A method for determining whether Poria cocos has been fumigated with sulfur.

Abstract

This invention provides a method for determining whether Poria cocos has been fumigated with sulfur. The method includes sample extraction, concentration, enrichment, detection, and fragment analysis. It is the first to discover a direct relationship between changes in relevant marker fragments and sulfur fumigation, and to identify the presence and relationship of Poria cocos sulfonic acid H and its isomers in sulfur-fumigated samples. Furthermore, by enriching specific components, the presence of these components was discovered in chromatograms and mass spectrometry analyses, laying the foundation for this method. It also overcomes the technical shortcomings of existing technologies, such as false negatives and large sample requirements, and has broad industrial applicability.

Description

Technical Field

[0001] This invention provides a method for determining whether Poria cocos has been fumigated with sulfur, which belongs to the field of traditional Chinese medicine and food testing. Background Technology

[0002] Sulfur fumigation is a common treatment method in the processing and storage of Chinese medicinal herbs and their food products, serving functions such as whitening, preventing mold and decay, and repelling insects. However, recent studies have found that sulfur-fumigated herbs and foods are not only prone to exceeding the standard for residual sulfur dioxide, which harms human health and can easily cause allergies, leading to symptoms such as difficulty breathing, diarrhea, and vomiting, but also alter the chemical composition of the herbs themselves, resulting in a decrease in their original medicinal and nutritional value. Furthermore, some heavy metals from industrial sulfur may be adsorbed onto the herbs, increasing their toxicity.

[0003] Poria cocos is a common Chinese medicinal herb used in clinical practice. Therefore, it is crucial to establish an accurate method to determine whether Poria cocos has been fumigated with sulfur for quality control.

[0004] Currently, the market and industry mainly rely on smelling the odor or detecting residual sulfur dioxide on the medicinal material to determine whether Poria cocos has been fumigated with sulfur. The method currently used in the Chinese Pharmacopoeia, specifically the 2020 edition, Part IV, General Chapter "2331 Determination of Sulfur Dioxide Residue," includes different methods for determining sulfur dioxide, such as acid-base titration, ion chromatography, and gas chromatography.

[0005] The disadvantages of the pharmacopoeia method are:

[0006] 1. Titration is relatively inexpensive and widely applicable, but its disadvantages are becoming increasingly apparent: low sensitivity (error 10 mg / kg), large sample requirement (10 g), cumbersome operation (reflux titration, distillation time 90 min), and low automation (single samples are processed sequentially). 2. Ion chromatography uses hydrogen peroxide to absorb sulfur dioxide and oxidize it to sulfate for determination. Thanks to the improved resolution of ion chromatography, the sensitivity has improved, but it still requires a long and cumbersome operation (distillation time of 30-40 min, additional settling time of 60 min). 3. Gas chromatography calculates residual sulfur dioxide by measuring sulfite in the sample and achieves automated sample injection, but the steps of sealing with wax and headspace injection are cumbersome and require a lot of time and effort from the operator.

[0007] More importantly, even after sulfur treatment, the residual sulfur dioxide content remains at an extremely low level (20-50 mg / kg) as the storage time is extended or desulfurization is carried out by heating. In some cases, the residual sulfur dioxide content of sulfur-fumigated samples is lower than the detection limit of this method (<10 mg / kg), resulting in false negative results. Therefore, the method cannot accurately determine whether Poria cocos has been fumigated with sulfur.

[0008] Therefore, there is an urgent need to establish stable and reliable rapid detection methods in the market. Summary of the Invention

[0009] To address the aforementioned technical issues, the applicant proposes a method for determining whether Poria cocos has been fumigated with sulfur, comprising the following steps:

[0010] 1) Take the Poria cocos sample to be tested, dissolve and extract it using an organic solvent, and concentrate it to obtain the sample solution;

[0011] 2) The sample solution described in step 1 is purified using a C18 solid-phase extraction column, and then concentrated to obtain a purified solution;

[0012] 3) The purified solution obtained in step 2) was analyzed using high performance liquid chromatography-time-of-flight coupling.

[0013] 4) Determine the presence of pamoate sulfonic acid H and its isomers in the sample to conclude whether the pamoate sample has been fumigated with sulfur. If the presence of pamoate sulfonic acid H and its isomers is detected, it is determined that the pamoate sample has been fumigated with sulfur; otherwise, it is determined that it has not been fumigated with sulfur.

[0014] In the above method, the organic solvent in step 1) is methanol, ethanol or an aqueous solution thereof.

[0015] In the above method, the organic solvent in step 1) is methanol or an aqueous methanol solution with a concentration greater than 70%.

[0016] In the above method, step 1) extraction is ultrasonic extraction.

[0017] In the above method, steps 1) and 2) involve concentration by rotary evaporation or boiling.

[0018] Other methods for removing solvents generally involve heating and evaporation or boiling.

[0019] In the above method, the C18 solid-phase extraction column mentioned in step 2 is a Waters Sep-Pak C18 solid-phase extraction column, preferably with a particle size of 55-105 micrometers (µm), pore size of 125 angstroms (Å), adsorbent weight of 200 milligrams (mg), pH range of 2-8, and tube size of 3 cc (cc: cubic centimeter, representing the volume of the extraction column body). The specifications of the extraction column can be adjusted according to the sample volume.

[0020] In the above method, the high-performance liquid chromatography (HPLC) conditions in step 3) are as follows: the chromatographic column is a Waters Acquity BEH C18 column, 100 Å, 1.8 µm, 2.1 mm × 100 mm, the column temperature is 35-45 °C, mobile phase A is 0.1% formic acid in water, and mobile phase B is 0.1% formic acid in acidified acetonitrile; the injection volume is 1-5 μL; the flow rate is 0.1-1 mL / min; the detection time is 2-10 min, and the elution gradient is: 0 min, 95% (v / v) mobile phase A: 5% (v / v) mobile phase B; 3 min, 40% (v / v) mobile phase A: 60% (v / v) mobile phase B; 5 min, 40% (v / v) mobile phase A: 60% (v / v) mobile phase B.

[0021] In the above method, during the flight time in step 3), the mass spectrometer uses an ESI ion source, selective ion scanning mode (Target MS / MS), target ion m / z 579.2, and scanning range 50-1700 m / z.

[0022] In the above method, the following parameters were collected during flight time: nitrogen gas was used as the atomizing gas, with a flow rate of 7 L / min; the atomizing gas temperature was 300 °C; the JetStream gas flow rate was 7 L / min; the sheath gas temperature was 300 °C; the atomizer pressure was 37 psi; the capillary voltage was 3000 V; the Skimmer voltage was 65 V; the Octopole voltage (RFV) was 600 V; the fragmentation voltage was 130 V; and the collision voltages were 25 V and 40 V, respectively, to obtain the chromatographic mass spectrum.

[0023] In the above method, the presence of pamoate sulfonic acid H and its isomers is determined by extracting and observing the characteristic ions at m / z 579.2→499.3 in the chromatogram and mass spectrum.

[0024] The purpose of step 1 is to dissolve and extract the sample components and concentrate the sample components.

[0025] The purpose of dissolving and extracting sample components from Poria cocos using organic solvents and concentrating them in a liquid sample is to prepare a sample for analysis using appropriate analytical techniques. Poria cocos contains a complex mixture of chemical components, including polysaccharides, triterpenoids, and other bioactive compounds. These components are present in small amounts and are often difficult to identify and quantify directly from a solid sample matrix. By using organic solvents to dissolve and extract sample components, we can separate the desired components from the solid sample matrix and concentrate them in a liquid sample. The concentrated sample can then be analyzed using analytical techniques such as liquid chromatography-mass spectrometry (LC-MS), which allows us to identify and quantify the individual components present in the sample.

[0026] The purpose of step 2 is to enrich the components to be detected.

[0027] In chemical analysis, the purpose of purifying and enriching samples is to remove unwanted components and concentrate the components of interest. The sample solution obtained from Poria cocos was purified using a C18 solid-phase extraction column, followed by concentration to obtain a purified solution. Solid-phase extraction (SPE) is a technique that purifies and concentrates a sample by selectively retaining the components of interest on a solid-phase extraction column while allowing unwanted components to pass through. In this case, a C18 column is used, a common SPE column that selectively retains hydrophobic compounds, such as triterpenoids and other bioactive components present in Poria cocos. After the sample solution passes through the C18 column, the desired components are retained on the column, while unwanted components are removed. The retained components are then eluted from the column using a solvent such as methanol or acetonitrile, thereby collecting them in a concentrated liquid sample. The purpose of this purification and enrichment step is to remove any unwanted components that may interfere with the analysis of the desired components and to concentrate the desired components. This improves the sensitivity and accuracy of analytical techniques used to detect and quantify the components of interest.

[0028] The purpose of step 3 is to perform rapid detection.

[0029] The described steps aim to rapidly and accurately detect components present in a sample by purifying it using a solid-phase extraction column and analyzing it using high-performance liquid chromatography-time-of-flight (UPLC-QTOF-MS / MS). UPLC-QTOF-MS / MS is a powerful analytical technique for separating, detecting, and quantifying individual components in complex mixtures, such as purified liquids obtained after solid-phase extraction. UPLC separation is based on the differential interactions of each component with the stationary and mobile phases. Individual components are then detected and quantified according to their mass ratios using a QTOF mass spectrometer. The purpose of purifying the sample using a solid-phase extraction column is to remove any unwanted components that might interfere with the analysis of the desired components. This improves the selectivity and sensitivity of UPLC-QTOF-MS / MS analysis, allowing for more accurate detection and quantification of target components. By rapidly and accurately detecting components present in a sample, valuable information about the chemical composition of the sample can be obtained, including the type and quantity of individual components present. This information can be used to assess the quality and consistency of traditional Chinese medicine products such as Poria cocos.

[0030] The purpose of step 4 is to locate key component fragments and ultimately confirm the existence of relevant facts.

[0031] To determine whether a Poria cocos sample has been fumigated with sulfur, it is necessary to detect the presence of pamoate H and its isomers. This can be accomplished using UPLC-QTOF-MS / MS analysis, which separates the components in the sample and detects them based on their mass-to-charge ratio. During UPLC-QTOF-MS / MS analysis, the key components of interest are broken down and their mass spectra are recorded. By comparing the mass spectra obtained from the sample with those obtained from a reference sample, the key components present in the sample, including pamoate H and its isomers, can be identified. If the presence of pamoate H and its isomers is detected in the sample (see...), the analysis will determine whether the sample has been treated with sulfur fumigation. Figure 5 and 6 If the result is positive, it can be concluded that the Poria cocos sample was fumigated with sulfur. This is because Poria cocos sulfonic acid H and its isomers are not naturally occurring compounds in Poria cocos, but are formed through sulfur fumigation.

[0032] Specifically, the above method consists of the following steps: In step (1), the amount of Poria cocos sample used is 0.1 g, the amount of 70% methanol used is 10 mL, the ultrasonic extraction time is 60 min, and the extract is concentrated to 1 mL using a rotary evaporator. Finally, the extract is filtered into a sample bottle using a 0.45 μm polytetrafluoroethylene filter membrane.

[0033] In step (2), the solid-phase extraction column is activated with 5 mL of methanol and then 5 mL of ultrapure water is added for equilibration. After equilibration, 1 mL of Poria cocos sample extract from step (1) is added to allow the sample to flow down drop by drop. After washing the impurities with 5 mL of 5% methanol aqueous solution, the target analyte is washed down with 5 mL of methanol and then concentrated to 1 mL for analysis.

[0034] In step (3), the conditions for high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS) were as follows: Agilent 1290 series HPLC system, Waters Acquity BEH C18 column (100 Å, 1.8 µm, 2.1 mm × 100 mm), column temperature 40 °C, mobile phase consisting of acidified acetonitrile containing 0.1% formic acid in water and 0.1% formic acid; injection volume 2 μL; flow rate 0.4 mL / min; detection time 5 min; and elution gradient as shown in Table 1 below.

[0035] Table 1 Mobile phase conditions

[0036]

[0037] Mass spectrometry system: Agilent 6540 series time-of-flight mass spectrometer, ESI ion source, selective ion scanning mode (Target MS / MS), target ion m / z 579.2, scan range 50-1700 m / z. Specific acquisition parameters: nebulizer gas (nitrogen) flow rate 7 L / min; nebulizer gas temperature 300 °C; JetStream gas flow rate 7 L / min; sheath gas temperature 300 °C; nebulizer pressure 37 psi; capillary voltage 3000 V; Skimmer 65 V; Octopole RFV 600 V; fragmentation voltage 130 V; collision voltages 25 V and 40 V. After acquisition, the chromatograms and mass spectra were analyzed using Agilent's accompanying software, MassHunter Qualitative Analysis (compatible with computers running Windows XP and later).

[0038] In step (4), the presence of stachyose H and its isomers is determined by extracting and observing the presence of characteristic ions at m / z 579.2→499.3 in the chromatogram and mass spectrum. If they are present, it proves that the sample has been fumigated with sulfur.

[0039] The prior art compared to this invention includes:

[0040] 1) This invention can supplement the current sulfur fumigation detection method in special cases where it is not applicable to Poria cocos;

[0041] 2) This invention is based on the fact that the structure of the triterpenic acids in Poria cocos is permanently altered after sulfur fumigation, thus avoiding false negative results;

[0042] 3) This invention has a simpler operation procedure than existing detection methods, greatly reducing experimental time and workload for experimental personnel;

[0043] 4) This invention, combined with SPE pretreatment, significantly improves the signal-to-noise ratio of the target compound. Combined with relevant spectra, it discovers target compounds that cannot be discovered by conventional means and reduces the matrix effect of other components in Poria cocos.

[0044] 5) The selective ion scanning mode (Target MS / MS) in this invention is a new method established for specific ions in Poria cocos, which is a method adjustment based on our previous research.

[0045] Beneficial effects:

[0046] This invention provides a method for determining whether Poria cocos has been fumigated with sulfur. Firstly, it detects specific compounds, a target discovered for the first time by the applicant, which is almost undetectable by HPLC. By adding a specific enrichment column, the components are specifically enriched, resulting in a method that requires minimal sample volume while accurately detecting the presence of relevant substances in the sample, thus accurately determining whether Poria cocos has been fumigated with sulfur. This method requires less sample volume, eliminates false negatives due to the volatilization of fumigation components, and enhances the detection method at the microscopic level, improving the detection rate of sulfur-treated Poria cocos and improving the quality of existing detection methods. This faster and more accurate method for determining whether Poria cocos has been fumigated with sulfur provides a new detection method for the quality control of Poria cocos medicinal materials and its related products and safe applications.

[0047] Specifically:

[0048] This invention is the first reported method for detecting pachymansia sulfonic acid (H). Firstly, compared to conventional methods, this method offers advantages such as smaller sample size (0.1 g), less solvent usage, simpler sample preparation, and automated batch sampling. In particular, it significantly reduces manual operation steps, lowers labor costs, and minimizes experimental errors; the average testing time for each sample is only 7-8 minutes. Furthermore, this method is highly specific and sensitive, requiring no reference standard for rapid testing and judgment. This method can directly detect the presence of pachymansia sulfonic acid (H), an irreversible change that occurs during sulfur fumigation, unaffected by other processing steps (e.g., volatilization during long-term storage and desulfurization after fumigation), resulting in more accurate detection results. This invention provides a new method for rapidly identifying sulfur-fumigated Poria cocos using high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS), offering a novel approach to distinguishing the quality of the commonly used medicinal material Poria cocos.

[0049] Regarding the reduction in time costs: the time data has been referenced from the processing time and steps in the determination method of sulfur dioxide residue in Chinese Pharmacopoeia 2331; the first method (titration method) requires that "after the water in the flask boils for 1.5 hours, heating should be stopped and the absorption liquid should be cooled", that is, each single sample requires a processing time of at least 90 minutes; the second method (gas chromatography method) specifies that the detector temperature is 250°C, with a programmed temperature increase; the initial temperature is 50°C, held for 2 minutes, then increased to 200°C at 20°C per minute, held for 2 minutes, and the equilibration time is 10 minutes. Adding the preparation time of the test solution and the headspace injection time, each single sample requires a processing time of at least 90 minutes. The third method (ion chromatography) states that (distillation is carried out until the total volume of the solution in bottle B is approximately 95 ml (time 30-40 minutes), the tail tube is washed with water and transferred to the absorption bottle, diluted to the mark, shaken well, and allowed to stand for 1 hour, then filtered through a microporous membrane to obtain the final product). This means that each single sample requires at least 90 minutes of processing time; in other words, processing and detecting a single sample using the above method takes at least 120-150 minutes.

[0050] In contrast, our method is relatively shorter, requiring 60 minutes of ultrasonic extraction, 5 minutes of rotary evaporation concentration, 10 minutes of solid-phase extraction purification, and 10 minutes of detection, which means each single sample only takes about 85 minutes. Attached Figure Description

[0051] Figure 1 The structural formula of pamoate sulfonic acid H and its isomers discovered in this invention;

[0052] Figure 2 Mass spectrum of pamoatenic acid H in sulfur-fumigated Poria cocos;

[0053] Figure 3A PCA score plot (A) of sulfur-fumigated (S) and non-fumigated (NS) Poria cocos samples based on UPLC-QTOF-MS / MS multivariate statistical analysis;

[0054] Figure 3B Based on UPLC-QTOF-MS / MS multivariate statistical analysis, OPLS-DA score plot (B) of sulfur-fumigated (S) and non-fumigated (NS) Poria cocos samples;

[0055] Figure 3C Volcano plots (C) of sulfur-fumigated (S) and non-fumigated (NS) Poria cocos samples based on UPLC-QTOF-MS / MS multivariate statistical analysis;

[0056] Figure 4 Extracted ion chromatograms and their signal-to-noise ratios of sulfur-fumigated Poria cocos (direct injection), sulfur-fumigated Poria cocos (solid-phase microextraction purification), and unfumigated Poria cocos;

[0057] Figure 5 The effect of different chromatographic mobile phases on experimental results;

[0058] Figure 6 Poria cocos acid H and its isomer (left) react with sulfur dioxide during sulfur fumigation to transform into poria cocos sulfonic acid H (right).

[0059] Figure 7 Representative chromatograms of Poria cocos samples: (A) Unfumigated Poria cocos; (B) Sulfur-fumigated Poria cocos. Detailed Implementation

[0060] The following examples are used to further illustrate the present invention, but are not limited to it.

[0061] Example 1

[0062] Screening of characteristic compounds from sulfur fumigation of Poria cocos

[0063] Six batches of fresh Poria cocos from different sources were collected. The dried sclerotia were from the fungus Poriacocos (Schw.) Wolf, a species specified in the Chinese Pharmacopoeia. After cutting, the sclerotia were either directly dried or dried after being fumigated with sulfur for 60 minutes. The outer skin was removed, resulting in six portions of Poria cocos and six portions of sulfur-fumigated Poria cocos; see Table 2.

[0064] Table 2. Origin of 6 batches of Poria cocos

[0065]

[0066] The above six batches of Poria cocos and sulfur-fumigated Poria cocos samples were collected and analyzed by solid-phase microextraction-high performance liquid chromatography-time-of-flight mass spectrometry using the following method:

[0067] (1) Weigh approximately 1 g of sample powder (80 mesh) and extract it ultrasonically with 10 mL of 70% methanol for 60 minutes. After centrifugation (4000 rpm, 10 minutes), obtain the supernatant. Concentrate the supernatant to 1 mL using a rotary evaporator. Filter all the obtained supernatant into a sample vial using a 0.45 μm polytetrafluoroethylene filter membrane.

[0068] (2) Activate the Waters Sep-Pak C18 solid phase extraction column (particle size: 55-105 μm; pore size: 125 Å; adsorbent weight: 200 mg; pH range: 2-8; tube size: 3 cc) with 5 mL of methanol, then add 5 mL of ultrapure water to equilibrate; after equilibration, add 1 mL of Poria cocos sample extract and let the sample drip down; wash the impurities with 5 mL of 5% methanol aqueous solution, then wash the target analyte with 5 mL of methanol and reconcentrate to 1 mL for analysis.

[0069] (3) The determination was performed by high performance liquid chromatography-time-of-flight mass spectrometry, with the following parameters:

[0070] The Agilent 1290 series high-performance liquid chromatography system was used, with a Waters Acquity BEH C18 column (100 Å, 1.8 µm, 2.1 mm × 100 mm), a column temperature of 40 °C, and a mobile phase consisting of acidified acetonitrile containing 0.1% formic acid in water and 0.1% formic acid. The injection volume was 2 μL, the flow rate was 0.4 mL / min, the detection time was 5 min, and the elution gradient is shown in Table 3 below.

[0071] Table 3 HPLC elution conditions

[0072]

[0073] Mass spectrometry system: Agilent 6540 series time-of-flight mass spectrometer. ESI ion source, fully automated scanning mode (AutoMS / MS), scan range 50-1700 m / z. Specific acquisition parameters: nebulizer gas (nitrogen) flow rate 7 L / min; nebulizer gas temperature 300 °C; JetStream gas flow rate 7 L / min; sheath gas temperature 300 °C; nebulizer pressure 37 psi; capillary voltage 3000 V; Skimmer 65 V; Octopole RFV 600 V; fragmentation voltage 130 V, collision voltages 25 V and 40 V.

[0074] (4) Chromatographic mass spectra were analyzed using Agilent’s MassHunter Qualitative Analysis software, and principal component analysis (PCA) scores and OPLC-DA scores were performed using SIMCA 14.1. Volcano plots were also performed using Mass Hunter Profiler B.02 to identify characteristic compounds of sulfur-fumigated Poria cocos.

[0075] The test data is as follows: see Figure 3.

[0076] Figure 3A The PCA score plot is shown (in the PCA plot, the x-axis represents the first principal component (t1), and the y-axis represents the second principal component (t2)). Figure 3B The OPLS-DA score graph is shown. Figure 3CThis is a volcano plot (in a volcano plot, the horizontal axis represents the Log2 (fold change) value. Log2 (fold change) is the logarithm of the change factor, base 2. The vertical axis represents the -Log10 (adjusted P-value) value. -Log10 (adjusted P-value) is the result of taking the negative logarithm of the adjusted P-value. The P-value is used to measure whether a difference is statistically significant. After negative logarithmic transformation, a larger value indicates a more significant difference.)

[0077] Among them, the PCA score plot, OPLS-DA score plot, and volcano plot are three commonly used data visualization tools in chemical analysis, used to help analysts better understand the performance of data and models. Their working principles and significance are as follows:

[0078] PCA Score Plot: Principal Component Analysis (PCA) is a commonly used dimensionality reduction technique used to transform high-dimensional data into lower-dimensional data for better visualization and analysis. A PCA score plot displays the scores of a sample on the first two principal components, which helps identify differences and similarities between samples, as well as potential clusters or outliers. This plot can help assess the structure and distribution of the data, and whether there are potential correlations and patterns. In our example, PCA is used to help visualize the level of difference between samples using sulfur-fumigated Poria cocos and those without, ensuring the reliability of the samples.

[0079] OPLS-DA Score Plot: OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis) is a supervised model used for classification and prediction. The OPLS-DA score plot displays the score of a sample in the OPLS-DA model, helping to identify the effectiveness of classification and the performance of the model. This plot can help assess the quality and predictive power of the model, as well as whether there are classification problems or model overfitting. In our example, OPLS-DA is used to help visualize the classification level between the sulfur-fumigated Poria cocos samples and the unfumigated Poria cocos samples (NS) used in our experiment.

[0080] Volcano plot: A volcano plot is a chart used to visualize the results of differential analysis. It shows the difference and significance level of each metabolite. The horizontal axis of the volcano plot shows the difference (usually a logarithmic fold change), and the vertical axis shows the significance (usually a negative logarithmic p-value). This chart can help identify differences and correlations between different treatment groups, and whether the expression differences between different features are significant. In our example, the volcano plot can be used to help visualize the differential metabolites between sulfur-fumigated Poria cocos and samples without sulfur-fumigated Poria cocos used in our experiment, thus obtaining information on key components.

[0081] Principal Component Analysis (PCA) is used to visualize the degree of clustering or separation between different sample groups by reducing the dimensionality of a dataset. (For simplicity, when we have a large amount of data and want to see if there are any patterns or differences between groups, we can use a technique called dimensionality reduction. This technique helps reduce the number of variables we need to look at. We can then plot the data on a less dimensional (usually 2- or 3-dimensional) graph, making it easier to see if there are any clusters or differences between groups. This can help us understand the relationships between different groups and whether they are similar or different.)

[0082] The chromatographic peaks of 6 sulfur-fumigated samples (S), 6 non-fumigated samples (NS), and 6 quality control samples (QC) were processed and then imported into SIMCA 14.1 for PCA and OPLS-DA chromatograms. Furthermore, volcano plot analysis was used to identify specific components with large fold changes (>3) and statistical significance (p<0.05), serving as a means to observe the reasons for chemical differences between sulfur-fumigated and non-fumigated Poria cocos samples.

[0083] exist Figure 3A , 3B In 3C, PCA and OPLS-DA models (3B) (R 2 X (cum.) or R 2 The value of Y (cum.) is close to 1, and Q... 2 A cum. value exceeding 0.5 indicates a sufficiently good fit and prediction to diagnose the differences between sulfur-fumigated and non-fumigated Poria cocos samples. The PCA and OPLS-DA score plots clearly show that sulfur-fumigated (S) and non-fumigated Poria cocos (NS) samples cluster in different regions. This further suggests that sulfur fumigation significantly alters the composition of Poria cocos, resulting in distinctly different overall chemical characteristics between sulfur-fumigated and non-fumigated Poria cocos samples.

[0084] Based on volcano plot (3C) screening, the component with a mass-to-charge ratio (m / z) of 579.26 was identified as a key component causing the difference between non-fumigated Poria cocos (NS) and sulfur-fumigated Poria cocos (S), namely, the newly formed compound during the sulfur fumigation process, shown in the right region of the volcano plot. According to the mass spectrometry fragmentation signal, the compound at m / z 579.26 was identified as pachymic acid H. Therefore, pachymic acid H was selected as the characteristic marker in subsequent detections. The structure is described in […]. Figure 1 and Figure 2 ,

[0085] Table 4. Chromatograms and mass spectrometry data of compounds in sulfur-fumigated Poria cocos samples identified by UPLC-QTOF-MS / MS.

[0086] Referring to Table 4, the main secondary metabolites detected were poricoic acids, namely poricoic acid A, poricoic acid B, and 25-hydroxyporicoic acid H. The results showed that the overall peak height detected in unfumigated Poria (NS-Poria) was significantly lower than that in sulfur-fumigated Poria (S-Poria), indicating a decrease in poricoic acid content. On the other hand, many new main peaks were detected in the sulfur-fumigated samples, while these new peaks were not detected in the non-fumigated samples, indicating that the main chemical components underwent chemical transformation during sulfur fumigation. Among them, 25-Hydroxyporicoic acid H sulfonate was confirmed as the detection peak. This confirmation was based on its mass-to-charge ratio (m / z 579) detected using selected ion mode and determined by secondary mass spectrometry fragmentation characteristics (m / z 499). Comparative Example 1: The Necessity of Solid-Phase Microextraction Purification

[0087] (1) Weigh approximately 0.1 g of Poria cocos powder (80 mesh) and extract it with 10 mL of 70% methanol using ultrasonic extraction for 60 minutes. After centrifugation (4000 rpm, 10 minutes), obtain the supernatant. Concentrate the supernatant to 1 mL using a rotary evaporator. Then filter it through a 0.45 μm polytetrafluoroethylene membrane into a sample vial to obtain sulfur-fumigated Poria cocos (direct injection) sample. Inject the sample directly into high performance liquid chromatography-time-of-flight mass spectrometry for analysis.

[0088] (2) The pretreatment was the same as in step (1), with the addition of a solid-phase microextraction purification step, as detailed below: Weigh approximately 0.1 g of Poria cocos powder (80 mesh) and extract it ultrasonically with 10 mL of 70% methanol for 60 minutes. After centrifugation (4000 rpm, 10 minutes), obtain the supernatant. Concentrate the supernatant to 1 mL using a rotary evaporator. Then filter it into a sample vial using a 0.45 μm polytetrafluoroethylene filter membrane. Activate the Waters Sep-Pak C18 solid-phase extraction column with 5 mL of methanol, and then add 5 mL of ultrapure water for equilibration; after equilibration, add 1 mL of Poria cocos sample extract, allowing the sample to drip down; wash the impurities with 5 mL of 5% methanol aqueous solution, then wash the target analyte with 5 mL of methanol, and reconcentrate to 1 mL to obtain the sulfur-fumigated Poria cocos (solid-phase microextraction purified) sample. Then perform high performance liquid chromatography-time-of-flight mass spectrometry analysis (conditions same as in Example 1).

[0089] The results are as follows Figure 4No chromatographic peaks were observed in the un-sulfur-fumigated Poria cocos sample, while chromatographic peaks only appeared in the sulfur-fumigated Poria cocos sample, proving that this compound is only present in sulfur-fumigated Poria cocos. Even if a slight signal was present in the sulfur-fumigated Poria cocos (direct injection) sample, it could not be considered a chromatographic peak due to its signal-to-noise ratio (S / N) being below 3. Therefore, HPLC detection alone cannot distinguish the relevant components. However, after purification using solid-phase microextraction, the matrix effect of other components in Poria cocos is reduced, increasing its S / N by approximately eight times (S / N>3), thereby increasing its detection accuracy and accurately reflecting the sulfur fumigation status of Poria cocos. Since the response of Poria cocos sulfonic acid H in mass spectrometry is weak, an effective signal cannot be directly detected by directly injecting Poria cocos samples. Therefore, it is necessary to use solid-phase microextraction purification combined with high-performance liquid chromatography-time-of-flight mass spectrometry for the detection of Poria cocos sulfonic acid H and its isomers.

[0090] Comparative Example 2: Results of different chromatographic mobile phases

[0091] See Figure 5 We will examine the effect of commonly used chromatographic mobile phases on the experimental results. Therefore, we will consider three commonly used combinations of chromatographic mobile phases: water and acetonitrile, and 0.1% formic acid water and 0.1% formic acid acidified acetonitrile, with other parameters being the same as those set in Example 1.

[0092] The results are as follows Figure 5 This method combines two commonly used chromatographic mobile phases to detect the presence of pamoate sulfonic acid H and its isomers in samples. Formic acid is a commonly used additive in reversed-phase liquid chromatography (RP-LC) mobile phases. In particular, the addition of 0.1% formic acid improves ionization efficiency, thereby improving peak shape, characterized by sharper peaks and clearer contours. Therefore, it is recommended to use 0.1% formic acid in water and 0.1% formic acid in acidified acetonitrile as the chromatographic mobile phase.

[0093] Comparative Example 3: Results of different collision energies

[0094] We will examine the impact of different collision energies in mass spectrometry on the experimental results. Since the recommended range of commonly used parameters varies among different manufacturers, it is difficult to define a specific range for all brands of mass spectrometers. Therefore, we can only examine the impact of collision energy on the experimental results. We will consider three collision energies: low, medium, and high, which are 25V, 40V, and 60V, respectively.

[0095] Results: Collision energies of varying degrees (25V, 40V, and 60V) all reflected characteristic fragments of Poria sulfonic acid H and its isomers (m / z 579→499). NP-Poria was non-sulfur-fumigated Poria, while S-Poria was sulfur-fumigated Poria. However, the results at 25V and 40V were better because higher collision energies promote more extensive fragmentation of precursor ions, resulting in a greater number of fragment ions and potentially further fragmentation of the characteristic fragments, thus weakening their signal. Therefore, it is recommended that the collision energies for mass spectrometry be set at 25V and 40V, as 60V cannot guarantee the accuracy of the characteristic fragment signal.

[0096] Comparative Example 4: Identification of Sulfur-Fumigated Poria Cocos Samples

[0097] Acid-base titration is the most commonly used method for detecting sulfur dioxide residue.

[0098] Weigh 10 g of finely powdered Poria cocos slices and place them in a 1000-mL two-necked round-bottom flask, adding 400 mL of water. Connect the round-bottom flask to a reflux condenser, and connect a rubber gas delivery tube to the bottom of a 100-mL conical flask at the upper end of the condenser. Add 50 mL of 3% (v / v) hydrogen peroxide solution to the conical flask, then add 3 drops of methyl red ethanol indicator (2.5 mg / mL), and titrate with 0.01 mol / L sodium hydroxide solution until a yellow color appears. Turn on nitrogen gas and adjust the gas flow rate to approximately 0.2 L / min using a flow meter. Open the stopcock of the separatory funnel and add 10 mL of hydrochloric acid solution (6 mol / L) to the round-bottom flask. Heat to boiling and maintain a gentle boil. After 1.5 h, stop heating. After the conical flask has cooled to room temperature, it is titrated with sodium hydroxide titrant (0.01 mol / L) until the yellow color persists for 20 seconds. The titration result is then corrected using a blank experiment. The residual sulfur dioxide in the sample is calculated using the following formula:

[0099]

[0100] V A The volume (mL) of sodium hydroxide titrant consumed for the test solution;

[0101] V B This represents the volume (mL) of sodium hydroxide titrant consumed during the blank test.

[0102] C NaOH The molar concentration (mol / L) of the sodium hydroxide titrant;

[0103] 32.23 is the milligram equivalent of sulfur dioxide;

[0104] 1000 is the conversion factor from milligram equivalents to microgram equivalents.

[0105] W represents the mass (g) of the test sample.

[0106] Table 5. Sulfur dioxide residue in 6 batches of Poria cocos

[0107]

[0108] As can be seen from the data in Table 5, the sulfur dioxide content in the six samples after sulfur fumigation in the examples ranged from 0 to 46.64 mg / kg.

[0109] Three of the sulfur-fumigated Poria cocos samples had a sulfur dioxide residue of 0 mg / kg, which was no different from the sulfur dioxide residue in the unfumigated samples of the same type. This indicates that the conventional titration method for detecting sulfur dioxide residue cannot distinguish between sulfur-fumigated and unfumigated Poria cocos.

[0110] To verify the applicability of this method, 50 commercially available Poria cocos samples randomly collected from the market were tested using both the method of this invention and acid-base titration. The results showed that pamoatenic acid H was detected in 28 samples, and residual sulfur dioxide was detected in only 13 samples, with levels ranging from 14.94 to 42.53 mg / kg (see Table 6). These findings indicate that 56% of the commercially available Poria cocos samples had undergone sulfur fumigation. Furthermore, 15 false negative results were observed in the determination of residual sulfur dioxide. This may be due to the instability of residual sulfur dioxide caused by long-term storage and heating, which has been verified in our related experiments.

[0111] Table 6. Test results of 50 commercially available Poria cocos samples from Hong Kong.

[0112]

Claims

1. A method for determining whether Poria cocos has been fumigated with sulfur, characterized in that, It consists of the following steps: 1) Take the Poria cocos sample to be tested, dissolve and extract it with an organic solvent, concentrate it to obtain the sample solution, and the organic solvent is methanol, methanol aqueous solution, ethanol or ethanol aqueous solution; 2) The sample solution described in step 1 is purified using a C18 solid-phase extraction column. After washing away impurities with a 5% methanol aqueous solution, the target analyte is washed off with methanol. After purification, the solution is concentrated to obtain a purified solution. The C18 solid-phase extraction column described in step 2) is a Waters Sep-Pak C18 solid-phase extraction column. 3) The purified solution obtained in step 2) was analyzed using high performance liquid chromatography-time-of-flight (HPLC-TOF). The HPLC conditions in step 3) were as follows: Waters Acquity BEH C18 column, 100 Å, 1.8 µm, 2.1 mm × 100 mm; column temperature 35-45 °C; mobile phase A was 0.1% formic acid in water; mobile phase B was 0.1% formic acid in acidified acetonitrile; injection volume 1-5 μL; flow rate 0.1-1 mL / min; elution gradient: 0 min, 95% v / v mobile phase A: 5% v / v mobile phase B; 3 min, 40% v / v mobile phase A: 60% v / v mobile phase B; 5 min, 40% v / v mobile phase A: 60% v / v mobile phase B. 4) Determine the presence of 25-Hydroxyporicoic acid H sulfonate in the sample to determine whether the Poria cocos sample has been fumigated with sulfur. If the presence of 25-Hydroxyporicoic acid H sulfonate is detected, it is determined that the Poria cocos sample has been fumigated with sulfur; otherwise, it is determined that the Poria cocos sample has not been fumigated with sulfur. The structure of 25-Hydroxyporicoic acid H sulfonate is as follows: , The presence of the 25-Hydroxyporicoic acid H sulfonate was determined based on the characteristic ion at m / z 579.2→499.3 in the extracted and observed chromatographic mass spectra.

2. The method according to claim 1, characterized in that, Step 1) The organic solvent is methanol or an aqueous methanol solution with a concentration greater than 70%.

3. The method according to claim 1, characterized in that, Step 1) Extraction is performed by ultrasonic extraction.

4. The method according to claim 1, characterized in that, In steps 1) and 2), the concentration is achieved by rotary evaporation or boiling evaporation.

5. The method according to claim 1, characterized in that, In step 3), during the flight time, the mass spectrometer uses an ESI ion source, selective ion scanning mode Target MS / MS, target ion m / z 579.2, and scanning range 50-1700 m / z.

6. The method according to claim 1, characterized in that, During flight, the following parameters were collected: nitrogen as the atomizing gas, with a flow rate of 7 L / min; atomizing gas temperature of 300 °C; JetStream gas flow rate of 7 L / min; sheath gas temperature of 300 °C; atomizer pressure of 37 psi; capillary voltage of 3000 V; Skimmer sampling cone voltage of 65 V; Octopole RFV octave voltage of 600 V; fragmentation voltage of 130 V; and collision voltages of 25 V and 40 V. Chromatographic mass spectra were obtained.

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