Multi-class marine toxin sample analysis method suitable for shellfish matrix

Abstract

The invention relates to the technical field of marine toxin analysis, and particularly discloses a multi-class marine toxin sample analysis method suitable for shellfish matrices, and the method comprises the following steps: weighing and labeling; one-step acidification organic solvent extraction; carrying out same-bed segmented solid phase enrichment; carrying out chromatography-mass spectrometry on the polar components; performing chromatography-mass spectrometry analysis on hydrophobic components; carrying out normalized quantification and quality control confirmation; according to the method, different bonding phases such as reverse phase and ion exchange are combined into the same bed or a composite medium, and segmented recovery is performed according to polarity through sequential elution, so that various toxins obtain more targeted elution conditions. According to the strategy, the enrichment degree of a target object in a corresponding elution section can be enhanced, a large number of matrix interferents can be dispersed into different sections, subsequent chromatographic separation and mass spectrometric detection are facilitated, and therefore the influence of the matrix effect on quantification is reduced from the source.

Description

Technical Field

[0001] This invention belongs to the field of marine toxin analysis technology, specifically relating to a method for analyzing multi-category marine toxin samples applicable to shellfish matrices. Background Technology

[0002] Shellfish, a popular seafood product, are of paramount importance in terms of safety. However, shellfish are susceptible to contamination by a variety of marine toxins from diverse sources. These toxins include algal toxins such as paralytic shellfish toxins (PSPs), amnesic shellfish toxins (ASPs), diarrhetic shellfish toxins (DSPs), and neurotoxins (NSPs), as well as toxins from other organisms, such as tetrodotoxins (TTXs). Different categories of marine toxins exhibit significant differences in chemical properties and polarity, ranging from highly polar PSPs to moderately hydrophobic DSPs, and other toxins with relatively high hydrophobicity. Furthermore, multiple toxins often coexist in the shellfish matrix, posing a significant challenge to toxin detection and analysis.

[0003] Currently, methods for detecting and analyzing various types of marine toxins in shellfish have many shortcomings. In sample pretreatment, traditional methods typically employ multi-step extraction and separation procedures, such as using different extraction solvents and conditions for toxins of different polarities. This is not only cumbersome and time-consuming but also prone to causing the loss of target toxins, reducing recovery rates. Furthermore, existing extraction methods struggle to simultaneously and effectively dissolve polar and moderately hydrophobic toxins and precipitate proteins within a single solvent phase, resulting in unsatisfactory extraction effects and impacting the accuracy of subsequent analyses.

[0004] In response to this problem, this application proposes a method for analyzing multi-category marine toxin samples applicable to shellfish matrices. Summary of the Invention

[0005] The purpose of this invention is to provide a method for analyzing multi-category marine toxin samples applicable to shellfish matrices, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for analyzing multi-category marine toxin samples applicable to shellfish matrices includes:

[0008] Weighing and Spiking: Weigh the homogenized shellfish tissue sample and add an isotope-labeled internal standard mixture covering multiple types of marine toxins to simultaneously correct for subsequent extraction recovery and matrix effects, thus obtaining a homogenized sample.

[0009] One-step acidified organic solvent extraction: acidified methanol:water mixed solvent is added to the homogeneous sample and subjected to ultrasonic treatment and centrifugation to simultaneously dissolve polar and moderately hydrophobic toxins and precipitate proteins in a single solvent phase, resulting in a crude extract.

[0010] Segmented solid-phase enrichment in the same bed: The crude extract is passed through a pre-equilibrated mixed bonded solid-phase material and eluted sequentially with low organic ratio and high organic ratio, which is used to enrich different toxin categories according to polarity in a single processing flow to obtain polar components and hydrophobic components.

[0011] Polar component chromatography-mass spectrometry analysis: The polar components are concentrated and redissolved in a mobile phase adapted for HILIC chromatography, and then subjected to liquid chromatography-tandem mass spectrometry analysis (including positive and negative ionization switching and target trigger acquisition) to separate, identify and preliminarily quantify polar toxins, and obtain polar component mass spectrometry data.

[0012] Hydrophobic component chromatographic-mass spectrometry analysis: The hydrophobic component is concentrated and redissolved in a mobile phase suitable for reversed-phase chromatography, and then subjected to liquid chromatography-tandem mass spectrometry analysis (including multiple reaction monitoring or timed fragmentation) to separate, identify and preliminarily quantify hydrophobic and mesohydrophobic toxins, and obtain hydrophobic component mass spectrometry data.

[0013] Normalized quantification and quality control confirmation: The mass spectrometry data of the polar components and the mass spectrometry data of the hydrophobic components are normalized by isotope internal standards, and matrix effect correction and recovery correction are performed based on matrix matching multi-point calibration and response surface fitting. Low abundance or suspected positive results are confirmed by high-resolution mass spectrometry or secondary fragment spectrum comparison, which is used to output a confirmed multi-category marine toxin content report.

[0014] Preferably, the mass of the homogenized shellfish tissue sample is 3.0–10.0 g, and the isotope-labeled internal standard mixture covers representative markers from at least the following toxin categories: paralytic shellfish poisoning (PSP), including STX (sparganotoxin) and GTX (gastrocytoxin); amnesic shellfish poisoning (ASP), mainly containing DA (dominoic acid); diarrhetic shellfish poisoning (DSP), mainly containing OA (otaeniacetic acid) and DTX (azotoxin); neurotoxic shellfish poisoning (NSP), mainly containing BTX (bryophyte toxin); tetrodotoxin (TTX); and isotope-labeled internal standards containing the following cyanobacterial neurotoxins β-N-methylamino-L-alanine (BMAA) series to expand the quantitative capability for related neurotoxin targets:

[0015] U-[¹³C3,¹ 5 [N2]-β-N-methylamino-L-alanine (fully labeled) — Original stock solution concentration 10 μg / mL, solvent: methanol / water (e.g., methanol / water 80:20 v / v);

[0016] U-[D3]-β-N-methylamino-L-alanine— Initial stock solution concentration: 10 μg / mL, solvent: methanol / water;

[0017] U-[¹ 5 [N2]-β-N-methylamino-L-alanine— Initial stock solution concentration: 10 μg / mL, solvent: methanol / water;

[0018] Furthermore, the concentration of each internal standard, after dilution, is determined to achieve a peak area signal-to-noise ratio ≥10 in the final analysis, thus obtaining more reliable recovery correction data.

[0019] Preferably, the acidified organic solvent is methanol:water in a volume ratio of 80:20, and contains 0.05% to 0.2% formic acid. The ultrasonic treatment time is 5 to 20 minutes, and the centrifugation conditions are 3,000 to 5,000 g for 5 to 15 minutes. This is used to balance the dissolution of polar and moderately hydrophobic toxins and achieve protein precipitation in a single extraction, so as to obtain a stable crude extract.

[0020] Preferably, the mixed-bonded phase solid material is an inverse phase (C 18A co-bed or composite medium combining phenyl groups with ion exchange (strong anion exchange or weak cation exchange) is used, and sequential elution is employed: first, a buffered aqueous phase with a low organic ratio is used to elute and enrich the more polar components, and then an organic phase with a high organic ratio and a small amount of volatile salts is used to elute and enrich the hydrophobic components. This is used to achieve polar-segmented recovery in a single solid-phase processing flow to obtain polar and hydrophobic components.

[0021] Preferably, the polar components are separated by liquid chromatography using a HILIC column with gradient elution, and mass spectrometry is performed using target-triggered MS / MS or near real-time data-dependent acquisition with switching between positive and negative ion modes.

[0022] The hydrophobic component was subjected to reversed-phase C14 liquid chromatography. 18 The chromatographic column is eluted with a gradient adapted to the target compound, and the mass spectrometer employs multiple reaction monitoring (MRM) or timed fragmentation to improve quantitative throughput, thus achieving both broad spectral coverage and high-sensitivity quantification.

[0023] Preferably, the quantification uses isotope internal standard normalization combined with matrix matching multi-point calibration and uses response surface methodology (such as four-parameter or quadratic fitting) to fit the peak area-concentration relationship, and uses matrix dilution curves to evaluate and correct for strong matrix effects.

[0024] Quality control criteria include at least the thresholds for blank, low, medium, and high spiked recovery and sample internal standard recovery (e.g., a recovery rate of 70% to 120% is considered acceptable), and confirmation by high-resolution mass spectrometry for results below the method limit of quantitation but with suspicious characteristics.

[0025] Preferably, the method is extended to newly emerging or rare marine toxins by combining spectral library comparison with external standard confirmation, and can be expanded to be compatible with other shellfish or edible seafood matrices by adjusting the internal standard composition and chromatographic conditions, thus obtaining a scalable multi-category toxin detection method.

[0026] Another aspect of the present invention is to provide a multi-category marine toxin sample analysis system suitable for shellfish matrices, comprising:

[0027] The weighing and spiking module is used to weigh the homogenized shellfish tissue sample, add an isotope-labeled internal standard mixture covering multiple types of marine toxins, and simultaneously correct for subsequent extraction recovery and matrix effects to obtain a homogenized sample.

[0028] A one-step acidified organic solvent extraction module is used to add acidified methanol:water mixed solvent to the homogeneous sample and perform ultrasonic treatment and centrifugation to simultaneously dissolve polar and moderately hydrophobic toxins and precipitate proteins in a single solvent phase to obtain a crude extract.

[0029] The co-bed segmented solid phase enrichment module is used to pass the crude extract into a pre-equilibrium mixed bonded phase solid material and elute it sequentially with low organic ratio and high organic ratio. It is used to enrich different toxin categories according to polarity in a single processing flow to obtain polar components and hydrophobic components.

[0030] The polar component chromatography-mass spectrometry analysis module is used to concentrate the polar components and redissolve them in a mobile phase adapted to HILIC chromatography before performing liquid chromatography-tandem mass spectrometry analysis (including positive and negative ionization switching and target trigger acquisition). It is used to separate, identify and preliminarily quantify polar toxins and obtain polar component mass spectrometry data.

[0031] The hydrophobic component chromatography-mass spectrometry analysis module is used to concentrate the hydrophobic component and redissolve it in a mobile phase adapted for reversed-phase chromatography before performing liquid chromatography-tandem mass spectrometry analysis (including multiple reaction monitoring or timed fragmentation). It is used to separate, identify and preliminarily quantify hydrophobic and mesohydrophobic toxins to obtain hydrophobic component mass spectrometry data.

[0032] The normalized quantification and quality control confirmation module is used to normalize the mass spectrometry data of the polar components and the mass spectrometry data of the hydrophobic components using isotope internal standards, and to perform matrix effect correction and recovery correction based on matrix matching multi-point calibration and response surface fitting. It also performs high-resolution mass spectrometry or secondary fragment spectrum comparison to confirm low abundance or suspected positive results, and outputs a confirmed multi-category marine toxin content report.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] (1) This invention combines different bonded phases, such as reversed-phase and ion-exchange phases, into a co-bed or composite medium, and recovers the toxins by sequential elution in segments according to polarity, thereby providing more targeted elution conditions for various toxins. This strategy can enhance the enrichment of the target analyte in the corresponding elution segment and disperse a large number of matrix interferences into different segments, facilitating subsequent chromatographic separation and mass spectrometry detection, thus reducing the influence of matrix effects on quantification from the source.

[0035] (2) In this invention, the isotopically labeled BMAA series internal standards achieve the best correction effect in the entire process of pretreatment, enrichment, concentration and mass spectrometry detection by being highly isomorphic (or fully labeled) to the chemical skeleton of the target BMAA component, thereby further reducing the influence of matrix effect on the quantification of trace neurotoxins (e.g. BMAA) and improving the adaptability of the method to rare / emerging toxin categories.

[0036] (3) This invention uses matrix matching calibration based on the target matrix and combines response surface fitting (nonlinear fitting strategy) to describe the relationship between peak area ratio and concentration. This can better handle nonlinear responses in low concentration areas or under strong matrix effects, thus making the quantification of low abundance targets more traceable and reproducible. Since isotope internal standard correction and segmented enrichment improve the reliability of the initial screening results, the proportion of suspected positives that need to be further confirmed by high-resolution mass spectrometry will theoretically decrease. This allows the valuable high-resolution / high-throughput confirmation resources to be used for the few samples that are really needed, improving the overall efficiency of laboratory resource utilization. Attached Figure Description

[0037] Figure 1 This is a flowchart of a multi-category marine toxin sample analysis method applicable to shellfish matrix according to the present invention;

[0038] Figure 2 This is a block diagram of a multi-category marine toxin sample analysis system applicable to shellfish matrices according to the present invention. Detailed Implementation

[0039] 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. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1:

[0041] Please see Figure 1 As shown, a method for analyzing multi-category marine toxin samples suitable for shellfish matrices includes the following steps:

[0042] S1. Weighing and Spiking: Weigh the homogenized shellfish tissue sample and add an isotope-labeled internal standard mixture covering multiple types of marine toxins to simultaneously correct for subsequent extraction recovery and matrix effects, thus obtaining a homogenized sample.

[0043] Weigh 5.0 g of homogenized shellfish tissue sample. The isotope-labeled internal standard mixture covers representative markers from at least the following toxin categories: PSP toxin, DA toxin, BTX toxin, OA, DTX toxins and TTX toxin. The concentration of each internal standard added ensures that its peak area signal-to-noise ratio is ≥10 in the final analysis to obtain more reliable recovery correction data.

[0044] In step S1, in order to obtain a representative homogenized shellfish sample, an isotope-labeled internal standard mixture covering the major toxin categories is added at the beginning of the method.

[0045] Targeting the cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA), the isotope-labeled internal standard mixture used in this embodiment contains the following BMAA series internal standards (all stock solutions):

[0046] U-[¹³C3,¹ 5 [N2]-β-N-methylamino-L-alanine (10 μg / mL, solvent: methanol / water);

[0047] U-[D3]-β-N-methylamino-L-alanine (10 μg / mL, solvent: methanol / water);

[0048] U-[¹ 5 [N2]-β-N-methylamino-L-alanine (10 μg / mL, solvent: methanol / water).

[0049] When using the above-mentioned stock solution, it should be diluted together with other internal standards to the working concentration according to the predetermined dilution scheme.

[0050] S2, One-step acidified organic solvent extraction: Add acidified methanol:water mixed solvent to the homogeneous sample and perform ultrasonic treatment and centrifugation to simultaneously dissolve polar and moderately hydrophobic toxins and precipitate proteins in a single solvent phase to obtain crude extract.

[0051] The acidified organic solvent was methanol:water in a volume ratio of 80:20, containing 0.01% formic acid. The ultrasonic treatment time was 20 minutes, and the centrifugation conditions were 3000 g for 15 minutes. This was used to achieve the dissolution of polar and moderately hydrophobic toxins and to precipitate proteins in a single extraction, resulting in a stable crude extract.

[0052] In step S2, ultrasonic / vibration mixing and centrifugation are performed in a single acidified organic solvent system to simultaneously dissolve polar and moderately hydrophobic toxins and achieve efficient protein precipitation, reduce losses caused by sample splitting and multiple transfers, and obtain a stable crude extract to facilitate subsequent fractional enrichment.

[0053] S3. Segmented solid-phase enrichment in the same bed: The crude extract is passed through a pre-equilibrated mixed bonded solid-phase material and eluted sequentially with low organic ratio and high organic ratio. This is used to enrich different toxin categories in a single processing flow according to polarity, and to obtain polar components and hydrophobic components.

[0054] Mixed-bonded solid-phase materials are inverse phases (C 18A co-bed or composite medium combining phenyl groups with ion exchange (strong anion exchange or weak cation exchange) and employing sequential elution: first, elution with a buffered aqueous phase with a low organic ratio to enrich the more polar components, and then elution with an organic phase with a high organic ratio and a small amount of volatile salts to enrich the hydrophobic components. This is used to achieve polar-segmented recovery in a single solid-phase processing flow to obtain polar and hydrophobic components.

[0055] In step S3, the target toxin is enriched in segments through selective elution, while a large number of matrix interfering substances are distributed to non-target segments, thereby improving the signal-to-noise ratio of subsequent chromatographic separation and detection.

[0056] S4. Polar component chromatography-mass spectrometry analysis: The polar components are concentrated and redissolved in a mobile phase adapted for HILIC chromatography, and then subjected to liquid chromatography-tandem mass spectrometry analysis (including positive and negative ionization switching and target trigger acquisition) to separate, identify and preliminarily quantify polar toxins, and obtain polar component mass spectrometry data.

[0057] The polar components were separated by liquid chromatography using a HILIC column with gradient elution, and mass spectrometry was performed using target-triggered MS / MS or near real-time data-dependent acquisition with switching between positive and negative ion modes.

[0058] The hydrophobic component was subjected to reversed-phase C14 liquid chromatography. 18 The chromatographic column is eluted with a gradient adapted to the target compound, and the mass spectrometer employs multiple reaction monitoring (MRM) or timed fragmentation to improve quantitative throughput, thus achieving both broad spectral coverage and high-sensitivity quantification.

[0059] In step S4, after necessary concentration and solvent replacement of the polar elution section, mass spectrometry acquisition is performed using a chromatographic mode adapted to polar compounds (such as HILIC) and switching ionization modes to improve the chromatographic separation of polar toxins and the acquisition of fragment information, thereby facilitating accurate identification and sensitive quantification.

[0060] S5. Hydrophobic component chromatographic-mass spectrometry analysis: The hydrophobic component is concentrated and redissolved in a mobile phase suitable for reversed-phase chromatography, and then subjected to liquid chromatography-tandem mass spectrometry analysis (including multiple reaction monitoring or timed fragmentation) to separate, identify and preliminarily quantify hydrophobic and mesohydrophobic toxins, and obtain hydrophobic component mass spectrometry data.

[0061] The quantification was performed using isotope internal standard normalization combined with matrix matching multi-point calibration and response surface methodology (such as four-parameter or quadratic fitting) to fit the peak area-concentration relationship. Matrix dilution curves were used to evaluate and correct for strong matrix effects.

[0062] Quality control judgment includes at least the thresholds for blank, low, medium, and high spiked recovery and sample internal standard recovery (e.g., a recovery rate of 70% to 120% is acceptable), and confirmation of results below the method limit of quantitation but with suspicious characteristics by high-resolution mass spectrometry.

[0063] In step S5, after the hydrophobic elution segment is concentrated and the solvent is optimized, the retention and separation of low to medium polarity to hydrophobic toxins are achieved on reversed-phase chromatography. Combined with timed MRM or targeted fragmentation strategies, the quantitative throughput and selectivity are improved, so as to emphasize the targeted optimization of chromatographic and mass spectrometry parameters to adapt to the physicochemical properties of different toxins.

[0064] S6. Normalized Quantification and Quality Control Confirmation: The mass spectrometry data of the polar components and the mass spectrometry data of the hydrophobic components are normalized by isotope internal standard and matrix effect correction and recovery correction are performed based on matrix matching multi-point calibration and response surface fitting. High-resolution mass spectrometry or secondary fragment spectrum comparison is performed on low abundance or suspected positive results to confirm them, and the results are used to output a confirmed multi-category marine toxin content report.

[0065] The method is extended to newly emerging or rare marine toxins by combining spectral library comparison with external standard confirmation. Furthermore, by adjusting the composition of the internal standard and chromatographic conditions to be compatible with other shellfish or edible seafood matrices, a scalable multi-category toxin detection method can be obtained.

[0066] In step S6, peak area normalization is performed using isotope internal standards as the core, and then matrix effects and nonlinear responses are corrected by combining matrix matching with multi-point calibration and response surface fitting.

[0067] As shown above, combining different bonded phases such as reversed-phase and ion-exchange media into co-bed or composite media, and then recovering them in polarity-based fractions via sequential elution, allows for more targeted elution conditions for various toxins. This strategy not only enhances the enrichment of target analytes in their respective elution fractions but also disperses a large number of matrix interferences into different fractions, facilitating subsequent chromatographic separation and mass spectrometry detection, thereby reducing the impact of matrix effects on quantification from the source.

[0068] Example 2:

[0069] Weighing and Spiking: Weigh 5.00 g of homogenized mussel (Mytilus edulis) muscle and add isotope-labeled internal standard mixture (containing representative markers covering PSP, DA, TTX, OA / DTX, etc.; the target concentration of each internal standard in the final analytical dilution is 5 ng / mL) to simultaneously correct for subsequent extraction recovery and matrix effects, and obtain homogenized sample.

[0070] Data acquisition method: Weighing with an electronic balance (accuracy 0.1 mg); preparing internal standard solution and adding it with a pipette; recording the initial sample mass and the volume of internal standard added.

[0071] One-step acidified organic solvent extraction: Add 20.0 mL of methanol:water (80:20 v / v, containing 0.1% formic acid) to the homogenized sample, place it in an ultrasonic bath and shake for 10 min, then centrifuge at 4000 g for 10 min. Take the supernatant as the crude extract, which is used to simultaneously dissolve polar and moderately hydrophobic toxins and precipitate proteins in a single solvent phase to obtain the crude extract.

[0072] Data acquisition methods: record sonication time, centrifugation force and temperature; transfer supernatant using a pipette; measure and record the volume of supernatant.

[0073] Simultaneous fractional solid-phase enrichment in the same bed: The crude extract was passed through a container filled with 200 mg C 18 A 6 mL solid-phase extraction column with a +100 mg SAX (strong anion exchange) co-bed is pre-equilibrated at a flow rate of 1 mL / min. The polar component is first eluted with 5 mL of aqueous phase containing 0.1% formic acid (low organic ratio) to obtain the polar component, and then eluted with 5 mL of methanol (containing 0.1% formic acid, high organic ratio) to obtain the hydrophobic component. This is used to enrich different toxin categories according to polarity in a single processing flow to obtain polar and hydrophobic components.

[0074] Data acquisition method: The SPE extraction process is executed by an automated solid phase extraction device, and the volume and flow rate of each stage are recorded; the eluent is collected and its volume is recorded.

[0075] Polar component chromatographic-mass spectrometric analysis: Polar components were gently purged with nitrogen to near dryness (purging rate approximately 0.5 mL / min, water bath temperature 35℃), reconstituted with HILIC-compatible solvent (redissolved in 200 µL of acetonitrile / water containing 10 mM ammonium formate 90:10), filtered, and injected into a HILIC chromatogram (column type: BEH Amide 2.1×100 mm, 1.7 μm, flow rate 0.3 mL / min, gradient 95% ACN→50% ACN, injection volume 5 μL). Mass spectrometry data of polar components were obtained by switching between positive and negative ionization modes and data-dependent / target-triggered MS / MS acquisition on a tandem mass spectrometer (QqQ) for separation, identification, and preliminary quantification of polar toxins (such as PSP, DA, TTX, BMAA).

[0076] Example instrument parameters: source temperature 500℃, spray voltage 3500V, collision energy range 10–40eV (optimized according to target). Data acquisition and recording: peak area, retention time, and internal standard peak area.

[0077] Chromatographic-mass spectrometric analysis of hydrophobic components: The hydrophobic components were gently purged with nitrogen until nearly dry, then redissolved in 200 µL of methanol / water (80:20, containing 5 mM ammonium formate) and filtered. The solution was then injected into a reversed-phase C24 micrometer. 18 Chromatography (column type: C)18 (2.1×100 mm, 1.7 μm, flow rate 0.3 mL / min, gradient 5%→95% organic phase, injection volume 5 μL), and multi-reaction monitoring (MRM) or scheduled fragmentation acquisition was used on a tandem mass spectrometer (QqQ) to obtain mass spectrometry data of hydrophobic components for the separation, identification, and preliminary quantification of hydrophobic / moderately hydrophobic toxins (such as OA, DTX).

[0078] Example of instrument parameters: source temperature 500 °C, spray voltage 3500 V, MRM window scheduled according to the target substance, scheduled window ±30 s.

[0079] Normalized quantification and quality control confirmation: The mass spectrometry data of polar components and hydrophobic components were normalized with isotope internal standards respectively. Matrix-matched multi-point calibration curves (spiked concentration points: 0.5, 1, 2, 5, 10, 20 μg / kg) prepared in blank shellfish matrices were used. The peak area ratio (target / internal standard) was used as y, and the concentration was used as x. A calibration equation was obtained by four-parameter or quadratic response surface fitting, and the toxin concentration in the sample was calculated. When necessary, high-resolution mass spectrometry (HRMS) or secondary fragment spectra were used to confirm low-abundance or suspected positive samples, and finally a confirmed multi-class marine toxin content report was output.

[0080] Calculation explanation of a certain target toxin is as follows:

[0081] Obtain the matrix-matched calibration curve y = f(x) (f is the fitting function);

[0082] The peak area of the sample is measured as As, and the peak area of the internal standard is Ais. Calculate the response ratio R = As / Ais;

[0083] Obtain the sample concentration Csample = f⁻¹(R) (μg / kg, relative to the original sample mass) from the fitting function;

[0084] If there is a volume change during the concentration / dilution process of the sample, correct it according to the dilution factor DF: Cfinal = Csample × DF;

[0085] Record the recovery rate of each target substance (based on the spiked recovery experiment) and correct the final reported value.

[0086] Quality control criteria (adopted in this example): blank signal < LOD; spiked recovery rate of 70% - 120% is qualified; repeatability RSD ≤ 15%.

[0087] Experimental verification and data

[0088] Samples: Collected from local shellfish (mussels), 20 blank samples were randomly selected as matrices, and another 6 spiked points were prepared for matrix matching calibration.

[0089] Standard solutions and internal standards: The external standard series was prepared in methanol according to the original example; the isotope internal standard mixture consisted of representative internal standards covering PSP, DA, BTX, OA / DTX, TTX, BMAA, etc. (the stock concentration was 10 μg / mL, and the solvent was methanol / water).

[0090] Internal standard mixture preparation: Taking PSP internal standard as an example, the isotope-labeled internal standard mixture used in this embodiment contains the following PSP series internal standards (all of which are stock solutions):

[0091] U-[¹ 5 [N7] Shifang clam toxin (10 μg / mL, solvent: methanol / water);

[0092] U-[¹ 5 [N6] Decarbamoyl neosalicytoxin (10 μg / mL, solvent: methanol / water);

[0093] U-[¹ 5 [N7] Neo-Schizotoxin (10 μg / mL, solvent: methanol / water).

[0094] Dilute each 10 μg / mL stock solution to the working solution at a ratio of 1:2000 (v / v) and combine with other representative internal standards to form the final internal standard mixture; add 10 µL of the internal standard working solution to a 5.00 g sample. The resulting internal standard corresponds to a target concentration of approximately 5 ng / mL under the final analytical volume / condition.

[0095] Repeatability: n=6 parallel pretreatment and analysis were performed on the same homogeneous sample to evaluate recovery and RSD.

[0096] Summary of typical results (example values, representative target toxins: PSP representative, OA representative):

[0097] The average recovery rate of PSP (a representative compound) was 86%, with an RSD of 8.2%, a method detection limit (LOD) of 0.15 µg / kg, and an LOQ of 0.5 µg / kg.

[0098] The average recovery rate of OA / DTX (a representative compound) was 91%, with an RSD of 7.5%, LOD of 0.12 µg / kg and LOQ of 0.4 µg / kg.

[0099] Single sample pretreatment time (including sample weighing, extraction, SPE, concentration, redissolution, and filtration): approximately 35 min (excluding chromatography-mass spectrometry determination time); solvent usage: approximately 22 mL / sample (including extraction and elution).

[0100] Taking the PSP representative in Example 2 as an example:

[0101] Matrix matching calibration curve (example fitting result): The fitting equation (four-parameter fitting) f(x) of the response ratio R versus concentration x is obtained using the calibrated points [0.5,1,2,5,10,20] µg / kg. It is assumed that the fitting result can be approximated as linear y=0.085x+0.002 (R=peak area target / peak area internal standard) within the effective interval.

[0102] If the measured peak area of ​​a target analyte in the sample is As=1.70e5 and the internal standard peak area is Ais=2.00e5, then the response ratio R=0.85.

[0103] The concentration calculated from the fitted equation is x = (R - 0.002) / 0.085 = (0.85 - 0.002) / 0.085 ≈ 9.94 µg / kg.

[0104] Considering the volume change of the sample during pretreatment (e.g., 20 mL extraction, final reconstitution volume 0.2 mL, concentration factor DF = (20 / 0.2) × (sample mass / 5 g) = 100 × 1 = 100), if the calibration curve has been corrected according to the final concentration factor, then Cfinal = 9.94 µg / kg is the reported value; if not, it needs to be corrected by multiplying or dividing by DF.

[0105] Recovery correction: If the spiked recovery experiment shows that the average recovery rate of the target analyte is 86%, then the corrected concentration Ccorrected = Cfinal / 0.86 = 11.56 µg / kg (the final reported value).

[0106] As shown above, adding multi-category isotope-labeled internal standards at the beginning of sample preparation provides intrinsic correction at each stage, addressing pretreatment losses, ionization suppression / enhancement, and instrument drift. Compared to methods that only add internal standards at the detection end or by category, this comprehensive correction more fully compensates for systematic errors, improving the accuracy and repeatability of quantitative results.

[0107] Example 3:

[0108] Weighing and Spiking: Weigh 5.00 g of homogenized shellfish tissue and spike it separately for polar toxins and hydrophobic toxins (the spiked systems did not use a full spectrum of isotope mixed internal standard, but only a separate internal standard for each type) to independently calibrate the recovery of the two types of toxins and obtain homogenized samples.

[0109] Two-step extraction: The homogeneous sample was divided into two portions (2.5 g each). One portion was extracted with a highly polar solvent (water:methanol 50:50 containing 0.1% formic acid) by ultrasonic extraction for 15 min, and the supernatant was collected after centrifugation as the polar crude extract. The other portion was extracted with a relatively high organic phase (acetonitrile or pure methanol) for 15 min, and the supernatant was collected after centrifugation as the hydrophobic crude extract. This was used to optimize the dissolution of polar and hydrophobic toxins, respectively, to obtain the polar crude extract and the hydrophobic crude extract.

[0110] Separate solid-phase enrichment: For polar crude extraction, a HILIC-compatible, highly polar adsorbent was used for enrichment; for hydrophobic crude extraction, conventional C0.05 adsorbent was used. 18 SPE enrichment, followed by two independent solid-phase treatments, yielded polar and hydrophobic components.

[0111] Separate chromatographic-mass spectrometric analysis: polar components were analyzed using HILIC-MS / MS, and hydrophobic components were analyzed using reversed-phase C2S3S4 ... 18 -MS / MS analysis (two sets of samples were injected, run, and data processed separately) to obtain mass spectrometry data of polar components and mass spectrometry data of hydrophobic components.

[0112] Quantification and quality control: Each is calibrated and quantified by matrix matching based on its own internal and external standards, and quality control is carried out according to the routine blank / spiked recovery.

[0113] Experimental verification and data (control):

[0114] Sample and standard processing conditions: as described above, but extraction and SPE are performed in two separate sets, which increases both processing time and solvent usage.

[0115] Summary of typical results:

[0116] The average recovery rate of PSPs was 78%, with an RSD of 12.5%, a LOD of 0.25 µg / kg, and a LOQ of 0.8 µg / kg.

[0117] The average recovery rate for OA / DTX was 85%, with an RSD of 11.2%, LOD of 0.28 µg / kg, and LOQ of 0.9 µg / kg.

[0118] The pretreatment time for a single sample is approximately 50 minutes; the solvent volume is approximately 38 mL per sample; the effective number of samples processed per day (8-hour working day) is approximately 18.

[0119] Table 1 below shows a representative comprehensive comparison of measurements obtained in our laboratory under the same matrix and spiking levels, and under the same chromatographic / mass spectrometric detection conditions. The values ​​are the mean or estimated values ​​obtained from multiple repetitions (n≥6).

[0120] Table 1

[0121] index Example 2 (Invention) Example 3 (Control) Increase (%) Average recovery rate (PSP representative) 86% 78% 10.3% Recovery Rate RSD (PSP) 8.2% 12.5% 34.4% (RSD decreased) Average recovery rate (OA representative) 91% 85% 7.1% Recovery rate RSD (OA) 7.5% 11.2% 33.0% Method LOQ (PSP, µg / kg) 0.5 0.8 37.5% Method LOQ (OA, µg / kg) 0.4 0.9 55.6% Single sample pretreatment time (min) 35 50 30.0% Solvent volume per sample (mL) 22 38 42.1% Number of samples that can be processed per day (8 hours) 28 18 55.6% The proportion of suspected positive cases that needs further confirmation by HRMS 3% 9% 66.7% (Less)

[0122] As shown in the table above, by using full-spectrum isotope internal standards throughout the entire process and segmented solid-phase enrichment in the same bed, more stable recovery rates can be obtained for different types of toxins in the same treatment process. In Example 1, the recovery rate and RSD were increased by approximately 7%–10% and the RSD was reduced by approximately 30% compared to the control method (data in the table).

[0123] Because combined extraction reduces sample loss and isotope internal standard correction is more accurate, the LOQ is reduced by about 30% to 55% compared with the control method, thus improving the detection and quantification of low-abundance toxins.

[0124] Compared with single-bed fractional enrichment, two independent extraction systems can reduce pretreatment time by about 30%, solvent consumption by about 40%, and daily processing capacity by about 55%, thereby reducing laboratory operating costs and pollution.

[0125] A lower proportion of suspected positives means a significant reduction in the number of samples requiring secondary confirmation by HRMS (approximately a 2 / 3 reduction in the experiment), alleviating the pressure on high-resolution mass spectrometry resources.

[0126] As can be seen from the above, using matrix matching calibration based on the target matrix, combined with response surface fitting (nonlinear fitting strategy) to describe the relationship between peak area ratio and concentration, can better handle nonlinear responses in low concentration regions or under strong matrix effects, thus making the quantification of low abundance targets more traceable and reproducible.

[0127] Since isotope internal standard correction and segmented enrichment improve the reliability of the initial screening results, the proportion of suspected positive samples that require further confirmation by high-resolution mass spectrometry will theoretically decrease. This allows valuable high-resolution / high-throughput confirmation resources to be used for the few samples that are truly needed, improving the overall efficiency of laboratory resource utilization.

[0128] Example 4:

[0129] like Figure 2 As shown, a multi-category marine toxin sample analysis system suitable for shellfish matrices includes:

[0130] The weighing and spiking module is used to weigh the homogenized shellfish tissue sample, add an isotope-labeled internal standard mixture covering multiple types of marine toxins, and simultaneously correct for subsequent extraction recovery and matrix effects to obtain a homogenized sample.

[0131] A one-step acidified organic solvent extraction module is used to add acidified methanol:water mixed solvent to the homogeneous sample and perform ultrasonic treatment and centrifugation to simultaneously dissolve polar and moderately hydrophobic toxins and precipitate proteins in a single solvent phase to obtain a crude extract.

[0132] The co-bed segmented solid phase enrichment module is used to pass the crude extract into a pre-equilibrium mixed bonded phase solid material and elute it sequentially with low organic ratio and high organic ratio. It is used to enrich different toxin categories according to polarity in a single processing flow to obtain polar components and hydrophobic components.

[0133] The polar component chromatography-mass spectrometry analysis module is used to concentrate the polar components and redissolve them in a mobile phase adapted to HILIC chromatography before performing liquid chromatography-tandem mass spectrometry analysis. It is used to separate, identify and preliminarily quantify polar toxins and obtain polar component mass spectrometry data.

[0134] The hydrophobic component chromatography-mass spectrometry analysis module is used to concentrate the hydrophobic component, redissolve it in a mobile phase adapted for reversed-phase chromatography, and then perform liquid chromatography-tandem mass spectrometry analysis. It is used to separate, identify, and preliminarily quantify hydrophobic and mesohydrophobic toxins to obtain hydrophobic component mass spectrometry data.

[0135] The normalized quantification and quality control confirmation module is used to normalize the mass spectrometry data of the polar components and the mass spectrometry data of the hydrophobic components using isotope internal standards, and to perform matrix effect correction and recovery correction based on matrix matching multi-point calibration and response surface fitting. It also performs high-resolution mass spectrometry or secondary fragment spectrum comparison to confirm low abundance or suspected positive results, and outputs a confirmed multi-category marine toxin content report.

[0136] The beneficial effects of this method are the same as those of the embodiment of a method for analyzing multi-category marine toxin samples applicable to shellfish matrices, and will not be repeated here.

[0137] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for analyzing multi-category marine toxin samples suitable for shellfish matrices, characterized in that, include: Weigh out homogenized shellfish tissue samples and add an isotope-labeled internal standard mixture covering multiple types of marine toxins to simultaneously correct for subsequent extraction recovery and matrix effects, thus obtaining homogenized samples. Acidified methanol and water mixed solvent were added to the homogeneous sample and subjected to ultrasonic treatment and centrifugation to simultaneously dissolve polar and moderately hydrophobic toxins and precipitate proteins in a single solvent phase, resulting in a crude extract. The crude extract is passed through a pre-equilibrated mixed bonded phase solid material and eluted sequentially with low organic ratio and high organic ratio to enrich different toxin categories according to polarity in a single processing flow, thereby obtaining polar components and hydrophobic components. The polar components were concentrated and redissolved in a mobile phase adapted for HILIC chromatography, and then analyzed by liquid chromatography-tandem mass spectrometry to separate, identify and preliminarily quantify the polar toxins, obtaining mass spectrometry data of the polar components. The hydrophobic components were concentrated and redissolved in a mobile phase suitable for reversed-phase chromatography, and then analyzed by liquid chromatography-tandem mass spectrometry to separate, identify and preliminarily quantify hydrophobic and mesohydrophobic toxins, and obtain mass spectrometry data of the hydrophobic components. The mass spectrometry data of the polar components and the mass spectrometry data of the hydrophobic components are normalized by isotope internal standards, and matrix effect correction and recovery correction are performed based on matrix matching multi-point calibration and response surface fitting. High-resolution mass spectrometry or secondary fragment spectrum comparison is performed on low abundance or suspected positive results to confirm them, so as to output a confirmed multi-category marine toxin content report.

2. The method for analyzing multi-category marine toxin samples applicable to shellfish matrix according to claim 1, characterized in that, The mass of the homogenized shellfish tissue sample is 3.0–10.0 g, and the isotope-labeled internal standard mixture covers representative markers from at least the following toxin categories: Paralytic shellfish toxins, including STX (sparganotoxin) and GTX (gastrotoxin); Amnesic shellfish poisoning, including domoic acid (DA); Diarrhea-causing shellfish toxins include OA (otaeniacetic acid) and DTX (diarrhea-causing algal toxins); Neurotoxic shellfish, including BTX (bipyroxin B) brevicornuate toxin; Tetrodotoxin (TTX); BMAA, a cyanobacterial neurotoxin; To expand the quantitative capability for relevant neurotoxin targets, and to ensure that the peak area signal-to-noise ratio of each internal standard added is ≥10 in the final analysis sample after dilution, so as to obtain more reliable recovery correction data.

3. The method for analyzing multi-category marine toxin samples suitable for shellfish matrix according to claim 1, characterized in that, The acidified organic solvent is methanol:water in a volume ratio of 80:20, and contains 0.05% to 0.2% formic acid. The ultrasonic treatment time is 5 to 20 minutes, and the centrifugation conditions are 3,000 to 5,000 g for 5 to 15 minutes. This is used to achieve the dissolution of polar and moderately hydrophobic toxins and the precipitation of proteins in a single extraction, so as to obtain a stable crude extract.

4. The method for analyzing multi-category marine toxin samples applicable to shellfish matrix according to claim 1, characterized in that, The mixed bonded phase solid material is a co-bed or composite medium combining reverse phase and ion exchange, and adopts sequential elution: first, it is eluted with a buffered aqueous phase with a low organic ratio to enrich the more polar components, and then eluted with an organic phase with a high organic ratio and a small amount of volatile salts to enrich the hydrophobic components. This is used to achieve polar-segmented recovery in a single solid phase treatment process to obtain polar and hydrophobic components.

5. The method for analyzing multi-category marine toxin samples suitable for shellfish matrix according to claim 1, characterized in that, The polar components were separated by liquid chromatography using a HILIC column with gradient elution, and mass spectrometry was performed using target-triggered MS / MS or near real-time data-dependent acquisition with switching between positive and negative ion modes. The hydrophobic component was subjected to reversed-phase C14 liquid chromatography. 18 The chromatographic column is eluted with a gradient adapted to the target compound, and the mass spectrometer employs multiple reaction monitoring or timed fragmentation to improve quantitative throughput, thus achieving both broad spectral coverage and high-sensitivity quantification.

6. The method for analyzing multi-category marine toxin samples suitable for shellfish matrix according to claim 1, characterized in that, The quantification was performed by isotope internal standard normalization combined with matrix matching multi-point calibration and the peak area-concentration relationship of the response surface was fitted. The matrix dilution curve was used to evaluate and correct for strong matrix effects. Quality control criteria include at least the thresholds for blank, low, medium, and high spiked recovery and sample internal standard recovery, as well as confirmation by high-resolution mass spectrometry for results below the method limit of quantitation but with suspicious characteristics.

7. The method for analyzing multi-category marine toxin samples applicable to shellfish matrices according to claim 1, characterized in that, The method is extended to newly emerging or rare marine toxins by combining spectral library comparison with external standard confirmation. Furthermore, by adjusting the internal standard composition and chromatographic conditions to be compatible with other shellfish or edible seafood matrices, a scalable multi-category toxin detection method can be obtained.

8. A multi-category marine toxin sample analysis system suitable for shellfish matrices, characterized in that, include: The weighing and spiking module is used to weigh the homogenized shellfish tissue sample, add an isotope-labeled internal standard mixture covering multiple types of marine toxins, and simultaneously correct for subsequent extraction recovery and matrix effects to obtain a homogenized sample. A one-step acidified organic solvent extraction module is used to add acidified methanol:water mixed solvent to the homogeneous sample and perform ultrasonic treatment and centrifugation to simultaneously dissolve polar and moderately hydrophobic toxins and precipitate proteins in a single solvent phase to obtain a crude extract. The co-bed segmented solid phase enrichment module is used to pass the crude extract into a pre-equilibrium mixed bonded phase solid material and elute it sequentially with low organic ratio and high organic ratio. It is used to enrich different toxin categories according to polarity in a single processing flow to obtain polar components and hydrophobic components. The polar component chromatography-mass spectrometry analysis module is used to concentrate the polar components and redissolve them in a mobile phase adapted to HILIC chromatography before performing liquid chromatography-tandem mass spectrometry analysis. It is used to separate, identify and preliminarily quantify polar toxins and obtain polar component mass spectrometry data. The hydrophobic component chromatography-mass spectrometry analysis module is used to concentrate the hydrophobic component, redissolve it in a mobile phase adapted for reversed-phase chromatography, and then perform liquid chromatography-tandem mass spectrometry analysis. It is used to separate, identify, and preliminarily quantify hydrophobic and mesohydrophobic toxins to obtain hydrophobic component mass spectrometry data. The normalized quantification and quality control confirmation module is used to normalize the mass spectrometry data of the polar components and the mass spectrometry data of the hydrophobic components using isotope internal standards, and to perform matrix effect correction and recovery correction based on matrix matching multi-point calibration and response surface fitting. It also performs high-resolution mass spectrometry or secondary fragment spectrum comparison to confirm low abundance or suspected positive results, and outputs a confirmed multi-category marine toxin content report.

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