Non-targeted identification method of fungal toxin metabolites in wine based on mass spectrometry coupled with chromatography

Abstract

This invention relates to a non-targeted identification method for mycotoxin metabolites in wine based on mass spectrometry coupled with chromatography, specifically for the detection and identification of ochratoxin A and its metabolites in wine. Through sample pretreatment, chromatographic separation, mass spectrometry detection, data screening, and fragment ion confirmation of wine samples, non-targeted detection of ochratoxin A and its metabolites can be achieved without relying on standards. In the sample pretreatment stage, the target analyte is extracted using acidified acetonitrile solution, and impurities are removed by salting out and centrifugation. Separation of ochratoxin A and its metabolites is achieved by coupling high-performance liquid chromatography with time-of-flight mass spectrometry using a reversed-phase C18 column for gradient elution. The mass spectrometry analysis uses full-scan mode and alternates between positive and negative ion modes to ensure high resolution and high sensitivity, making it suitable for the detection of mycotoxins in wine and other foods, meeting food safety regulatory requirements.

Description

Technical Field

[0001] This invention belongs to the field of wine technology, and specifically relates to a non-targeted identification method for wine fungal toxin metabolites based on mass spectrometry coupled with chromatography. Background Technology

[0002] Mycotoxins are secondary metabolites produced by molds in plants and their products, posing a threat to human and animal health. Ochratoxin A (OTA) is one of the most common mycotoxins, widely found in foods such as wine, and its ingestion may lead to kidney damage and other health problems. Traditional OTA detection methods mostly rely on targeted detection technologies, such as enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). While these methods are accurate, they are cumbersome in sample pretreatment and have long analysis times.

[0003] With the advancement of mass spectrometry technology, mass spectrometry-chromatography (MS-GC) has gradually become an important tool in the field of food safety testing. Although existing MS-GC techniques can provide high resolution and sensitivity, most existing methods are still focused on targeted detection, i.e., relying on known standards and databases for quantitative analysis, and cannot effectively identify unknown fungal toxins or their metabolites. Summary of the Invention

[0004] The purpose of this invention is to provide a non-targeted identification method for mycotoxin metabolites in wine based on mass spectrometry and chromatography, which can use mass spectrometry-chromatography to perform non-targeted identification of ochratoxin A, a mycotoxin metabolite in wine. This invention provides a non-targeted identification method for wine fungal toxin metabolites based on mass spectrometry coupled with chromatography, comprising the following steps: (1) Sample pretreatment: Take the wine sample to be tested, add an organic solvent to extract the fungal toxins and their metabolites, the organic solvent being an acidified acetonitrile solution; add salting-out reagent to the sample and shake thoroughly to mix, then centrifuge to obtain the extract to be tested; (2) Chromatography-mass spectrometry detection: After the extract obtained in step (1) is separated by chromatography, it is entered into a mass spectrometry detection device, and mass spectrometry data of each component in the wine are collected in the full scan mode of mass spectrometry. (3) Data screening and analysis: The mass spectrometry data obtained in step (2) is compared and screened with the database of fungal toxins and their metabolites. The chromatographic peaks of suspected fungal toxins and their metabolites are detected based on the mass-to-charge ratio information. The database of fungal toxins and their metabolites comes from existing public databases. (4) Fragment ion confirmation: Mass spectrometry fragment ion analysis is performed on the chromatographic peaks corresponding to the suspected fungal toxins and their metabolites obtained in step (3) to obtain the product ion spectrum, and the obtained product ions are compared with the standard spectra in the fungal toxins and their metabolites database to confirm the molecular structure and identity of the suspected compound.

[0005] Preferably, the specific extraction method in step (1) is as follows: an equal volume of acetonitrile solution containing 0.1% formic acid is added to the wine sample, followed by the addition of anhydrous magnesium sulfate and sodium chloride as salting-out reagents. After shaking and mixing, the mixture is centrifuged. The obtained supernatant organic phase is separated and the solid impurities are removed by filtration membrane to obtain a sample extract enriched with ochratoxin A and its metabolites.

[0006] Preferably, the chromatographic separation in step (2) is performed by high performance liquid chromatography, and a reversed-phase C18 column is used for gradient elution; the mobile phase is a gradient system of water and organic solvent, and mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid; during the gradient elution process, the proportion of mobile phase B is gradually increased from the initial 5-10% to 90-100% to elute the target compound, and the gradient elution is completed within 10-15 minutes.

[0007] Preferably, the mass spectrometry detection device in step (2) is a time-of-flight mass spectrometer, and data is acquired alternately in positive and negative ion modes using an electrospray ionization interface; wherein the mass spectrometer acquires full-scan mass spectrometry data of the wine sample. Preferably, the mass spectrometer has a mass resolution of not less than 10,000. Preferably, the quality measurement error is controlled within ±5ppm.

[0008] Preferably, step (3) is as follows: by comparing the database, the mass spectrometry precision mass of the unknown component detected in the sample is matched with the theoretical mass of ochratoxin A and its metabolites in the database. When the mass spectrometry error is within the preset threshold range and the isotope distribution is consistent, the suspected target of ochratoxin A or its corresponding metabolites is determined, and its chromatographic retention time and intensity information are extracted for subsequent verification.

[0009] Preferably, the fragment ion analysis in step (4) employs tandem mass spectrometry to perform collision-induced dissociation of the suspect target, obtaining characteristic product ions; the obtained product ion mass spectrum is compared with the standard fragment spectra of ochratoxin A and its metabolites in the database to confirm the compound structure; if a signal corresponding to the characteristic fragment ions of ochratoxin A appears in the product ion spectrum, the target can be confirmed as ochratoxin A; for the dechlorination metabolites of ochratoxin A, such as ochratoxin B, if their product ion spectra are compared with the corresponding standard spectra... Figure 1 This can also be used to confirm the conclusion. Beneficial effects

[0010] This invention employs high-performance liquid chromatography coupled with time-of-flight mass spectrometry (TOF-MS). Through gradient elution and full-scan mass spectrometry data acquisition, it enables efficient separation and analysis of ochratoxin A and its metabolites in a short time. The coupling of chromatography and mass spectrometry improves the resolution and sensitivity of the detection, and significantly reduces the analysis time and operational complexity of traditional methods. Unlike traditional targeted detection methods, the method of this invention does not rely on existing standards or known toxin information; by comparing with public databases, it can detect and identify unknown fungal toxins and their metabolites in wine samples, thus expanding the scope of detection. Furthermore, this invention employs a time-of-flight mass spectrometer for high-resolution full-scan mass spectrometry data acquisition, and combines mass spectrometry fragment ion analysis to confirm the molecular structure of the target compound; through precise mass comparison and fragment ion confirmation, it ensures highly accurate identification of ochratoxin A and its metabolites. Attached Figure Description

[0011] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a flowchart of the method of the present invention; Detailed Implementation

[0012] See details Figure 1 Example 1 provides a non-targeted identification method for wine fungal toxin metabolites based on mass spectrometry coupled with chromatography. The specific implementation method is as follows: Take 200 mL of the red wine sample to be tested, add an equal volume of 0.1% formic acid-acetonitrile solution, and then add anhydrous magnesium sulfate and sodium chloride as salting-out reagents; shake the mixture vigorously for 1 minute to fully extract ochratoxin A (OTA) and its metabolites (e.g., ochratoxin B (OTB)) into the organic phase; after centrifugation, separate the supernatant organic phase; filter the supernatant through a 0.22 µm organic phase needle filter membrane to remove particulate impurities, and obtain a clear extract enriched with OTA and its metabolites; the treated extract can be directly used for subsequent chromatographic-mass spectrometry analysis; The extract was separated using high-performance liquid chromatography (HPLC); a reversed-phase C18 column was used at 35℃; mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid; a gradient elution program was used as follows: the initial mobile phase B was 5%, linearly increased to 95% B within 12 minutes, and maintained for 1 minute to fully elute the target compound, followed by restoring the column to the initial 5% B equilibrium at 15 minutes; the flow rate was 0.3 mL / min, and the injection volume was 10 µL; this gradient elution program could complete the separation of OTA and its metabolites within 10–15 minutes. The column effluent enters the time-of-flight mass spectrometer via the electrospray ionization interface. Mass spectrometry acquisition employs alternating positive and negative ion modes, scanning ion signals within the m / z range of 100-600. The instrument's mass resolution is set to no less than 10,000 to ensure sufficient mass accuracy. The mass spectrometry resolution of this method is approximately 30,000, and the mass measurement error is controlled within ±5 ppm. During data acquisition, the ion mode is switched every 1 second to achieve alternating scanning of positive and negative ions. The obtained full-scan mass spectrometry data were imported into a mycotoxin database for comparison and screening. This database contains molecular information on ochratoxin A and its related metabolites, such as theoretical molecular formula, accurate mass-to-charge ratio, isotope distribution, and reference fragment spectra. First, the suspected target analytes were matched based on the accurate mass of the mass spectrometer: in negative ion mode, the theoretical m / z of the deprotonated ion of OTA in the database was 402.08, and the theoretical m / z of the deprotonated ion of OTB (dechlorinated metabolite) was 368.12. Two unknown peaks were found in the total ion chromatogram of the sample: one peak had a retention time of about 7.5 minutes, corresponding to an ion at m / z 402.081; the other peak had a retention time of about 6.8 minutes, corresponding to an ion at m / z 368.113. The measured accurate masses of both were very close to the theoretical mass-to-charge ratios of OTA and OTB, with a mass deviation of less than 3%. Within ppm; at the same time, the isotopic distribution of these ions is consistent with the molecular structure characteristics of chlorine-containing (for OTA) or chlorine-free (for OTB), further increasing the reliability of the identification; when the mass spectrometry error is within ±5 ppm of the preset threshold and the isotopic mode matches, the presence of ochratoxin A or its corresponding metabolite in the sample can be determined; therefore, the above two chromatographic peaks are labeled as suspected OTA and suspected OTB, and their retention time, mass-to-charge ratio and response intensity information are recorded for subsequent confirmatory analysis.

[0013] To further confirm the molecular structure of the suspected peaks, mass spectrometry fragmentation analysis was performed on the chromatographic peaks of suspected ochratoxin A and ochratoxin B. Tandem mass spectrometry was used, and the corresponding precursor ions were selected for collision-induced dissociation on time-of-flight mass spectrometry. In positive ion mode, the precursor ion at m / z 404.09 generated a series of product ions via CID, including characteristic fragments at m / z 239 and m / z 358. m / z 239 is a typical fragment ion of OTA, corresponding to the fluorinyl isocoumarin fragment with a chlorine atom in the ochratoxin A molecule, while m / z 358 originates from the fragment after the OTA precursor ion loses its carboxyl group. Literature reports that selective monitoring of OTA fragment ions 404→239 and 404→358 in positive ion ESI mode can achieve qualitative and quantitative analysis of OTA. Simultaneously, in negative ion mode, m / z 358 and m / z 358 frequently appear in the fragments of the OTA deprotonated ion (m / z 402.08). Ions in the range of 238-240 all match the standard mass spectrum. The product ion spectrum of the suspected OTA peak obtained in this embodiment was compared with the standard OTA spectrum in the database. It was found that the characteristic fragment ions and their abundance ratios matched (e.g., the relative abundance deviations of fragment ions m / z 239 and m / z 358 were both within 20%). Thus, it was confirmed that the target substance was ochratoxin A. For the suspected ochratoxin B peak (OTB, a dechlorinated OTA metabolite), characteristic ion signals consistent with the reported OTB fragment spectra were observed in tandem mass spectrometry analysis. Since the OTB molecule does not contain chlorine, its fragment spectrum differs slightly from that of OTA: for example, the chlorinated fragment m / z 239 produced by OTA corresponds to approximately m / z 205 in OTB (losing the same structural unit but without chlorine), and another major fragment is m / z 221 (corresponding to the fragment after the removal of phenylalanine from the OTB molecule). Comparing the suspected OTB product ion spectrum obtained in this experiment with the database standard, it was found that the fragment ion mass number and abundance matched the spectral characteristics of OTB. Therefore, the chromatographic peak can be confirmed as ochratoxin B (i.e., the dechlorinated product of ochratoxin A). Furthermore, the fragment ion analysis strategy of this method is also applicable to the confirmation of other potential OTA metabolites, such as ochratoxin α (OTα) produced by the hydrolysis of OTA. For example, the product spectrum of the suspected target analyte detected in the sample and its corresponding standard spectrum... Figure 1 By identifying the structure, its identity can be confirmed.

[0014] In summary, the above embodiments have achieved a non-targeted identification method for ochratoxin A and its metabolites in wine based on chromatography-mass spectrometry (GC-MS). This method encompasses sample pretreatment, full-scan mass spectrometry screening, and fragment ion confirmation, with results from each step supporting the described technical solution. In practical applications, this method demonstrates high sensitivity and reliable qualitative confirmation capabilities for OTA and related metabolites: OTA and its metabolites can be rapidly screened and confirmed from complex wine matrices without the need for prior establishment of specific detection methods for a single toxin. This non-targeted identification strategy provides a high-throughput, accurate, and reliable technical means for the safe monitoring of mycotoxins in wine and other foods.

[0015] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A non-targeted identification method for wine fungal toxin metabolites based on mass spectrometry coupled with chromatography, characterized in that, The fungal toxin and its metabolites are ochratoxin A and its metabolites. The method includes the following steps: (1) Sample pretreatment: Take the wine sample to be tested, add an organic solvent to extract the fungal toxins and their metabolites, the organic solvent being an acidified acetonitrile solution; add salting-out reagent to the sample and shake thoroughly to mix, then centrifuge to obtain the extract to be tested; (2) Chromatography-mass spectrometry detection: After the extract obtained in step (1) is separated by chromatography, it is entered into a mass spectrometry detection device, and mass spectrometry data of each component in the wine are collected in the full scan mode of mass spectrometry. (3) Data screening and analysis: The mass spectrometry data obtained in step (2) is compared and screened with the database of fungal toxins and their metabolites. The chromatographic peaks of suspected fungal toxins and their metabolites are detected based on the mass-to-charge ratio information. The database of fungal toxins and their metabolites comes from existing public databases. (4) Fragment ion confirmation: Mass spectrometry fragment ion analysis is performed on the chromatographic peaks corresponding to the suspected fungal toxins and their metabolites obtained in step (3) to obtain the product ion spectrum, and the obtained product ions are compared with the standard spectra in the fungal toxins and their metabolites database to confirm the molecular structure and identity of the suspected compound.

2. The method for non-targeted identification of wine fungal toxin metabolites based on mass spectrometry coupled with chromatography according to claim 1, characterized in that, The specific extraction method in step (1) is as follows: Add an equal volume of acetonitrile solution containing 0.1% formic acid to the wine sample, then add anhydrous magnesium sulfate and sodium chloride as salting-out reagents, shake and mix, and centrifuge; separate the obtained supernatant organic phase and remove solid impurities through a filter membrane to obtain a sample extract enriched with ochratoxin A and its metabolites.

3. The method for non-targeted identification of wine fungal toxin metabolites based on mass spectrometry coupled with chromatography according to claim 1, characterized in that, In step (2), the chromatographic separation is performed using high performance liquid chromatography (HPLC) with a reversed-phase C18 column for gradient elution. The mobile phase is a gradient system of water and organic solvent, with mobile phase A being an aqueous solution containing 0.1% formic acid and mobile phase B being an acetonitrile solution containing 0.1% formic acid. During the gradient elution process, the proportion of mobile phase B is gradually increased from 5-10% to 90-100% to elute the target compound, and the gradient elution is completed within 10-15 minutes.

4. The method for non-targeted recognition of wine fungal toxin metabolites based on mass spectrometry coupled with chromatography according to claim 1, characterized in that, The mass spectrometry detection device mentioned in step (2) is a time-of-flight mass spectrometer, which uses an electrospray ionization interface to alternately acquire data in positive and negative ion modes; wherein the mass spectrometer acquires full-scan mass spectrometry data of the wine sample. Preferably, the mass spectrometer has a mass resolution of not less than 10,000. Preferably, the quality measurement error is controlled within ±5ppm.

5. The method for non-targeted identification of wine fungal toxin metabolites based on mass spectrometry coupled with chromatography according to claim 1, characterized in that, The specific process of step (3) is as follows: by comparing the database, the precise mass spectrometry mass of the unknown component detected in the sample is matched with the theoretical mass of ochratoxin A and its metabolites in the database. When the mass spectrometry error is within the preset threshold range and the isotope distribution is consistent, the suspected target of ochratoxin A or its corresponding metabolites is determined, and its chromatographic retention time and intensity information are extracted for subsequent verification.

6. The method for non-targeted identification of wine fungal toxin metabolites based on mass spectrometry coupled with chromatography according to claim 1, characterized in that, In step (4), the fragment ion analysis uses tandem mass spectrometry to perform collision-induced dissociation of the suspected target to obtain characteristic product ions. The obtained product ion mass spectrum is compared with the standard fragment spectra of ochratoxin A and its metabolites in the database to confirm the compound structure. If a signal corresponding to the characteristic fragment ion of ochratoxin A appears in the product ion spectrum, the target can be confirmed as ochratoxin A. For the dechlorination metabolite of ochratoxin A, such as ochratoxin B, if its product ion spectrum is consistent with the corresponding standard spectrum, it can also be confirmed accordingly.

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