure the samples at different times under the above-mentioned chromatographic-mass spectrometry conditions, record the peak area of ​​each ion pair, and calculate the peak area RSD value. The results are shown in Table 11.

[0125] Table 11 Results of intermediate precision experiments (peak area)

[0126]

[0127] The results showed that the peak areas of each ion pair could be clearly detected. The peak areas of each ion pair in samples prepared by different personnel were somewhat different, but this did not affect the identification of characteristic ions.

[0128] (4) Stability test

[0129] Samples from the same batch (standard decoction of nine-fragrant insects) were prepared into test solutions according to the test solution preparation method. 2 μl of each solution was injected at 0, 2, 4, 8, 12, 18, and 24 h. The stability of the solution was evaluated by the peak area of ​​each ion pair. The results are shown in Table 12.

[0130] Table 12 Stability test results (peak area)

[0131]

[0132] The results showed that after the test solution was left at room temperature for 24 hours, the peak areas of each ion pair could be clearly detected. After the test solution was left for 8 hours, the peak areas of some ion pairs fluctuated, but this did not affect the identification of characteristic ions.

[0133] (5) Repeated examination

[0134] Take samples from the same batch (standard decoction of nine-fragrant insects), 6 parallel groups, prepare test solutions according to the test solution preparation method, inject 2µl of each sample, record the peak area of ​​each ion pair, calculate the peak area RSD value, and the results are shown in Table 13.

[0135] Table 13 Repeatability test results (peak area)

[0136]

[0137] The results showed that the RSD values ​​of the peak areas of each ion pair were all less than 2%, indicating good repeatability.

[0138] (6) Durability test

[0139] 1) Investigation of different chromatographic columns

[0140] Samples from the same batch (St. 'Nine-Fragrance Insect' standard decoction) were prepared into test solutions according to the test solution preparation method. Three different chromatographic columns—Agilent Eclipse Plus C18 RRHD (2.1 mm × 100 mm, 1.8 μm), Agilent Poroshell EC-C18 (2.1 mm × 100 mm, 1.9 μm), and Waters ACQUITY HSS T3 (2.1 mm × 100 mm, 1.8 μm)—were used to investigate their influence on the characteristic peptide profiles of the Nine-Fragrance Insect standard decoction. Results are shown below. Figure 10 Table 14.

[0141] Table 14 Results of experiments conducted using different chromatographic columns (peak areas)

[0142]

[0143] The results showed that the specified characteristic ion pairs could be clearly detected by chromatographic columns of different brands, indicating that different models of chromatographic columns had no effect on the identification of characteristic peptide spectra of the Nine-Fragrance Insect Standard Decoction.

[0144] 2) Investigation at different column temperatures

[0145] Samples from the same batch (St. 'Nine-Fragrance Insect Standard Decoction) were prepared into test solutions according to the test solution preparation method. An Agilent Poroshell EC-C18 (2.1 mm × 100 mm, 1.9 μm) column was used to compare the effects of column temperatures of 25℃, 30℃, and 35℃ on the characteristic peptide spectra of the St. 'Nine-Fragrance Insect Standard Decoction.' Results are shown below. Figure 11 Table 15.

[0146] Table 15 Results of experiments at different column temperatures (peak area)

[0147]

[0148] The results showed that the specified characteristic ion pairs could be clearly detected within the column temperature range of 25℃-35℃, indicating that small column temperature variations had no effect on the identification of characteristic peptide spectra of the Nine-Fragrance Insect Standard Decoction.

[0149] 3) Investigation of different flow velocities

[0150] Samples from the same batch were used to prepare test solutions according to the test solution preparation method. An Agilent Poroshell EC-C18 (2.1 mm × 100 mm, 1.9 μm) column was used to compare the effects of flow rates of 0.25 ml / min, 0.30 ml / min, and 0.35 ml / min on the characteristic peptide profile of the *Corydalis yanhusuo* standard decoction. Results are shown below. Figure 12 Table 16.

[0151] Table 16 Results of experiments at different flow velocities (peak area)

[0152]

[0153] The results showed that the specified characteristic ion pairs could be clearly detected within a flow rate range of 0.25-0.35 ml / min, indicating that small flow rate variations had no effect on the identification of characteristic peptide spectra in the Nine-Fragrance Insect Standard Decoction.

[0154] Example 4: Characteristic Peptide Profile Identification Study of Genuine and Counterfeit Nine-Fragrance Insects

[0155] To verify the ability of the established characteristic peptide profiling method to identify *Corydalis yanhusuo* and its common adulterants, this embodiment conducted parallel determination and comparative analysis on multiple batches of genuine and adulterant standard decoctions of *Corydalis yanhusuo*.

[0156] The chromatographic-mass spectrometry conditions and the preparation method of the test solution are the same as in Example 1, and will not be repeated here.

[0157] 1. Sample testing and data analysis

[0158] Seventeen batches of standard decoctions of *Symplocos jiunifolia*, three batches of *Symplocos jiunifolia* (a counterfeit), three batches of *Symplocos septum* (a counterfeit), and one batch of *Symplocos rubra* (a counterfeit) were taken respectively, and their characteristic peptide spectra were determined. The authenticity of the *Symplocos jiunifolia* samples was compared and analyzed based on the differences in the peak area of ​​each ion pair.

[0159] Combination Figure 13 The authenticity of the sample to be tested was determined by the characteristic peptide spectrum obtained in step (3). The characteristic peptide spectra of the standard decoction of *Symplocos rubescens* and its adulterants (*Sawtooth Bug*, *Small Wrinkled Bug*, and *Hemp Skin Bug*) were compared. There were significant differences between the characteristic peptide spectra of *Symplocos rubescens* and its adulterants. *Sawtooth Bug*: only peak 3 was detected, and the other 5 characteristic peaks were missing. *Small Wrinkled Bug*: peaks 4, 5, and 6 were detected, but peaks 1–3 were not detected. *Hemp Skin Bug*: none of the 6 characteristic peaks were detected.

[0160] Example 5: Drug specificity study in different animal species

[0161] To examine whether the established characteristic peptide profiling method for *Stachys chinensis* possesses species specificity, this embodiment conducts parallel determinations and comparative analyses of standard decoctions of *Stachys chinensis* and various commonly used animal-derived traditional Chinese medicines (including mantis egg case, scorpion, male silkworm moth, centipede, and roasted gecko).

[0162] The chromatographic-mass spectrometry conditions and the preparation method of the test solution are the same as in Example 1, and will not be repeated here.

[0163] 1. Sample testing and data analysis

[0164] Standard decoctions of nine-fragrant insects, mantis egg case, scorpion, male silkworm moth, centipede, and gecko were taken respectively, and the characteristic peptide spectra of the above-mentioned animal drug standard decoctions were determined. The different animal drug samples were compared and analyzed based on the differences in the peak area of ​​each ion pair.

[0165] Combination Figure 14 The comparison results of the characteristic peptide spectra shown indicate that the characteristic peptide spectra of the *Nine-Fragrance Insect* standard decoction differ significantly from those of standard decoctions of other species (including *Mantis Egg Case*, *Scorpion*, male silkworm moth, centipede, and gecko). Furthermore, the six characteristic ion peaks presented in the *Nine-Fragrance Insect* characteristic peptide spectra were not detected in other animal drugs. Therefore, this method can effectively and accurately distinguish *Nine-Fragrance Insect* from other easily confused animal drugs and identify the species.

[0166] In summary, the characteristic peptide spectrum detection method for *Corydalis yanhusuo* samples provided in the above embodiments can simultaneously identify genuine and counterfeit products as well as different types of animal drugs. It is convenient, rapid, highly specific, and stable, enabling quality control from multiple aspects, including species and adulterant identification, thus providing stronger support for the industrialization of *Corydalis yanhusuo* formula granules.

Claims

1. A method for constructing a characteristic peptide profile of Blattella germanica, characterized in that, It comprises the following steps: (1) separately preparing a test sample solution and a reference solution of the Aspongopus control medicinal material; (2) analyzing the test sample solution and the reference solution prepared in step (1) by using an ultra-high performance liquid chromatography-mass spectrometry instrument to obtain corresponding peptide spectra; (3) taking the peptide spectrum of the control medicinal material as a control, determining the following six characteristic ion peaks with double-charged parent ions from the peptide spectrum of the test sample to constitute the characteristic peptide spectrum of Aspongopus: Peak 1: parent ion m / z 469.263 (double charge), daughter ion m / z 522.312; Peak 2: parent ion m / z 617.811 (double charge), daughter ion m / z 731.402; Peak 3: parent ion m / z 559.780 (double charge), daughter ion m / z 760.400; Peak 4: parent ion m / z 649.800 (double charge), daughter ion m / z 785.350; Peak 5: parent ion m / z 623.840 (double charge), daughter ion m / z 430.260; Peak 6: parent ion m / z 789.926 (double charge), daughter ion m / z 1167.

596.

2. The method for constructing the characteristic peptide spectrum of Spilarctia spp. according to claim 1, characterized in that, The analysis conditions of the ultra-high performance liquid chromatography-mass spectrometry instrument are as follows: stationary phase: a chromatographic column with octadecylsilane-bonded silica gel as the filler; mobile phase: acetonitrile or 0.05-0.15% formic acid acetonitrile as the mobile phase A, and 0.05%-0.15% formic acid as the mobile phase B, using gradient elution: 0-10 min, 10-25% A, 10-11 min, 25-10% A; mass spectrometry detection: electrospray ionization (ESI) positive ion mode, multiple reaction monitoring (MRM).

3. The method for constructing the characteristic peptide spectrum of Spilosoma sp. according to claim 3, characterized in that, The flow rate is 0.25-0.35 mL per minute; the column temperature is 25-35°C.

4. The method for constructing the characteristic peptide profile of *Symplocos jirunica* according to claim 3, characterized in that, The chromatographic column is selected from EC-C18, HSS T3, and EP-C18 chromatographic columns.

5. The method for constructing the characteristic peptide spectrum of *Symplocos jirunica* according to claim 3, characterized in that, The injection amount of the test sample solution and the reference solution is 1-3 μl.

6. The method for constructing the characteristic peptide profile of *Symplocos jirunica* according to claim 1, characterized in that, The test sample in step (1) is selected from Aspongopus medicinal materials, Aspongopus standard decoctions, Aspongopus preparation intermediates or finished products, and Aspongopus adulterants.

7. The method for constructing the characteristic peptide profile of *Symplocos jirunica* according to claim 1, characterized in that, The preparation of the test sample solution and / or the reference solution in step (1) comprises extraction and enzymolysis steps.

8. The method for constructing the characteristic peptide spectrum of *Symplocos jirunica* according to claim 7, characterized in that, The extraction is performed by ultrasonic extraction or heating reflux extraction, and the extraction time is 15-60 minutes; the enzymolysis is performed by using trypsin, and the amount of trypsin used is 100-300 μL, and the enzymolysis time is 2-8 hours.

9. The method for constructing the characteristic peptide profile of *Symplocos jirunica* according to claim 8, characterized in that, The extracting agent in the extraction step is 1 wt% ammonium bicarbonate solution.

10. Use of the characteristic peptide spectrum of any one of claims 1-9 in identifying Aspongopus and distinguishing its adulterants.

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