A high-resolution mass spectrometry method for screening ganoderma lucidum adulterated with ganoderma applanatum

Abstract

The application provides a high-resolution mass spectrometry method for screening Ganoderma lucidum adulterated with Ganoderma amphiacep. The method belongs to the technical field of medicine analysis and comprises the following steps: preparing a test sample solution from a to-be-tested Ganoderma lucidum sample; using an ultra-high performance liquid chromatography-electric field orbitrap high-resolution mass spectrometry technology to detect the test sample solution under a negative ion mode and with 0.1% formic acid-acetonitrile gradient elution; extracting characteristic ions with mass-to-charge ratios of 357.17 and 599.32 from a total ion chromatogram obtained through detection; and determining that the to-be-tested Ganoderma lucidum sample contains Ganoderma amphiacep if a chromatographic peak with a mass-to-charge ratio of 357.17 and two isomer chromatographic peaks with a mass-to-charge ratio of 599.32 are detected simultaneously. The application uses high-resolution mass spectrometry to screen specific characteristic compounds of Ganoderma amphiacep, establishes a unified screening method suitable for medicinal materials, decoction pieces and Chinese patent medicines, and solves the problem that the prior art cannot distinguish Ganoderma amphiacep from genuine Ganoderma lucidum.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a high-resolution mass spectrometry method for screening for adulterated Ganoderma lucidum products such as sessile Ganoderma lucidum. Background Technology

[0002] Reishi mushroom is a traditional Chinese medicine with extremely high medicinal value and broad application prospects. With increasing clinical use, wild resources can no longer meet the demand, leading to a surge in cultivated varieties entering the market and gradually dominating. However, while reishi production has increased year by year, problems have also arisen, including a chaotic mix of cultivated varieties, unclear varietal characteristics of commercially available reishi, and inconsistent quality of reishi-related products. Recent studies have discovered that a sessile reishi (Ganoderma lucidum) species with highly similar morphological characteristics to reishi mushroom has been mixed into cultivated varieties. Ganoderma sessile Developing a method to effectively screen for sessile Ganoderma lucidum is of great significance for ensuring the quality of Ganoderma lucidum medicinal materials and their preparations.

[0003] The 2025 edition of the Chinese Pharmacopoeia uses thin-layer chromatography (TLC) with Ganoderma lucidum reference material as a reference for identification, and ultraviolet spectrophotometry (UV) is used to determine the content of polysaccharides and triterpenes. However, experiments have shown that the TLC behavior of sessile Ganoderma lucidum is consistent with that of Ganoderma lucidum, and the polysaccharide and triterpene content in sessile Ganoderma lucidum is also very close to that in Ganoderma lucidum. Therefore, the current Chinese Pharmacopoeia standard for Ganoderma lucidum cannot effectively distinguish between sessile Ganoderma lucidum and Ganoderma lucidum. The United States Pharmacopeia (USP) 41 stipulates that the high-performance liquid chromatogram of Ganoderma lucidum should show the same chromatographic peaks as the 10 ganoderic acid reference standards, including ganoderic acid C, ganoderic acid C2, ganoderic acid G, and ganoderic acid B. Studies have found that the number of such chromatographic peaks in sessile Ganoderma lucidum is significantly less than that in Ganoderma lucidum (both Ganoderma lucidum and Ganoderma sinense are legal sources of Ganoderma lucidum in the Chinese Pharmacopoeia). This method can effectively distinguish between Ganoderma lucidum and sessile Ganoderma lucidum, but the above compounds cannot be detected simultaneously in Ganoderma sinense. Therefore, when using this method for screening, Ganoderma sinense is easily misidentified as sessile Ganoderma lucidum.

[0004] High-performance liquid chromatography-mass spectrometry (HPLC-MS) is a commonly used technique for chemical composition analysis and species identification of Ganoderma lucidum. Existing research has collected 26 species of the Ganoderma genus and constructed a phylogenetic tree based on the ITS+tef1 sequence matrix for molecular biological identification, classifying them as Ganoderma lucidum (…). Ganoderma lingzhi ), Ganoderma lucidum ( Ganoderma sinense ) and stalkless Ganoderma ( Ganoderma sessileThis study also used ultra-high performance liquid chromatography-quadrupole mass spectrometry (UHPLC-QQQ-MS) to analyze triterpenoid compounds in Ganoderma lucidum mycelium. Specific components of Ganoderma lucidum mycelium (specifically, those of Ganoderma sinense and Ganoderma purpurea) were obtained, providing a new approach for chemical taxonomy identification. However, the method has the following limitations: First, it only obtains specific components of Ganoderma sinense and Ganoderma purpurea, not those of Ganoderma sessileum. While it can be used to screen for the presence of Ganoderma sinense or Ganoderma purpurea in samples, it cannot screen for the presence of Ganoderma sessileum. Second, these components are from the mycelium, requiring the mycelium to be isolated and cultured before compound extraction. This method is suitable for medicinal materials or processed medicinal slices, but it is difficult to screen medicinal materials used in traditional Chinese medicine preparations because these materials often undergo extraction (e.g., decoction), making it difficult to obtain the mycelium, and the components in the mycelium differ significantly from those in the fruiting body. Third, the method is complex, involving multiple steps such as mycelial isolation, culture, DNA extraction, and PCR amplification, resulting in a long processing time.

[0005] In summary, sessile Ganoderma lucidum, as a counterfeit of Ganoderma lucidum, has been circulating in the market. Existing methods cannot effectively distinguish sessile Ganoderma lucidum from Ganoderma lucidum, or are complicated to operate and have limited applicability. There is currently no screening method that is easy to operate, highly sensitive, has high resolution, and can be applied to various sample types such as medicinal materials, processed slices, and traditional Chinese medicine preparations. Summary of the Invention

[0006] To address the problems of existing methods being unable to effectively distinguish between sessile Ganoderma lucidum and genuine Ganoderma lucidum, or being complex to operate and having limited applicability, this invention provides a high-resolution mass spectrometry method for screening sessile Ganoderma lucidum, a counterfeit product. By using high-resolution mass spectrometry to screen for the specific characteristic compounds of sessile Ganoderma lucidum, a unified screening method applicable to medicinal materials, processed medicinal materials, and traditional Chinese medicine preparations is established, solving the problem that existing technologies cannot distinguish between sessile Ganoderma lucidum and genuine Ganoderma lucidum.

[0007] The embodiment provides a high-resolution mass spectrometry method for screening Ganoderma lucidum adulterants, specifically the sessile Ganoderma lucidum, comprising the following steps: (1) Prepare a test solution from the Ganoderma lucidum sample to be tested; (2) The detection was performed using ultra-high performance liquid chromatography-electrostatic field orbital trap high resolution mass spectrometry in negative ion mode with gradient elution of 0.1% formic acid-acetonitrile; (3) Extract characteristic ions with mass-to-charge ratios of 357.17 and 599.32 from the total ion chromatogram obtained by detection; if the chromatographic peak with a mass-to-charge ratio of 357.17 and the two isomer chromatographic peaks with a mass-to-charge ratio of 599.32 are detected at the same time, it is determined that there is sessile Ganoderma in the sample to be tested.

[0008] Preferably, step (1) specifically includes: taking the Ganoderma lucidum sample to be tested, adding methanol for ultrasonic extraction, filtering, and taking the filtrate as the test solution.

[0009] More preferably, the Ganoderma lucidum sample to be tested is Ganoderma lucidum medicinal material, Ganoderma lucidum decoction pieces, or traditional Chinese medicine made from Ganoderma lucidum.

[0010] Preferably, the chromatographic conditions in step (2) are as follows: the chromatographic column is a Waters Acquity UPLC HSS T3 column; the gradient elution program is 0~18 min for acetonitrile to increase from 25% to 50%, and 18~22 min for acetonitrile to increase from 50% to 90%; the flow rate is 0.3 mL / min; the column temperature is 30℃; and the injection volume is 1 µL.

[0011] Preferably, the mass spectrometry conditions in step (2) are as follows: using an LTQ-Orbitrap electrostatic field orbital trap high-resolution mass spectrometer, an electrospray ion source, a sheath gas flow rate of 50 arb, an auxiliary gas flow rate of 15 arb, a spray voltage of 2.8 kV, an ion transmission tube temperature of 300℃, an ion source temperature of 350℃, a scan range mass-to-charge ratio of 50 to 1500, a primary resolution of 60,000, a secondary resolution of 30,000, and a collision voltage of 35 eV.

[0012] Preferably, when determining by mass-to-charge ratio, the chromatographic peak with a mass-to-charge ratio of 357.17 is used as the reference peak, and the relative retention times of the two isomer chromatographic peaks with a mass-to-charge ratio of 599.32 are 1.58 and 1.72, respectively, and their relative retention times should be within ±10% of the specified value.

[0013] Preferably, in the determination step, if the three chromatographic peaks are detected simultaneously in the Ganoderma lucidum sample to be tested, it is determined that the Ganoderma lucidum sample to be tested is mixed with sessile Ganoderma lucidum.

[0014] On the other hand, the embodiments also provide the application of the high-resolution mass spectrometry method for screening Ganoderma lucidum adulterants, Ganoderma lucidum sessileum, in the qualitative detection of traditional Chinese medicine preparations.

[0015] Compared with the prior art, the beneficial effects of the present invention include at least the following: (1) The characteristic compound mass spectrometry information of Ganoderma lucidum adulterant Ganoderma lucidum was obtained, which can effectively screen out Ganoderma lucidum adulterant in medicinal materials, decoction pieces and traditional Chinese medicine.

[0016] (2) Based on the mass spectrometry information of characteristic compounds, a screening method was established by using ultra-high performance liquid chromatography-electrostatic field orbital trap high resolution mass spectrometry. This method can perform qualitative analysis on Ganoderma lucidum raw materials, Ganoderma lucidum slices, and Ganoderma lucidum adulterants in traditional Chinese medicine prescriptions containing Ganoderma lucidum. It has high sensitivity and strong specificity, overcoming the shortcomings of previous chemical methods and high performance liquid chromatography methods, which cannot effectively screen Ganoderma lucidum. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0018] Figure 1 This is a schematic flowchart of a high-resolution mass spectrometry method for screening Ganoderma lucidum adulterants, specifically the sessile Ganoderma lucidum, provided as an example.

[0019] Figure 2 Chromatograms of characteristic components extracted from Ganoderma lucidum from Ganoderma lucidum, Ganoderma sinense, and Ganoderma sessileum, respectively, are provided for the examples.

[0020] Figure 3 The ion current characteristic spectrum of Ganoderma lucidum purchased online is provided as an example.

[0021] Figure 4 The ion current spectrum of Ganoderma lucidum slices extracted using sessile Ganoderma lucidum as feed is provided for the example. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and given in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0023] The samples used in the examples were all purchased from the market.

[0024] This embodiment provides a high-resolution mass spectrometry method for screening for adulterants such as sessile Ganoderma lucidum. By using high-resolution mass spectrometry to screen for characteristic compounds of sessile Ganoderma lucidum, it is possible to effectively screen for sessile Ganoderma lucidum in medicinal materials, processed slices, and traditional Chinese medicine preparations, thereby achieving the purpose of distinguishing genuine from counterfeit products and providing a basis for the quality control and supervision of Ganoderma lucidum. Figure 1 As shown, the details are as follows: (1) Prepare the test solution from the Ganoderma lucidum sample: Take about 0.5 g of the powdered Ganoderma lucidum sample to be tested, place it in a stoppered conical flask, add 40 mL of methanol, seal tightly, sonicate (power 300 W; frequency 40 kHz) for 30 minutes, cool, filter, and take the filtrate as the test solution.

[0025] The Ganoderma lucidum samples to be tested included: Ganoderma lucidum (Red Ganoderma) Ganoderma lucidum ), Ganoderma lucidum ( Ganoderma sinense ) and stalkless Ganoderma ( Ganoderma sessile ).

[0026] (2) Using ultra-high performance liquid chromatography (UHPLC) coupled with low-frequency electrostatic field orbital trap mass spectrometry (LTQ-Orbitrap-MS), a UHPLC-LTQ-Orbitrap-MS method was established to perform chromatographic separation and mass spectrometric analysis of components in Ganoderma lucidum sessileum, obtaining the accurate mass of the parent and daughter ions of each separated component, and screening for differential components among different Ganoderma lucidum species. Details are as follows: The instrument used was a Thermo Fisher UHPLC-LTQ-Orbitrap liquid chromatography-mass spectrometry system.

[0027] The chromatographic conditions in the examples (Table 1) included: a Waters Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm); gradient elution with 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B, as specified in Table 1; a flow rate of 0.3 mL / min; a column temperature of 30 °C; and an injection volume of 1 µL.

[0028] Table 1 The mass spectrometry conditions in the embodiments include: the mass spectrometry analysis was performed using an LTQ-Orbitrap electrostatic field orbital trap high-resolution mass spectrometer, the ion source was an electrospray (ESI) ion source, the sheath gas flow rate was 50 arb, the auxiliary gas flow rate was 15 arb, the spray voltage was 2.8 kV in negative ion mode, the ion transmission tube temperature was 300℃, the ion source temperature was 350℃, the negative ion mode was used for scanning, the acquisition method was full scan / data-dependent two-stage scan, the mass-to-charge ratio (m / z) of the scanning range was 150~1500, the first-stage resolution was 60000, the second-stage resolution was 30000, and the collision voltage was 35 eV.

[0029] (3) Extract characteristic ions with mass-to-charge ratios of 357.17 and 599.32 from the total ion chromatogram obtained from the detection; if a chromatographic peak with a mass-to-charge ratio of 357.17 and two isomer chromatographic peaks with a mass-to-charge ratio of 599.32 are detected simultaneously, it is determined that sessile Ganoderma lucidum exists in the sample to be tested. Specifically: By means of the assay method, 1 μL of each test solution is injected into a UHPLC-LTQ-Orbitrap liquid chromatography-mass spectrometry instrument for analysis. Judgment criteria: In the examples, characteristic ions were extracted from the obtained total ion chromatogram of the sample. In the ion chromatogram of *Ganoderma lucidum* extract, peak 1 had the highest response, with a mass-to-charge ratio (m / z) of 357.17. Peaks 2 and 3 were isomers, with a mass-to-charge ratio (m / z) of 599.32. If all three chromatographic peaks of *Ganoderma lucidum* were extracted from the sample simultaneously, the sample was considered adulterated with *Ganoderma lucidum*, which did not meet the requirements and was deemed unqualified.

[0030] First, the specificity of the high-resolution mass spectrometry method was validated, and a method for screening specific compounds of Ganoderma lucidum in different Ganoderma lucidum species (Ganoderma sinense, Ganoderma aurantium, and Ganoderma sessileum) was provided. Specific spectra can be found in [link to relevant data]. Figure 2 The results showed that the characteristic components of Ganoderma lucidum were only present in the Ganoderma lucidum sample, indicating that the mass spectrometry information of the characteristic compounds of Ganoderma lucidum obtained by this method has good specificity.

[0031] Based on the mass spectrometry information of characteristic compounds, a screening method was established using ultra-high performance liquid chromatography-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-EEG ... Figure 3 The other 37 batches of samples were all Ganoderma lucidum, which met the requirements.

[0032] In response to the current market situation where sessile Ganoderma lucidum is being used to impersonate Ganoderma lucidum, this practice has been found not only in raw medicinal materials and processed medicinal slices but also in prepared Chinese medicines. Sessile Ganoderma lucidum contains extremely low levels of the active ingredient, triterpenoid acids, compared to Ganoderma lucidum, severely impacting the efficacy of the medicine. Based on the same inventive concept, the embodiments also provide the application of the high-resolution mass spectrometry method for screening sessile Ganoderma lucidum adulterants in the qualitative detection of prepared Chinese medicines.

[0033] This example collected 14 batches of Ganoderma lucidum tablets, which are tablets made by boiling Ganoderma lucidum in water. The method described in this paper was used to detect the Ganoderma lucidum tablets. The results showed that two batches of Ganoderma lucidum tablets from one company contained characteristic components of Ganoderma lucidum sessile (such as...). Figure 4As shown in the image, the sample is consistent with the sessile Ganoderma lucidum, proving that the Ganoderma lucidum used by the company is sessile Ganoderma lucidum. Using raw materials that differ from the standard regulations violates the Drug Administration Law and seriously harms consumer interests. Therefore, the method of this invention can effectively screen out Ganoderma lucidum slices made with sessile Ganoderma lucidum, providing a strong methodological basis for supervision. Furthermore, the company claims to use Ganoderma lucidum var. rubrum as its raw material. If the company has not concealed the truth, it suggests that the company itself may lack the ability to distinguish between genuine Ganoderma lucidum and counterfeit products. The method established by this invention is applicable to both raw materials and traditional Chinese medicine preparations, providing a powerful method for manufacturers to control the quality of raw materials and thus produce high-quality medicines.

[0034] In summary, the method established in this invention can effectively screen for sessile Ganoderma lucidum in Ganoderma lucidum raw materials, Ganoderma lucidum decoction pieces, and traditional Chinese medicines containing Ganoderma lucidum.

[0035] Finally, it should be noted that the above-described embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and not to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the technical scope disclosed in the present invention, or make equivalent substitutions for some of the technical features; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A high-resolution mass spectrometry method for screening Ganoderma lucidum adulterants, specifically sessile Ganoderma lucidum, characterized in that, Includes the following steps: (1) Prepare a test solution from the Ganoderma lucidum sample to be tested; (2) The detection was performed using ultra-high performance liquid chromatography-electrostatic field orbital trap high resolution mass spectrometry in negative ion mode with gradient elution of 0.1% formic acid-acetonitrile; (3) Extract characteristic ions with mass-to-charge ratios of 357.17 and 599.32 from the total ion chromatogram obtained by detection; if the chromatographic peak with a mass-to-charge ratio of 357.17 and the two isomer chromatographic peaks with a mass-to-charge ratio of 599.32 are detected at the same time, it is determined that there is sessile Ganoderma in the sample to be tested.

2. The high-resolution mass spectrometry method for screening Ganoderma lucidum adulterants, specifically the sessile Ganoderma lucidum, according to claim 1, is characterized in that... Step (1) specifically includes: taking the Ganoderma lucidum sample to be tested, adding methanol for ultrasonic extraction, filtering, and taking the filtrate as the test solution.

3. The high-resolution mass spectrometry method for screening Ganoderma lucidum adulterants, specifically the sessile Ganoderma lucidum, according to claim 2, is characterized in that... The Ganoderma lucidum sample to be tested is Ganoderma lucidum medicinal material, Ganoderma lucidum decoction pieces, or traditional Chinese medicine made from Ganoderma lucidum.

4. The high-resolution mass spectrometry method for screening Ganoderma lucidum adulterants, specifically the sessile Ganoderma lucidum, according to claim 1, is characterized in that... The chromatographic conditions in step (2) are as follows: the column is a Waters Acquity UPLC HSS T3 column; the gradient elution program is 0~18 min acetonitrile from 25% to 50%, and 18~22 min acetonitrile from 50% to 90%; the flow rate is 0.3 mL / min; the column temperature is 30℃; and the injection volume is 1 µL.

5. The high-resolution mass spectrometry method for screening Ganoderma lucidum adulterants, specifically the sessile Ganoderma lucidum, according to claim 1, is characterized in that... The mass spectrometry conditions in step (2) are as follows: LTQ-Orbitrap electrostatic field orbital trap high-resolution mass spectrometer, electrospray ion source, sheath gas flow rate 50 arb, auxiliary gas flow rate 15 arb, spray voltage 2.8 kV, ion transmission tube temperature 300℃, ion source temperature 350℃, scan range mass-to-charge ratio 50~1500, primary resolution 60000, secondary resolution 30000, collision voltage 35eV.

6. The high-resolution mass spectrometry method for screening Ganoderma lucidum adulterants, specifically the sessile Ganoderma lucidum, according to claim 1, is characterized in that... When determining the mass-to-charge ratio, the chromatographic peak with a mass-to-charge ratio of 357.17 is used as the reference peak. The relative retention times of the two isomer chromatographic peaks with a mass-to-charge ratio of 599.32 are 1.58 and 1.72, respectively, and their relative retention times should be within ±10% of the specified value.

7. The high-resolution mass spectrometry method for screening Ganoderma lucidum adulterants, specifically the sessile Ganoderma lucidum, according to claim 1, is characterized in that... In the determination step, if the three chromatographic peaks are detected simultaneously in the Ganoderma lucidum sample to be tested, it is determined that the Ganoderma lucidum sample to be tested is mixed with sessile Ganoderma lucidum.

8. The application of the high-resolution mass spectrometry method for screening Ganoderma lucidum adulterants, as described in any one of claims 1 to 7, in the qualitative detection of traditional Chinese medicine preparations.

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