Method for accurate and comparable detection of low concentrations of intracellular and extracellular prymnesium parvum toxins

Abstract

This invention discloses an accurate and comparable method for detecting low-concentration intracellular and extracellular *Streptocytoxin*, belonging to the field of environmental monitoring technology. The method includes the following steps: S1. Calibration of *Streptocytoxin* standards and construction of cross-platform standard curves; S2. Separation and pretreatment of intracellular and extracellular *Streptocytoxin* in water samples; S3. Solid-phase extraction to concentrate and enrich *Streptocytoxin*; S4. LC-MS / MS detection and quantitative calculation. This invention binds the LC-MS / MS results to the built-in standards of widely commercially available and validated ELISA kits, using the kit's built-in standards as a benchmark for standard calibration. This fundamentally avoids systematic errors between standards from different brands and enhances the correlation between ELISA and LC-MS / MS results, providing reliable methodological support for integrating long-term monitoring data from multiple reservoirs, comparing pollution levels across regions, and reanalyzing historical monitoring data.

Description

Technical Field

[0001] This invention relates to the field of environmental monitoring technology, specifically to a method for accurate and comparable detection data of low-concentration intracellular and extracellular columniform toxins. Background Technology

[0002] Cylindrospermopsin (CYN) is a potent multi-organ toxicity cyanobacterial toxin that primarily targets and damages the liver and kidneys. In freshwater environments, this toxin is mainly produced by the dominant algal bloom species *Rhizophora raspevillei*, which is one of the main cyanobacterial species causing frequent algal blooms in lakes and reservoirs. Cylindrospermopsin exists in water in two forms: intracellular bound and extracellular dissolved. Accurate quantification of intracellular and extracellular cylindrospermopsin concentrations is the foundation and prerequisite for conducting early warning and precise control of cylindrospermopsin pollution risks.

[0003] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is a core technology for the precise quantification, contamination mechanism research, and risk assessment of *Streptocytoxin* due to its combination of high detection sensitivity and high target specificity. The quantitative accuracy and comparability of this method depend entirely on the reliability of the quantitative standard curve constructed from *Streptocytoxin* standards. Currently, *Streptocytoxin* standards used in LC-MS / MS detection are mainly divided into two categories: one is commercially available *Streptocytoxin* standard powders or solutions from different brands; the other is standards integrated into commercially validated ELISA kits.

[0004] Existing quantitative detection methods for *Streptocytoxin* based on LC-MS / MS cannot achieve uniformity and cross-platform comparability of detection results, and cannot simultaneously meet the requirements for accurate quantification of low concentrations of *Streptocytoxin* in natural water bodies and the low-cost requirements for large-scale routine monitoring.

[0005] Specifically, the labeled and actual quality of commercially available *Strombyx mori* toxin standards from different brands varies significantly. LC-MS / MS standard curves built upon these labeled values ​​contain inherent systematic errors, directly hindering cross-platform comparisons of test results from different laboratories and batches. This severely interferes with the integration of long-term monitoring data from multiple lakes and reservoirs, cross-regional pollution comparisons, and historical data reanalysis. While high-quality ELISA kits with reliable metrological values ​​can be used as the metrological benchmark for LC-MS / MS detection, these kits are expensive, and the total amount of built-in standards is limited. Long-term use as routine testing standards would significantly increase testing costs, making large-scale routine monitoring economically impractical. Furthermore, the concentration of *Strombyx mori* toxins in natural water bodies is mostly in the range of several ng / L. -1Due to the low level of concentration, existing LC-MS / MS methods, while controlling detection costs, struggle to accurately quantify low concentrations of *Strombocytoxin*, further limiting the applicability of these methods and exacerbating quantitative biases and incomparability in the detection results.

[0006] Therefore, we propose a method for detecting low concentrations of intracellular and extracellular columniform toxins that provides accurate and comparable data, in order to alleviate or resolve the aforementioned problems.

[0007] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a method for accurate and comparable detection data of low-concentration intracellular and extracellular *Pyroxypyr* toxins. This method solves the problems in the prior art, such as inconsistent detection results of *Pyroxypyr* toxins, poor cross-platform comparability, high cost of large-scale routine monitoring, and difficulty in accurately quantifying trace low-concentration *Pyroxypyr* toxins in natural water bodies.

[0009] To achieve the above objectives, the present invention provides a method for accurate and comparable detection data of low-concentration intracellular and extracellular columniform toxins, comprising the following steps:

[0010] S1. Using the series of gradient concentration standards built into the commercially available *Streptococcus pluvialis* toxin ELISA kit with proven accuracy as the metrological baseline, a baseline standard curve was obtained by LC-MS / MS detection. A series of standard solutions of gradient concentrations to be calibrated were prepared using *Streptococcus pluvialis* toxin standard powder. The standard solutions to be calibrated were detected under LC-MS / MS conditions identical to those used to construct the baseline standard curve. The actual concentration of the standard solutions to be calibrated was calculated based on the baseline standard curve. After metrological calibration of the standards, a quantitative standard curve for LC-MS / MS detection was constructed using the calibrated actual concentration as the baseline.

[0011] S2. The water sample to be tested is filtered to separate the filtrate and the filter membrane carrying algal cells. The filtrate is used as the test precursor solution for extracellular columniformis toxin. The filtrate obtained after eluting algal cells, disrupting cells, and filtering impurities from the filter membrane is used as the test precursor solution for intracellular columniformis toxin. The pH of the two test precursor solutions is adjusted to obtain the sample solution to be enriched.

[0012] S3. The graphitized carbon black (PGC) solid-phase extraction column was used to activate, load, enrich, dry and elute the sample solution to be enriched in sequence. The collected eluent was dried by nitrogen blowing and the residue was dissolved with a reconstitution solution to obtain the solution to be tested on the instrument.

[0013] S4. The test solution is detected using the same LC-MS / MS conditions as in step S1. Based on the quantitative standard curve constructed in step S1, the concentration of *Pterocytoxin* in the test solution is obtained. Combined with the concentration factor and recovery rate of solid-phase extraction, the actual concentrations of intracellular and extracellular *Pterocytoxin* in the water sample are calculated.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] This invention binds LC-MS / MS measurement results to built-in standards in ELISA kits that have been widely commercialized and whose accuracy has been fully verified. Standard calibration is performed using the built-in standards as a benchmark, which not only avoids systematic errors between standards from different brands at the source, but also enhances the correlation between ELISA detection results and LC-MS / MS measurement results. This provides reliable methodological support for the integration of long-term sequence monitoring data from multiple reservoirs, the comparison of pollution levels across regions, and the reanalysis of historical monitoring data.

[0016] This invention uses a standard solution prepared by oneself from a pterostilbene toxin standard powder. Only a small amount of ELISA kit standard is needed for one-time calibration. There is no need to purchase expensive brand kits or commercial pterostilbene toxin standard solutions as routine standards for LC-MS / MS detection. While ensuring the accuracy of the values, it reduces the detection cost of pterostilbene toxin and is suitable for the routine monitoring needs of large-scale environmental water samples.

[0017] This invention achieves efficient separation of intracellular and extracellular *Streptocytoxin* through membrane filtration, enabling quantitative detection of the two existing forms of the toxin. Simultaneously, solid-phase extraction is used to concentrate *Streptocytoxin* in water by 400-500 times, raising the detection limit and solving the problem of accurate quantification of low concentrations of *Streptocytoxin* at the ng / L level using existing LC-MS / MS methods.

[0018] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0019] Figure 1 This is a flowchart of the quantitative detection and correction method for columnar cytotoxins of the present invention.

[0020] Figure 2The figures show the LC-MS / MS peak chromatograms and standard curves of the ELISA built-in standards of the present invention; wherein, Figures (a)-(g) represent the LC-MS / MS 414.4 / 272.1 ion pair peak chromatograms of CYN standards at 2 μg / L, 1 μg / L, 0.5 μg / L, 0.2 μg / L, 0.1 μg / L, 0.05 μg / L and 0 μg / L, respectively, and Figure (h) is a linear fitting graph of peak area versus standard concentration. Detailed Implementation

[0021] 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. It should be noted that the drawings are schematic and not illustrated to scale. For clarity and convenience, the relative sizes and proportions of the parts shown in the drawings have been exaggerated or reduced in size. Any size is only illustrative and not limiting.

[0022] Instruments: Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS, with Analyst workstation), 5L water sampler, vacuum pump, porous vacuum solid-phase extraction device, nitrogen blower, ultrasonic cleaner, centrifuge, 0.2μm polycarbonate (PC) filter membrane (47mm diameter), 0.22μm needle filter, 50mL centrifuge tubes, 1.5mL brown glass sample vials, 40mL brown glass bottles, and numbered polyethylene plastic buckets.

[0023] Reagents and materials: ABRAXIS Cylindrospermopsin ELISA kit (PN522011, USA), Cylindrospermopsin (CYN) standard powder, chromatographic grade methanol, chromatographic grade formic acid, chromatographic grade dichloromethane, NaOH, ultrapure water, PGC solid phase extraction column (500 mg, 6 mL, Thermo).

[0024] Example 1

[0025] A method for detecting low concentrations of intracellular and extracellular columniform toxins that provides accurate and comparable data includes the following steps:

[0026] Take the series of gradient concentrations of CYN standards built into the ABRAXISE LISA kit, with concentrations of 2 μg / L. -1 1 μgL -1 0.5 μg / L -1 0.2 μg / L -1 0.1 μg / L -1 0.05 μg / L -1 0 μgL -1Each gradient solution has a volume of 1 mL and is used directly as a primary reference standard for LC-MS / MS detection.

[0027] Taking 100 μg CYN standard powder as an example, add 1 mL of 2.5% methanol aqueous solution to a glass bottle containing CYN standard powder, and fully dissolve the powder to form a theoretical concentration of 100 μg / mL. -1 CYN stock solution; take 1 μL of CYN stock solution into a 1.5 mL brown glass sample vial, add 999 μL of 2.5% methanol aqueous solution, and gently pipette the solution 30 times to mix, forming 100 μg / L of CYN stock solution. -1 CYN intermediate solution; using 100 μg / L -1 Prepare 10 μg / L CYN solution using the same stepwise dilution method. -1 1 μgL -1 0.5 μg / L -1 0.2 μg / L -1 The standard solution to be calibrated has a concentration gradient that can be adjusted as needed.

[0028] LC-MS / MS detection conditions

[0029] Chromatographic conditions: Kinetex@ 2.1 μm C18 100A column (100×2.1 mm); column temperature 40℃, injection volume 50 μL, flow rate 0.3 mL / min. -1 Mobile phase A is an aqueous solution containing 0.5% formic acid by volume, and mobile phase B is methanol.

[0030] Mass spectrometry conditions: electrospray ionization (ESI), multiple reaction monitoring (MRM) negative ion mode; CYN scan MRM quantitative ion pair 414.4 / 272.1, qualitative ion pair 414.4 / 302.1, 414.4 / 97.0.

[0031] Following the instructions in the LC-MS / MS assay kit, each concentration was measured in triplicate using the primary reference standard. The CYN peak diagram was confirmed and calibrated using Analyst software to obtain the 414.4 / 272.1 ion pair peak areas for each concentration of the standard. Specific data are as follows:

[0032] 2μgL -1 Peak area 3.37×10 4 counts; 1 μg / L -1 Peak area 1.68×10 4 counts; 0.5 μg / L -1 Peak area 8.57×10 3 counts; 0.2 μg / L -1Peak area 3.36×10 3 counts; 0.1 μg / L -1 Peak area 1.18×10 3 counts; 0.05 μg / L -1 Peak area 4.58×10 2 counts; 0 μg / L -1 No target peak response. The peak area values ​​mentioned above may vary due to normal fluctuations in detection conditions such as instrument status and mobile phase ratio, and do not constitute a limitation on the detection results of this method.

[0033] Using the known concentration of CYN as the ordinate (y) and the peak area of ​​the 414.4 / 272.1 ion pair as the abscissa (x), a linear regression analysis was performed to obtain the regression equation for the baseline standard curve: y = a × x + b, where y is the CYN concentration, x is the peak area of ​​the 414.4 / 272.1 ion pair, a is the slope of the regression equation, and b is the intercept of the regression equation. In this embodiment, the linear correlation coefficient r = 0.99998, within the range of 0.05-2 μg / L. -1 It exhibits excellent linearity within the concentration range and can be used as a benchmark for calibration.

[0034] The secondary standard solutions to be calibrated were tested under the same LC-MS / MS conditions as described above. Each concentration was tested in parallel three times. The average peak area was substituted into the regression equation of the reference standard curve to calculate the actual concentration of each solution to be calibrated. Then, based on the dilution ratio of the solution preparation, the actual concentration of the CYN stock solution was deduced to complete the calibration of the standard values ​​and eliminate the systematic error between the labeled value and the actual value of the standard powder.

[0035] Using the corrected actual concentration of the CYN mother liquor as a baseline, a series of CYN standard working solutions with gradient concentrations were prepared by stepwise dilution with 2.5% methanol aqueous solution. The solutions were then detected under the same LC-MS / MS conditions described above to construct an LC-MS / MS quantitative standard curve for subsequent water sample testing.

[0036] Example 2

[0037] This embodiment verifies the enrichment effect and recovery stability of the solid-phase extraction process for CYN of the present invention. The specific operation steps include:

[0038] Experimental Design

[0039] Using the CYN stock solution with a known corrected concentration from Example 1, preparations were made with a concentration of 5 μg / L. -1 1 μg / L -1 0.2 μg / L -1 400 mL of CYN aqueous solution was prepared for each concentration, with 3 parallel samples for each concentration. An ultrapure water blank control group was also prepared. The concentration gradient can be adjusted as needed.

[0040] Take 950 μL of solution from each concentration water sample into a 1.5 mL brown glass vial, add 50 μL of methanol, and store at 4℃ in the dark for determination of the initial concentration before solid-phase extraction; add 1% of the filtrate volume of methanol to the remaining water sample, shake gently to mix, add 2 M NaOH solution to adjust the pH to 11, and obtain the sample solution to be enriched.

[0041] Subsequently, solid-phase extraction enrichment was performed, including column activation: the PGC solid-phase extraction column (500 mg, 6 mL, Thermo) was placed on a porous vacuum device, and 6 mL of dichloromethane, 6 mL of methanol, and 6 mL of ultrapure water with pH=11 were added to the column in sequence, 3 mL of each reagent was added each time, in two portions, and the liquid was allowed to drain naturally.

[0042] Sample loading and enrichment: Insert one end of the acid-soaked Teflon capillary into the reagent bottle containing the sample through the small hole in the cap. Place the sample bottle at a high position, connect the connector to the syringe through the silicone tubing, and siphon water. Quickly disconnect the silicone tubing and syringe, insert the capillary through the connector into the activated PGC column, and after the water sample overflows, assemble the connector and PGC column, suspend the column, and load the sample by gravity filtration.

[0043] Drying: After filtration, place the PGC column on a vacuum device and vacuum dry for 5-10 minutes until there is no visible moisture in the column;

[0044] Elution and redissolution: After confirming that there is no visible water in the column, add 2-3 mL of eluent (dichloromethane:methanol = 40:60 (v / v), containing 0.5% formic acid) each time, for a total of 10 mL; collect the eluent in a clean 40 mL brown glass bottle; dry the eluent by blowing with nitrogen, then add 1 mL of methanol:water = 5:95 (v / v) solution to dissolve the residue, sonicate for 10 minutes to promote the dissolution of the residue, and obtain the enriched test solution.

[0045] The initial solution before solid-phase extraction and the analyte solution after extraction were analyzed according to the LC-MS / MS conditions of Example 1. The corresponding concentrations were obtained based on the quantitative standard curve, and the recovery rate was calculated according to the following formula:

[0046] Solid-phase extraction recovery rate = Concentration after extraction ÷ Concentration before extraction

[0047] The average value of three parallel samples for each concentration was taken as the final recovery rate for that concentration; simultaneously, linear regression analysis was performed on the concentrations before and after extraction, and the recovery rate was calculated based on R0. 2 The value is used to determine the stability of the recovery rate at different concentrations.

[0048] Experimental results

[0049] This embodiment demonstrates that the solid-phase extraction process of the present invention has a stable enrichment and recovery rate of CYN with good repeatability, and can achieve a 400-fold high-efficiency concentration of CYN in water samples, meeting the detection requirements of low-concentration samples.

[0050] Example 3

[0051] This embodiment is used to achieve simultaneous and accurate quantification of intracellular and extracellular CYN in actual lake and reservoir water samples. The specific operation steps include:

[0052] A 5L water sampler was used to collect surface, middle and bottom water samples from the target lake / reservoir. The samples were then poured into polyethylene plastic buckets with pre-labeled sample numbers. Before filling the buckets, the buckets were rinsed three times with in-situ water samples of the corresponding depth to prevent sample contamination. The collected water samples were transported back to the laboratory for immediate processing within 1 hour, or stored in a vehicle-mounted low-temperature freezer at 4°C and transported back to the laboratory for processing as soon as possible.

[0053] Take 500 mL of the water sample to be tested and filter it through a 0.2 μm 47 mm diameter PC membrane with a negative filtration pressure of less than 0.3 MPa. Collect the filtrate in a 500 mL clean glass bottle for extracellular toxin determination. Collect the filtered membrane in a 50 mL centrifuge tube, add 50 mL of ultrapure water, shake the centrifuge tube vigorously to completely detach the algal cells from the filter membrane, sonicate for 10-20 minutes to break the cells, filter out cell debris with a 0.22 μm needle filter, and collect the filtrate for intracellular toxin determination.

[0054] Add 1% of the filtrate volume of methanol to the extracellular toxin filtrate and the intracellular toxin filtrate respectively, shake gently to mix the samples, then add 2 M NaOH solution to adjust the pH to 11 to obtain the sample solution to be enriched.

[0055] Following the solid-phase extraction procedure in Example 2, the sample solution to be enriched was activated, loaded, dried, eluted, and reconstituted to obtain the solution to be tested. This solution was stored at 4°C in the dark and analyzed by LC-MS / MS within one week. In this example, the volume of water sample filtered by solid-phase extraction was 500 mL, and the final volume was 1 mL, resulting in a concentration factor of 500 times.

[0056] The LC-MS / MS test solution was analyzed according to the conditions described in Example 1. Based on the quantitative standard curve constructed in Example 1, the CYN concentration in the test solution was determined. The actual concentration of CYN in the water sample was calculated using the following formula: Actual concentration = Measured concentration ÷ Concentration ratio ÷ Recovery rate

[0057] The recovery rate used is the average recovery rate measured in Example 2.

[0058] Test results

[0059] This embodiment can realize the concentration of ngL in lake and reservoir water samples. -1Simultaneous and accurate quantification of low-concentration intracellular and extracellular CYN can clearly distinguish the occurrence form and concentration distribution of CYN in water samples from different water layers, and accurately obtain the proportion of extracellular toxins.

[0060] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0061] 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 method for detecting low-concentration intracellular and extracellular *Streptocytoxin* with accurate and comparable data, comprising the steps of pretreatment of the *Streptocytoxin*-containing water sample, enrichment and concentration of the target analyte, and quantitative detection by liquid chromatography-tandem mass spectrometry, characterized in that, It also includes the following steps: S1. Using the series of gradient concentration standards built into the commercially available *Streptococcus pluvialis* toxin ELISA kit with proven accuracy as the metrological baseline, a baseline standard curve was obtained by LC-MS / MS detection. A series of standard solutions of gradient concentrations to be calibrated were prepared using *Streptococcus pluvialis* toxin standard powder. The standard solutions to be calibrated were detected under LC-MS / MS conditions identical to those used to construct the baseline standard curve. The actual concentration of the standard solutions to be calibrated was calculated based on the baseline standard curve. After metrological calibration of the standards, a quantitative standard curve for LC-MS / MS detection was constructed using the calibrated actual concentration as the baseline. S2. The water sample to be tested is filtered to separate the filtrate and the filter membrane carrying algal cells. The filtrate is used as the test precursor solution for extracellular columniformis toxin. The filtrate obtained after eluting algal cells, disrupting cells, and filtering impurities from the filter membrane is used as the test precursor solution for intracellular columniformis toxin. The pH of the two test precursor solutions is adjusted to obtain the sample solution to be enriched. S3. The graphitized carbon black (PGC) solid-phase extraction column was used to activate, load, enrich, dry and elute the sample solution to be enriched in sequence. The collected eluent was dried by nitrogen blowing and the residue was dissolved with a reconstitution solution to obtain the solution to be tested on the instrument. S4. The test solution is detected using the same LC-MS / MS conditions as in step S1. Based on the quantitative standard curve constructed in step S1, the concentration of *Pterocytoxin* in the test solution is obtained. Combined with the concentration factor and recovery rate of solid-phase extraction, the actual concentrations of intracellular and extracellular *Pterocytoxin* in the water sample are calculated.

2. The method for detecting low-concentration intracellular and extracellular columniform toxins according to claim 1, characterized in that, In step S1, the commercial cylindrospermopsin ELISA kit is ABRAXIS Cylindrospermopsin ELISA Kit; the gradient concentrations of the standard product built-in the kit are 2 μg / L -1 , 1 μg / L -1 , 0.5 μg / L -1 , 0.2 μg / L -1 , 0.1 μg / L -1 , 0.05 μg / L -1 , 0 μg / L -1 , respectively, and the volume of each gradient standard solution is 1 mL.

3. The method for detecting low-concentration intracellular and extracellular columniform toxins according to claim 1, characterized in that, In step S1, the standard solution to be calibrated is prepared by adding 2.5% methanol aqueous solution to the *Strombocytoxin* standard powder to dissolve it, resulting in a theoretical concentration of 100 μg / mL. -1 The mother liquor was then serially diluted using a 2.5% methanol aqueous solution.

4. The method for detecting low-concentration intracellular and extracellular columniform toxins according to claim 1, characterized in that, In steps S1 and S4, the conditions for LC-MS / MS detection are as follows: The chromatographic column used was a Kinetex@ 2.1 μm C18 100A column, with dimensions of 100 × 2.1 mm. The column temperature was 40℃, the injection volume was 50 μL, and the flow rate was 0.3 mL / min. -1 Mobile phase A was an aqueous solution containing 0.5% formic acid (v / v), and mobile phase B was methanol. Mass spectrometry was performed using an electrospray ionization source in negative ion mode with multiple reaction monitoring. The quantitative ion pair for *Strombyx mori* toxin was 414.4 / 272.1, and the qualitative ion pairs were 414.4 / 302.1 and 414.4 / 97.

0. Peak diagrams were confirmed and corrected using Analyst software. A linear regression was performed with *Strombyx mori* toxin concentration as the ordinate (y) and the peak area of ​​the quantitative ion pair as the abscissa (x), yielding the regression equation y = a × x + b, where y is the *Strombyx mori* toxin concentration, x is the peak area of ​​the quantitative ion pair 414.4 / 272.1, a is the slope of the regression equation, and b is the intercept of the regression equation.

5. The method for detecting low-concentration intracellular and extracellular columniform toxins according to claim 1, characterized in that, In step S2, before adjusting the pH of the extracellular and intracellular test precursor fluids, 1% of the filtrate volume of methanol is added to the filtrate, mixed well, and then the pH is adjusted to 11.

6. The method for detecting low-concentration intracellular and extracellular columniform toxins according to claim 1, characterized in that, In step S3, the PGC solid-phase extraction column has a specification of 500 mg / 6 mL; The activation steps are as follows: add 6 mL of dichloromethane, 6 mL of methanol, and 6 mL of ultrapure water with pH=11 in sequence. Add each reagent in two portions of 3 mL each time and let it drain naturally. The enrichment of samples is achieved using a siphon gravity filtration method: One end of an acid-soaked Teflon capillary is inserted through a small hole in the cap into the reagent bottle containing the sample solution to be enriched. The sample bottle is placed at a high position, and the connector is connected to a syringe via a silicone tubing. Water is siphoned in. The silicone tubing and syringe are quickly disconnected, and the capillary is inserted through the connector into the activated PGC column. After the water sample overflows, the connector and PGC column are reassembled, the column is suspended, and the sample is loaded via gravity filtration. After loading, the sample is vacuum dried for 5-10 minutes. The elution step is as follows: 10 mL of eluent is added in portions, wherein the eluent is a dichloromethane-methanol mixture with a volume ratio of 40:60 and contains 0.5% formic acid by volume. The reconstitution procedure is as follows: after the eluent is dried by nitrogen blowing, 1 mL of methanol-water solution with a volume ratio of 5:95 is added, and the solution is dissolved by sonication for 10 min to obtain the test solution. The solution is stored at 4℃ and LC-MS / MS detection is performed within one week.

7. The method for detecting low-concentration intracellular and extracellular columniform toxins according to claim 1, characterized in that, The recovery rate of solid-phase extraction in step S3 was determined by preliminary experiments: using the known concentration of *Strombyx mori* toxin stock solution after correction in step S1, a concentration of 5 μg / L was prepared. -1 1 μg / L -1 0.2 μg / L -1 The aqueous solution of *Pterocytoxin* was prepared, with three parallel samples for each concentration. The concentrations before and after solid-phase extraction were measured, and the recovery rate was calculated as the concentration after extraction divided by the concentration before extraction. The average value of each parallel sample was taken.

8. The method for detecting low-concentration intracellular and extracellular columniform toxins according to claim 1, characterized in that, In step S4, the formula for calculating the actual concentration of *Pterocytoxin* in the water sample to be tested is: actual concentration = measured concentration ÷ concentration ratio ÷ recovery rate, where the concentration ratio = volume of water sample filtered by solid-phase extraction ÷ volume of the solution to be tested.

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