A detection method for adulterated leeches of whonophlebopterx in wide-body gold leech medicinal materials, decoction pieces and traditional Chinese patent medicines

Abstract

The application discloses a detection method of adulterated Limnatis humilis in wide-body Limnatis humilis medicinal materials, decoction pieces and Chinese patent medicines. With the characteristic chemical component Bdelline B of the adulterated Limnatis humilis as an index, whether Limnatis humilis is adulterated in the wide-body Limnatis humilis medicinal materials, decoction pieces and Chinese patent medicines is quickly and accurately detected through liquid chromatography-mass spectrometry. The method is simple in operation, fast in detection speed, good in specificity, not affected by the complex process of samples, effectively fills the technical blank that DNA barcoding cannot detect Chinese patent medicines, is a technology for detecting wide-body Limnatis humilis adulterated Limnatis humilis based on chemical components, and provides a new method for quality control of Limnatis humilis.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine adulteration detection and quality control technology, specifically relating to a method for detecting adulterated Hirudo medicinalis in Hirudo medicinal materials, processed slices and prepared Chinese medicines. Background Technology

[0002] The 2020 edition of the Chinese Pharmacopoeia includes, in Part I, the dried whole bodies of the leeches *Whitmania pigra* Whitman (hereinafter referred to as broad-bodied golden-thread leech), *Hirudo niponica* Whitman (hereinafter referred to as Japanese medicinal leech), or *Whitmania acranulata* Whitman (hereinafter referred to as slender golden-leaf leech) of the Hirudo family for medicinal use. In recent years, wild leech resources have become increasingly scarce, and most leeches on the market are currently artificially bred. The main variety circulating is *Whitmania pigra*, with a small amount of *Hirudo niponica*, while *Whitmania acranulata* is extremely rare. With the increasing market demand for leeches, the mixing of genuine leeches with adulterants is extremely common, with the adulterant *Hirudo niponica* being the most prevalent.

[0003] Currently, *Hirudo medicinalis* is only included in the local standards of Yunnan Province and Guangxi Zhuang Autonomous Region, and is not yet included in the 2020 edition of the *Chinese Pharmacopoeia*. Due to the high economic value of leeches and the fact that their anticoagulant activity is significantly lower than that of *Hirudo medicinalis*, *Hirudo medicinalis* is often mixed with *Hirudo scabra* to enhance its activity and, to some extent, alleviate the scarcity of leech resources. Leeches have strong blood-activating and stasis-removing effects and are widely used clinically. Prescription drugs containing leeches, such as Huoxue Tongmai Capsules, Tongxinluo Capsules, and Naoxuekang Capsules, are widely used and in high demand. Studies have shown that the anticoagulant pathways of *Hirudo medicinalis* and *Hirudo scabra*, as well as their mechanisms of action on thrombin, differ to some extent. Therefore, the confusion surrounding the origin of leeches poses significant challenges to the safety and efficacy of their clinical use, as well as to the quality control of traditional Chinese medicines containing leeches. Thus, determining the origin of medicinal leeches is particularly necessary.

[0004] CN108866205A discloses specific primers for identifying Hirudo medicinalis based on DNA barcoding, with the upstream primer being 5'-TCTGGCTCTTAGGCTTCG-3' and the downstream primer being 5'-GTTGTCTTCCTGGCTG TT-3'. Ding Yuezhu used SDS-PAGE to detect the effects of dried leeches, dried Hirudo medicinalis, and fresh Hirudo medicinalis on fibrinogen. The results showed that leeches hydrolyze fibrinogen, affecting both its α and β chains, while Hirudo medicinalis did not hydrolyze fibrinogen. The difference in protein bands can be used to distinguish between leeches and Hirudo medicinalis. CN102914612A provides a method for detecting the fingerprint spectrum of Hirudo medicinalis, which contains 23 common peaks and can be used for the quality control of Hirudo medicinalis. Currently, molecular identification techniques are the most widely used for leech authentication, but this technology is only suitable for rapid and accurate identification of raw medicinal materials and processed medicinal slices. In traditional Chinese medicine preparations, the complex preparation process leads to the degradation of leech nucleic acids and proteins, making this technique unsuitable for identification. In addition, some studies differentiate between *Hirudo nipponia* and *Hirudo scabra* based on differences in chemical composition, mainly focusing on protein peptides, amino acids, and nucleoside components. However, the identified components lack specificity, and quality control is based solely on differences in content. Therefore, to ensure the quality and efficacy of leeches, it is urgent to find a new characteristic component and establish a method to identify *Hirudo scabra* and its adulterant, *Hirudo nipponia*. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the inventors, based on the ligand-fishing technique used by their research group to screen the pteridine component Bdelline B from Hirudo medicinalis using affinity ultrafiltration with thrombin as a ligand, verified whether it was a differentiating component between Hirudo medicinalis and Hirudo maxima using liquid chromatography-mass spectrometry (LC-MS) and established a detection method for this component. This method effectively identifies adulterated Hirudo medicinalis in Hirudo maxima medicinal materials, processed slices, and traditional Chinese medicine preparations.

[0006] The purpose of this invention is to provide a method for detecting adulteration of *Hirudo medicinalis* (also known as broad-bodied golden thread leech), its processed forms, and prepared Chinese medicines, thereby ensuring the quality of the raw materials and prepared medicines. The detection method of this invention uses Bdelline B, a characteristic chemical component of adulterated *Hirudo medicinalis*, as an indicator, and employs liquid chromatography-mass spectrometry (LC-MS) technology to rapidly and accurately detect whether *Hirudo medicinalis* is adulterated in *Hirudo medicinalis*, its processed forms, and prepared Chinese medicines.

[0007] The structural formula of the Bdelline B is as follows:

[0008]

[0009] The method for detecting adulterated Hirudo medicinal material, processed slices and traditional Chinese medicine preparations of Hirudo macrocarpa, provided by the present invention, includes the following steps:

[0010] 1) Preparation of reference solution

[0011] Preparation of reference solution: Take reference Bdelline B, add methanol to prepare a 1 mg / mL stock solution, accurately pipette 1 μL of the stock solution, add 999 μL of methanol to prepare a reference solution with a Bdelline B concentration of 1 μg / mL;

[0012] 2) Preparation of test solution: The medicinal material or slices of the broad-bodied golden thread leech to be tested are pulverized and passed through a 50-mesh sieve, or the contents of the traditional Chinese medicine to be tested are taken out, 0.1g of the medicinal material powder is added to 1mL of 80% methanol and vortexed to mix well, weighed, and ultrasonicated for 1h. Then, 80% methanol is added to make up to the original weight, centrifuged at 4000r for 15min, the supernatant is collected, methanol is added for dilution, and centrifuged at 14000r for 10min. The supernatant is collected.

[0013] 3) Inject the reference solution obtained in step 1) into an ultra-high performance liquid chromatography-mass spectrometry instrument to obtain the ion chromatogram of Bdelline B;

[0014] 4) Inject the supernatant obtained in step 2) into an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) instrument to obtain the ion chromatogram of the test sample. Compare it with the ion chromatogram of Bdelline B obtained in step 3). If a chromatographic peak with the same retention time as the Bdelline B reference standard appears in the ion chromatogram of the test sample, it indicates that phenanthrene leech has been added to the test sample. If no chromatographic peak with the same retention time as the Bdelline B reference standard appears (S / N < 3), it indicates that Bdelline B was not detected in the test sample, and it is considered that phenanthrene leech has not been added.

[0015] In steps 3) and 4) of the above method, the ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) detection conditions are as follows:

[0016] Chromatographic conditions: The chromatographic column was a Waters BEH C18 column (1.7 μm, 2.1 mm ID × 100 mm);

[0017] Mobile phase A: water (containing 0.1% (volume ratio) formic acid), phase B: acetonitrile (containing 0.1% (volume ratio) formic acid);

[0018] Gradient elution (Table 1):

[0019] Table 1 Gradient elution program

[0020]

[0021] Flow rate: 400 μL / min; Injection volume: 3 μL;

[0022] Mass spectrometry conditions: A triple quadrupole mass spectrometer detector and an electrospray ionization source (ESI ionization source) were used in positive ion scanning multiple reaction monitoring (MRM) mode. Specific mass spectrometry operating parameters are shown in Table 2.

[0023] Table 2 Mass Spectrometry Operating Parameters

[0024]

[0025] The application of the above method in the quality control of Hirudo medicinal materials, processed slices and traditional Chinese medicine preparations is also within the scope of protection of this invention.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The method established in this invention can be used to detect adulterated Hirudo medicinal materials, processed slices, and traditional Chinese medicine preparations containing leeches containing fennel. This method is simple to operate, fast in detection, and has good specificity. It is not affected by the complex process of the sample and effectively fills the technical gap that DNA barcoding cannot detect traditional Chinese medicine preparations. It is a technology based on chemical composition detection of adulterated Hirudo medicinal materials containing leeches containing fennel, providing a new method for the quality control of leeches. Attached Figure Description

[0028] Figure 1 Extraction ion chromatograms of Bdelline B at m / z 523.1→435.9 and m / z 523.1→320.9.

[0029] Figure 2 This is a secondary mass spectrum of Bdelline B in positive ion mode.

[0030] Figure 3 The extraction ion chromatograms of Hirudo medicinalis F1-F7 at m / z 523.1→320.9 are shown.

[0031] Figure 4 Extraction ion chromatograms of broad-bodied golden leeches K1-K7 at m / z 523.1→320.9. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] Example 1: Verification that Bdelline B is a differential component between Hirudo nipponia and Hirudo floribunda.

[0035] 1. Experimental Instruments and Materials

[0036] 1.1 Instruments

[0037] The following equipment was used: an API 6500Qtrap liquid chromatography-mass spectrometry system from Applied Biosystems (including a G4220A pump, G1316C column oven, G4226A injector, G1330B thermostat, ESI and APCI ion sources, and Analyst 1.6.3 mass spectrometry workstation software); a KQ-500E ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); a DV215CD electronic balance (Ohaus Instruments (Shanghai) Co., Ltd.); and a BY-R18 medical centrifuge (Beijing Baiyang Medical Instruments Co., Ltd.).

[0038] 1.2 Reagents

[0039] Mass spectrometry grade methanol; mass spectrometry grade acetonitrile; mass spectrometry grade formic acid, all purchased from Fisher Scientific; Watson's purified water; Bdelline B (obtained from Hirudo medicinalis by Baoji Chenguang Biotechnology Co., Ltd., with a purity of 98.42%).

[0040] The structural formula for Bdelline B is shown below:

[0041]

[0042] 1.3 Sample Information

[0043] Table 3 Information on Hirudo medicinalis Samples

[0044]

[0045]

[0046] Table 4. Information on samples of broad-bodied golden thread leeches

[0047]

[0048] All of the above samples were identified by DNA barcoding, and their origins were accurate.

[0049] 2. Experimental Methods

[0050] 2.1 Preparation of reference solution: Take reference Bdelline B, add methanol to prepare a 1 mg / mL stock solution, accurately pipette 1 μL of the stock solution, add 999 μL of methanol to prepare a reference solution with a Bdelline B concentration of 1 μg / mL.

[0051] 2.2 Preparation of test solution: The medicinal materials of Hirudo medicinalis and Hirudo medicinalis were pulverized and passed through a 50-mesh sieve. 0.1 g of the medicinal material powder was added to 1 mL of 80% methanol and vortexed to mix well. The mixture was weighed and sonicated for 1 h. Then, 80% methanol was added to make up to the original weight. The mixture was centrifuged at 4000 r for 15 min. The supernatant was collected, diluted with an appropriate amount of methanol, and centrifuged at 14000 r for 10 min. The supernatant was then injected into a liquid chromatography-mass spectrometry (LC-MS) instrument.

[0052] 2.3 Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) detection:

[0053] Chromatographic conditions: Waters BEH C18 column (1.7 μm, 2.1 mm ID × 100 mm); mobile phase A: water (containing 0.1% (v / v) formic acid), mobile phase B: acetonitrile (0.1% (v / v) formic acid); gradient elution program: 0–1 min, 25% B, 1–2.5 min, 25% B–100% B, 2.5 min–4 min, 100% B, 4–4.1 min, 100% B–25% B; flow rate 400 μL / min; injection volume 3 μL.

[0054] Mass spectrometry conditions: A triple quadrupole mass spectrometer detector and an electrospray ionization source (ESI ionization source) were used in multiple reaction monitoring (MRM) mode with positive ion scanning. Specific mass spectrometry operating parameters are shown in Table 2.

[0055] 3. Experimental Results

[0056] Figure 1 Extraction ion chromatograms of Bdelline B at m / z 523.1→435.9 and m / z 523.1→320.9.

[0057] Figure 2 This is a secondary mass spectrum of Bdelline B in positive ion mode.

[0058] The results of Bdelline B determination in each batch of Hirudo nipponia and Hirudo floribunda are shown in Table 5.

[0059] Table 5. Results of Bdelline B determination in Hirudo medicinalis and Hirudo macrocarpa.

[0060]

[0061]

[0062] Figure 3 The extraction ion chromatograms of Hirudo medicinalis F1-F7 at m / z 523.1→320.9 are shown.

[0063] Figure 4Extraction ion chromatograms of broad-bodied golden leeches K1-K7 at m / z 523.1→320.9.

[0064] Therefore, it can be seen that in the ion chromatograms of 18 batches of Hirudo nipponia extracted at m / z 523.1→435.9 and m / z 523.1→320.9, chromatographic peaks with retention times consistent with Bdelline B reference standard were observed. However, in the ion chromatograms of 18 batches of Hirudo floribunda extracted at m / z 523.1→435.9 and m / z 523.1→320.9, the signal-to-noise ratio of the chromatographic peaks was less than 3, meaning that no chromatographic peaks with retention times consistent with Bdelline B reference standard were observed, and the results showed that Bdelline B was not detected. This indicates that Bdelline B is a differential component in Hirudo nipponia and can be used to distinguish between Hirudo nipponia and Hirudo floribunda.

[0065] Example 2, Methodological Investigation

[0066] 2.1 Linear Relationship

[0067] Accurately weigh an appropriate amount of Bdelline B reference standard, add methanol to prepare a 1 mg / mL stock solution, and serially dilute the stock solution to prepare Bdelline B reference standard solutions of 1000 ng / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, 1 ng / mL, and 0.5 ng / mL. Determine the Bdelline B reference standard under the chromatographic and mass spectrometric conditions described in the above detection method. Plot a standard curve with the peak area of ​​the chromatographic peak in the Bdelline B m / z 523.1→320.9 extraction ion chromatogram as the ordinate and the concentration of the reference standard solution as the abscissa. The linear regression equation for Bdelline B is Y = 632X - 45.7, R = 0.9952, indicating good linearity.

[0068] 2.2 Stability

[0069] Quality control (QC) values ​​of Bdelline B at low, medium, and high concentrations were measured at 0h, 4h, 8h, 12h, and 24h after preparation, respectively, under the chromatographic and mass spectrometric conditions described in the above detection method. The RSDs of the concentrations calculated by substituting the peak areas into the Bdelline B standard curve were 2.12%, 2.78%, and 3.76%, respectively, indicating that the component has good stability within 24h.

[0070] 2.3 Precision

[0071] Six quality control (QC) samples at three concentrations (low, medium, and high) of Bdelline B were taken and analyzed repeatedly under the same chromatographic and mass spectrometric conditions. The intra-day precision RSD (n=6) was calculated to be 2.83%, 2.10%, and 1.89%, respectively. The six QC samples at the three concentrations were analyzed for three consecutive days, and the inter-day precision RSD (n=6) was calculated to be 1.59%, 1.79%, and 1.87%, respectively, indicating that the instrument precision was good.

[0072] 2.4 Detection Limit

[0073] The Bdelline B reference solution was serially diluted. The limit of detection for the compound B was calculated to be 0.5 ng / mL based on the signal-to-noise ratio of the chromatographic peak in the extract ion chromatogram of Bdelline B m / z 523.1→320.9 with a peak signal-to-noise ratio of 3:1.

[0074] 2.5 Repeatability

[0075] Six F14 samples were taken and prepared into test solutions according to the above method. They were then detected under the same chromatographic and mass spectrometric conditions. The RSD (n=6) of the content was calculated by substituting the peak area into the Bdelline B standard curve and found to be 1.79%, indicating that the method has good repeatability.

[0076] 2.6 Recovery rate

[0077] Approximately 0.1 g of the pre-concentrated F14 sample was accurately weighed, and appropriate amounts of reference solutions at three levels (80%, 100%, and 120%) of the corresponding concentration of Bdelline B compound were accurately added to prepare the test solution. The content of this component was determined, and the recoveries were calculated to be 106.39%, 98.44%, and 100.56%, respectively, with RSDs of 3.86%, 2.07%, and 3.98%, respectively, indicating that the method has good accuracy.

[0078] Example 3: Detection of processed medicinal materials and prepared Chinese medicines

[0079] 1. Sample Information

[0080] This embodiment collects leech formula granules and commonly used traditional Chinese medicines containing leeches. Specific information is shown in Table 6.

[0081] Table 6 Information on Traditional Chinese Medicine Samples

[0082]

[0083] 2. The experimental method is the same as in Example 1.

[0084] 3. Experimental Results

[0085] Table 7 Results of Bdelline B determination in traditional Chinese medicine preparations

[0086]

[0087] Results: The ion chromatograms of Huoxue Tongmai Capsules extracted at m / z 523.1→435.9 and m / z 523.1→320.9 both showed peaks with retention times consistent with Bdelline B reference standard, indicating that both traditional Chinese medicines may have been adulterated with Hirudo medicinalis. The signal-to-noise ratios of the peaks in the ion chromatograms of Naoxuekang Capsules, Dahuang Zhechong Pills, and Hirudo medicinalis granules extracted at m / z 523.1→435.9 and m / z 523.1→320.9 were all less than 3, meaning no peaks with retention times consistent with Bdelline B reference standard appeared, and the results indicated no adulteration.

[0088] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for detecting adulteration of Hirudo medicinalis, processed medicinal slices, and prepared Chinese medicines with Hirudo medicinalis, comprising the following steps: 1) Preparation of reference solution: Take reference Bdelline B, add methanol to prepare a 1 mg / mL stock solution, accurately pipette 1 μL of the stock solution, add 999 μL of methanol to prepare a reference solution with a Bdelline B concentration of 1 μg / mL; 2) Preparation of test solution: The medicinal material or slices of the broad-bodied golden thread leech to be tested are pulverized and passed through a 50-mesh sieve, or the contents of the traditional Chinese medicine to be tested are taken out. Take 0.1g of the medicinal material powder, add 1 mL of 80% methanol and vortex mix well, weigh, sonicate for 1 hour, add 80% methanol to make up to the original weight, centrifuge for 15 min, take the supernatant, add methanol to dilute, centrifuge for 10 min, and take the supernatant. 3) Inject the reference solution obtained in step 1) into an ultra-high performance liquid chromatography-mass spectrometry instrument to obtain the ion chromatogram of Bdelline B; 4) Inject the supernatant obtained in step 2) into an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) instrument to obtain the ion chromatogram of the test sample. Compare it with the ion chromatogram of Bdelline B obtained in step 3). If a chromatographic peak with the same retention time as the Bdelline B reference standard appears in the ion chromatogram of the test sample, it indicates that phenanthrene leech has been added to the test sample. If no chromatographic peak with the same retention time as the Bdelline B reference standard appears, it indicates that Bdelline B was not detected in the test sample, and it is considered that phenanthrene leech has not been added. The structural formula of Bdelline B is: Ultra-high performance liquid chromatography, chromatographic conditions: Waters BEH C18 column; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile containing 0.1% formic acid. Gradient elution: 0-1 min 25%B, 1-2.5 min 25%B-100%B, 2.5 min-4 min 100%B, 4-4.1 min 100%B-25%B; Mass spectrometry conditions: A triple quadrupole mass spectrometer detector and an electrospray ionization source were used in multiple reaction monitoring mode with positive ion scanning. The mass spectrometry operating parameters were as follows: Qualitative ion pairs of Bdelline B: the parent ion is m / z 523.1, daughter ion is m / z 435.9, CE is 30 eV; Quantitative ion pairs: the parent ion is m / z 523.1, daughter ion is m / z 320.9, CE is 45 eV.

2. The method according to claim 1, characterized in that: Mobile phase flow rate: 400 µL / min; injection volume: 3 μL.

3. The application of the method described in claim 1 or 2 in the quality testing of Hirudo medicinal materials, processed slices and traditional Chinese medicine preparations.

Images