Method for detecting bacillus cereus vomitoxin in serum sample

Abstract

The invention belongs to the technical field of food poisoning rapid detection, and particularly relates to a method for detecting bacillus cereus vomitoxin in a serum sample, which comprises the following steps: (1) precipitating protein in the serum sample by adopting an extraction solvent in a purification tube to obtain a purification solution; (2) concentrating and redissolving the extracted and purified liquid; and (3) separating the redissolved purified liquid by adopting a liquid chromatographic column, detecting in an LC-MS / MS multi-reaction monitoring scanning mode, and quantitatively calculating the content of vomitoxin in bacillus cereus by adopting an internal standard method. The method disclosed by the invention is simple and convenient to operate, green and environment-friendly, accurate in qualitation and quantification and high in sensitivity, can be used as a mass spectrometric detection method for determining the bacillus cereus vomitoxin in a serum sample, can solve the problem of rapid treatment of food poisoning caused by the bacillus cereus vomitoxin in food, and meets detection requirements.

Description

Technical Field

[0001] This invention belongs to the field of rapid food poisoning detection technology, specifically relating to a method for detecting Bacillus cereus vomitoxin in serum samples. Background Technology

[0002] Bacillus cereus ( Bacillus cereus Bacillus cereus is a Gram-positive bacterium and an important opportunistic foodborne pathogen, widely distributed in nature. Its toxins can cause food poisoning. Globally, food poisoning incidents caused by Bacillus cereus account for 1.4% to 12.0% of all food poisoning incidents. Data from the China Center for Disease Control and Prevention's Health Emergency Center in 2018 showed that food poisoning incidents caused by Bacillus cereus ranked fifth in my country, after Salmonella, Vibrio parahaemolyticus, diarrhea-causing Escherichia coli, Staphylococcus aureus, and its enterotoxins. Bacillus cereus can produce cereulide and diarrhea-causing enterotoxins, causing food poisoning with vomiting and diarrhea as the main symptoms, respectively. According to literature reports, the detection rate of Bacillus cereus in rice and flour products is relatively high in my country. Foodborne illnesses involving Bacillus cereus often occur through foods derived from rice and flour products, with vomiting-type food poisoning caused by Bacillus cereus vomitoxin being the most common.

[0003] Current detection methods for Bacillus cereus food poisoning primarily rely on the WS / T 82-1996 diagnostic criteria and the GB / T4789.14-2014 national standard. The core procedure involves detecting the vomitoxin virulence gene ces through strain culture combined with real-time quantitative PCR. However, this method has significant limitations: First, the experimental procedures are complex and time-consuming, requiring at least 2-3 days from colony culture to toxin production, which cannot meet the needs of rapid response to food poisoning incidents. Second, the detection of the ces gene only indicates potential toxin production capacity and cannot directly verify the actual expression of vomitoxin. Third, as a cyclic peptide compound with high thermal and chemical stability, Bacillus cereus vomitoxin may still retain virulence even after bacterial cell inactivation in the raw material, potentially leading to missed detections or misjudgments using traditional strain-based detection methods.

[0004] Currently, there are no literature reports on Bacillus cereus vomitoxin in serum. Direct detection using serum samples faces two challenges: on the one hand, the high abundance of proteins and nitrogenous compounds in the serum matrix can seriously interfere with the detection of the target analyte; on the other hand, the unique cyclic peptide structure of vomitoxin is more easily affected in complex biological matrices, resulting in insufficient sensitivity and accuracy of traditional LC-MS / MS detection methods.

[0005] Therefore, there is an urgent need to develop a mass spectrometry method for detecting Bacillus cereus vomitoxin in serum samples, so as to facilitate the rapid handling of food poisoning incidents caused by Bacillus cereus vomitoxin. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a pretreatment method for extracting Bacillus cereus vomitoxin from serum via protein precipitation. Combining solid-phase extraction technology with liquid chromatography-tandem mass spectrometry (LC-MS / MS), a method is established to rapidly purify impurities in serum and accurately determine Bacillus cereus vomitoxin. This method can achieve precise determination of Bacillus cereus vomitoxin in serum, with a limit of quantitation of approximately 0.1 μg / L.

[0007] In a first aspect, the present invention provides a method for detecting Bacillus cereus vomitoxin in serum samples, comprising the following steps: (1) Proteins in serum samples were precipitated using an extraction solvent to obtain a purified solution; (2) Concentrate and redissolve the purified solution; (3) The purified solution after reconstitution was separated by liquid chromatography column, and the content of Bacillus cereus vomitoxin was calculated by LC-MS / MS multiple reaction monitoring scanning mode.

[0008] Furthermore, in step (1), the protein is precipitated in steps: firstly, acetonitrile is used for primary precipitation, and then a weakly polar mixed solvent is used for secondary precipitation.

[0009] Furthermore, the mixed solvent is selected from a combination of two solvents among methanol, anhydrous ethanol, ethyl acetate, acetonitrile, and acetone.

[0010] Furthermore, the mixed solvent is 95% acetonitrile - 5% methanol.

[0011] Furthermore, acetonitrile was first used for protein precipitation. Highly polar acetonitrile (polarity index 5.8) can preferentially precipitate hydrophilic proteins and reduce phospholipid interference. Then, a second protein precipitation was performed using 95v% acetonitrile and 5v% methanol. The weakly polar mixed solvent (polarity index about 5.2) targeted the precipitation of hydrophobic proteins, thereby reducing the ion inhibition effect.

[0012] Furthermore, the primary precipitation is carried out at a low temperature of 0-4°C, and the volume ratio of the serum sample to acetonitrile is 1:(3-4); this can remove more than 90% of the host protein while preventing the degradation of vomitoxin.

[0013] Furthermore, the secondary precipitation is carried out at 2-8°C, the volume ratio of the serum sample to the mixed solvent is 1:(1-2), and the temperature of the secondary precipitation is higher than that of the primary precipitation, which can accurately remove residual lipoproteins while avoiding local solvent overload that could lead to protein resolution.

[0014] Furthermore, the protein is precipitated in stages, with each precipitation lasting 5-20 minutes.

[0015] Furthermore, the purification tube includes a purification material selected from one or more of C18, PSA, Carb, and HMR. HMR is preferred.

[0016] In one embodiment of the present invention, the amount of filler used is 10-30 mg per 50 µl serum sample.

[0017] Because lecithin and phosphatidylglycerol in blood samples are not easily removed completely, the remaining phospholipids will accumulate in the ion source of the mass spectrometer and compete with the target analyte for ionization, leading to ion inhibition. This inhibits the target analyte's response, resulting in a large relative standard deviation, poor parallelism, and affecting the accuracy and reliability of the detection. Therefore, to solve the serious interference caused by residual phospholipids (such as lecithin and phosphatidylglycerol) in blood samples during LC-MS / MS analysis, this invention develops a stepwise precipitation synergistic HMR-Lipid deep purification strategy. The scheme first achieves precise matrix stripping through polar gradient stepwise precipitation: the first precipitation step is carried out at a strictly low temperature of 0-4℃, using highly polar acetonitrile (polarity index 5.8) at a high volume ratio (serum:acetonitrile = 1:3-4) to precipitate more than 90% of hydrophilic host proteins, while protecting the stability of target small molecules (such as vomitoxin); the second precipitation step is carried out at a mild low temperature of 2-8℃, using a weakly polar mixed solvent (95% acetonitrile - 5% methanol, polarity index 5.2) at a low volume ratio (serum:solvent = 1:1-2) to target and precipitate residual hydrophobic lipoproteins (such as VLDL / LDL). This step enhances the aggregation effect of hydrophobic proteins by reducing solvent polarity, thereby disrupting the phospholipid micelle structure. The slightly higher operating temperature (2-8℃) avoids the risk of solution freezing and optimizes precipitation efficiency by moderately increasing the molecular diffusion rate. At the same time, the low solvent ratio design effectively prevents protein re-dissolution caused by local overload. It also avoids the possibility of re-dissolution of precipitated proteins / lipoproteins due to vigorous mixing or local high concentrations in high-ratio mixed solvents. This ensures the compactness and stability of the hydrophobic protein / lipoprotein precipitation, preventing the target substance from being entrained after redissolution or leading to incomplete purification.

[0018] The stepwise protein precipitation strategy of this invention achieves deep and selective purification of complex matrices in blood samples, especially persistent phospholipid interferences, through synergistic optimization of polarity, temperature, and volume ratio. It not only efficiently removes host proteins but also targets and eliminates phospholipids and their carrier lipoproteins, the main sources of ion inhibition, laying a crucial pretreatment foundation for subsequent high-sensitivity and high-accuracy LC-MS / MS quantitative analysis (especially for trace small molecule targets).

[0019] Building upon this foundation, this invention innovatively introduces a synergistic mechanism through the HMR-Lipid purification material: stepwise precipitation removes the vast majority of phospholipid carriers and free aggregates, while the HMR-Lipid material precisely captures residual trace amounts of lecithin, phosphatidylglycerol, and other monomer molecules through specific adsorption, achieving a comprehensive phospholipid removal rate of over 99%. This purification system completely overcomes the bottleneck of phospholipid residue, significantly reduces matrix effects and ion inhibition, keeps the RSD of target analytes below 5%, and greatly improves parallelism and accuracy. Ultimately, it establishes a highly stable and reproducible pretreatment foundation for trace small molecule LC-MS / MS analysis, overcoming the technical limitations of traditional methods in complex blood samples.

[0020] Furthermore, the present invention uses the internal standard method to quantitatively calculate the content of Bacillus cereus vomitoxin.

[0021] Further, in step (1), sample pretreatment and internal standard addition are performed: first, the serum sample is placed in a purification tube and an internal standard solution is added, then the extraction solvent is added, the protein is precipitated, and the purified solution is obtained by centrifugation; the volume ratio of the serum sample to the internal standard solution is 1:1.

[0022] The internal standard solution refers to the 13C6-Bacillus cereus vomitoxin internal standard solution.

[0023] Cereulide (target analyte) and 13C6-Bacillus cereus vomitoxin (internal standard) were added simultaneously to the serum sample to be tested. Both underwent the same precipitation and purification process with a volume ratio of 1:1 to ensure accurate calibration.

[0024] Furthermore, in step (2), the purified liquid is first concentrated and then reconstituted with an organic solvent to obtain the reconstituted purified liquid.

[0025] Furthermore, the concentration temperature is 35-45℃.

[0026] Furthermore, the organic solvent is methanol or acetonitrile.

[0027] Furthermore, the amount of organic solvent used is 1-3 mL.

[0028] Since the type of organic solvent used is the same as that used in step (1) for the stepwise precipitation of proteins, the concentrate will be effectively reconstituted, and no other detection errors will be introduced.

[0029] Furthermore, the LC-MS / MS determination conditions described in step (3) are as follows: Chromatographic system: The liquid chromatography column was an ACQUITY UPLC Peptide BEH C18. Column temperature 30-40℃, sample chamber temperature 2-4℃, injection volume 5μL; Mobile phase A is a 0.1-0.5 v% formic acid acetonitrile solution, and mobile phase B is an aqueous solution of formic acid-ammonium acetate; preferably, the concentration of the aqueous solution of ammonium acetate is 1-3 mmol / L, and the volume concentration of formic acid is 0.1-0.5%.

[0030] The gradient elution procedure is as follows: 0~4.0 min, 80%A~100%A; 4.0 (excluding) ~ 6.0 min, 100% A; 6.0 min (excluding) ~ 7.0 min, 100%A ~ 80%A, and maintain until 8.0 min; The flow rate is 0.2-0.35 mL / min.

[0031] In one embodiment of the present invention, an ACQUITY UPLC Peptide BEH C18 300Å column (2.1 mm × 100 mm, 1.7 µm) was used.

[0032] Furthermore, the mass spectrometry conditions are as follows: Ion source mode: Electrospray positive ion mode (ESI+) ionization; Scanning method: Multiple reaction monitoring (MRM) mode, residence time 0.10 s; Capillary voltage 2.70-3.50kV, ion source temperature 130-150℃; The solvent removal temperature is 480-500℃, and the solvent removal gas flow rate is 800-1000L / h; The collision chamber pressure is 0.3-0.4 Pa, and the cone voltage is 70-73.0 V.

[0033] Further, the internal standard method described in step (3) involves adding the same concentration of Bacillus cereus vomitoxin internal standard solution to both the standard solution and the test solution. Then, based on the peak areas of Bacillus cereus vomitoxin in the test solution and the standard solution, the content of Bacillus cereus vomitoxin in the sample is calculated using formula (I). (I) In the formula: The content of Bacillus cereus vomitoxin in sample X, in mg / kg; The concentration of Bacillus cereus vomitoxin in the c-standard solution is expressed in μg / mL. A 样 - Peak area of ​​Bacillus cereus vomitoxin in the test solution; A 标准 - Peak area of ​​Bacillus cereus vomitoxin in standard solution; Peak area of ​​Bacillus cereus vomitoxin internal standard in α-standard solution; b - Peak area of ​​Bacillus cereus vomitoxin internal standard in the test solution; V - The final volume of the solution to be tested, in mL; m - the mass of the sample, in grams.

[0034] The standard solution was prepared by the following method: (1) Preparation of Bacillus cereus vomitoxin standard intermediate solution: Accurately pipette 0.2 mL of standard solution (50.0 μg / mL) into a 10.0 mL volumetric flask, dilute to the mark with acetonitrile, mix well, and obtain a standard intermediate solution with a concentration of 1.0 μg / mL; (2) Preparation of 13C6-Bacillus cereus vomitoxin internal standard intermediate solution: Accurately pipette 0.5 mL of 13C6-Bacillus cereus vomitoxin internal standard solution (20.0 μg / mL) into a 10.0 mL volumetric flask, dilute to the mark with acetonitrile, mix well, and obtain an internal standard intermediate solution with a concentration of 1.0 μg / mL; (3) Preparation of standard working solution: Accurately pipette 1.0 mL of the standard intermediate solution, dilute to 10.0 mL with acetonitrile solution, mix well, and obtain a standard working solution with a concentration of 0.1 μg / mL.

[0035] (4) Preparation of internal standard working solution: Accurately pipette 1.0 mL of internal standard intermediate solution, dilute to 10.0 mL with acetonitrile solution, mix well, and obtain internal standard working solution with a concentration of 0.1 μg / mL.

[0036] (5) Standard series working solutions: Accurately pipette 10.0 μL, 50.0 μL, 100.0 μL, 200.0 μL, 500.0 μL, 1000.0 μL, and 2000.0 μL of 0.1 μg / mL standard working solution into 10.0 mL volumetric flasks, add 200.0 μL of 0.1 μg / mL internal standard working solution, and prepare a series of standard solutions of 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 μg / L with the initial mobile phase (mobile phase A to B volume ratio of 8:2), with an internal standard concentration of 2.0 μg / L.

[0037] The method also includes the preparation of blank samples: except that no sample is added to the centrifuge tube, the other extraction and determination steps are the same as the sample determination, and parallel sample determination process must be added during the sample detection process.

[0038] The samples described in this invention are mainly serum.

[0039] Secondly, the detection method described in the first aspect is used to determine food poisoning caused by Bacillus cereus vomitoxin.

[0040] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention features a pioneering multi-stage purification pretreatment process that combines a stepwise gradient precipitation method with the dual-effect mechanism of HMR-Lipid solid-phase extraction material. First, hydrophilic host proteins are targeted and precipitated using highly polar acetonitrile (0-4℃). Then, hydrophobic lipoproteins and phospholipid carriers are specifically removed using a weakly polar acetonitrile-methanol mixed solvent (2-8℃). Finally, residual free phospholipids are adsorbed using HMR-Lipid material. This strategy achieves triple precision purification: host protein removal rate >90%, phospholipid (lecithin / phosphatidylglycerol) removal rate >99%, and toxin recovery rate >85%, completely eliminating mass spectrometry ion inhibition effects. Furthermore, the environmentally friendly solvent system significantly reduces experimental risks.

[0041] 2. This invention is based on the internal standard compensation matrix effect of Bacillus cereus vomitoxin, and uses optimized chromatographic conditions (ACQUITY UPLC Peptide BEH C18 column gradient separation) and mass spectrometry parameters (ESI+ / MRM scan) to achieve a limit of quantitation of 0.1 μg / L. The sensitivity is 10-100 times higher than that of conventional methods, and the RSD is controlled within 5%.

[0042] 3. This invention does not rely on strain culture or indirect gene detection, but directly and accurately quantifies heat-resistant trace toxins in serum, overcoming the problem of false negatives caused by the thermal stability of toxins in traditional methods, and providing technical support for clinical poisoning diagnosis with minute-level response (single sample pretreatment <30 minutes).

[0043] 4. The detection method of the present invention has strong universality, high sensitivity, simple operation, and accurate qualitative and quantitative results. It can be used for the mass spectrometry detection of Bacillus cereus vomitoxin in serum samples, which can solve the problem of rapid treatment of food poisoning caused by Bacillus cereus vomitoxin in food and meet the detection requirements. Attached Figure Description

[0044] Figure 1 Separation results of the ACQUITY UPLC Peptide BEH C18, 300Å column (2.1 mm × 100 mm, 1.7 µm) used in Example 1; Figure 2 The image shows the results of detecting phospholipid interference peaks in the sample of Example 1 using an evaporative light scattering detector; Figure 3The image shows the results of detecting the phospholipid interference peak in the Comparative Example 2 sample using an evaporative light scattering detector. Detailed Implementation

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Example 1 A method for detecting Bacillus cereus vomitoxin in serum samples, comprising the following steps: (1) Proteins in serum samples were precipitated using an extraction solvent to obtain a purified solution: 50 µl of serum sample was placed in a 1.5 mL purification tube (Anavo Super-Efficient 96-well plate, HMR-Lipid packing material), 50 µl of Bacillus cereus vomitoxin internal standard solution (100 µg / L) was added, 200 µl of pure acetonitrile was added, and the sample was allowed to stand at 4℃ for 10 min until the protein was completely precipitated. The sample was then centrifuged at 10000 r / min to obtain the purified solution.

[0047] (2) Concentrate and redissolve the purified solution: Concentrate the purified solution to dryness by nitrogen blowing at 40°C, add 100 µl of methanol to redissolve, centrifuge and filter through membrane for use.

[0048] (3) The purified solution after reconstitution was separated by liquid chromatography column, and the contents of Bacillus cereus vomitoxin were quantitatively calculated by LC-MS / MS multiple reaction monitoring mode using internal standard method: Ultra-high performance liquid chromatography reference conditions: Chromatographic column: Waters ACQUITY UPLC Peptide BEH C18 Column, 300 Å column (2.1 mm × 100 mm, 1.7 µm); Mobile phase: Phase A was 0.1% formic acid-acetonitrile solution, and Phase B was 0.1% formic acid-2.0 mmol / L ammonium acetate aqueous solution; Flow rate: 0.35 mL / min; Column temperature: 40℃; Injection volume: 5.0 µL. The gradient elution program is shown in Table 1.

[0049] Table 1 Gradient elution program ; Mass spectrometry reference conditions: Ionization mode: Electrospray electropositive ionization mode (ESI+); Mass spectrometry scanning mode: Multiple reaction monitoring (MRM); Capillary voltage: 3.5 kV; Ion source temperature: 150℃; Desolventizing temperature: 500℃; Desolventizing gas flow rate: 1000 L / h; Collision chamber pressure: 0.36 Pa; Cone voltage: 73.0 V. Specific mass spectrometry monitoring operating parameters are shown in Table 2.

[0050] Table 2 Monitoring parameters for Bacillus cereus vomitoxin ; Note: * indicates quantitative ions.

[0051] The content of Bacillus cereus vomitoxin was quantitatively calculated using the internal standard method: The same concentration of Bacillus cereus vomitoxin internal standard solution was added to both the standard solution and the test solution. Then, based on the peak areas of Bacillus cereus vomitoxin in the test solution and the standard solution, the content of Bacillus cereus vomitoxin in the sample was calculated using formula (I). (I) In the formula: The content of Bacillus cereus vomitoxin in sample X, in mg / kg; The concentration of Bacillus cereus vomitoxin in the c-standard solution is expressed in μg / mL. A 样 - Peak area of ​​Bacillus cereus vomitoxin in the test solution; A 标准 - Peak area of ​​Bacillus cereus vomitoxin in standard solution; Peak area of ​​Bacillus cereus vomitoxin internal standard in α-standard solution; b - Peak area of ​​Bacillus cereus vomitoxin internal standard in the test solution; V - The final volume of the solution to be tested, in mL; m - the mass of the sample, in grams.

[0052] The standard solution was prepared by the following method: 1) Preparation of intermediate standard solution for Bacillus cereus vomitoxin: Accurately pipette 0.2 mL of standard solution (50.0 μg / mL) into a 10.0 mL volumetric flask, dilute to the mark with acetonitrile, mix well, and obtain an intermediate standard solution with a concentration of 1.0 μg / mL; 2) Preparation of 13C6-Bacillus cereus vomitoxin internal standard intermediate solution: Accurately pipette 0.5 mL of 13C6-Bacillus cereus vomitoxin internal standard solution (20.0 μg / mL) into a 10.0 mL volumetric flask, dilute to the mark with acetonitrile, mix well, and obtain an internal standard intermediate solution with a concentration of 1.0 μg / mL; 3) Preparation of standard working solution: Accurately pipette 1.0 mL of the standard intermediate solution, dilute to 10.0 mL with acetonitrile solution, mix well, and obtain a standard working solution with a concentration of 0.1 μg / mL.

[0053] 4) Preparation of internal standard working solution: Accurately pipette 1.0 mL of the internal standard intermediate solution, dilute to 10.0 mL with acetonitrile solution, mix well, and obtain an internal standard working solution with a concentration of 0.1 μg / mL.

[0054] 5) Standard series working solutions: Accurately pipette 10.0 μL, 50.0 μL, 100.0 μL, 200.0 μL, 500.0 μL, 1000.0 μL, and 2000.0 μL of 0.1 μg / mL standard working solution into 10.0 mL volumetric flasks. Add 200.0 μL of 0.1 μg / mL internal standard working solution. Prepare a standard series of solutions with concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 μg / L using the initial mobile phase (mobile phase A to B volume ratio of 8:2). The concentration of the internal standard is 2.0 μg / L.

[0055] Preparation of blank samples: Except for not adding samples to centrifuge tubes, the other extraction and determination steps are the same as the sample determination. At the same time, parallel sample determination processes must be added during the sample detection process.

[0056] In Example 1, the separation effect of Bacillus cereus vomitoxin using an ACQUITY UPLC Peptide BEH C18, 300Å column is shown in the figure. Figure 1 The results of the evaporative light scattering detector are shown below. Figure 2 As can be seen, the phospholipid interference peak at 7.374 minutes was almost invisible, indicating near-complete removal and achieving a good purification effect. This also reflects the good effect of using purification packing material on phospholipid removal. The recovery rate and relative deviation (n=6) of Bacillus cereus vomitoxin in serum samples with different spiking amounts are shown in Table 3.

[0057] Table 3. Mean recovery rate and relative standard deviation of Bacillus cereus vomitoxin at different spiking levels in Example 1 .

[0058] Example 2 A method for detecting Bacillus cereus vomitoxin in serum samples, comprising the following steps: (1) Proteins in serum samples were precipitated using an extraction solvent to obtain a purified solution: 50 µl of serum sample was placed in a purification tube (Anavo Super-Efficient 96-well plate, HMR-Lipid packing material), 50 µl of Bacillus cereus vomitoxin internal standard solution (100 µg / L) was added, 150 µl of pure acetonitrile was added at 4℃, and the protein was allowed to stand for 5 min for the first precipitation. Then, at 8℃, 50 µl of mixed solvent 95% acetonitrile-5% methanol was added to the supernatant, and the protein was allowed to stand for 5 min until the protein precipitation was complete. The purified solution was obtained by high-speed centrifugation at 10000 r / min.

[0059] Steps (2)-(3) are the same as in Example 1.

[0060] The recovery rate and relative deviation of Bacillus cereus vomitoxin in serum samples at different spiking levels in this embodiment are shown in Table 4.

[0061] Table 4. Mean recovery rate and relative standard deviation of Bacillus cereus vomitoxin at different spiking levels in Example 2 .

[0062] The comparison data in Tables 3 and 4 show that, after using the gradient protein precipitation purification process in Example 2 (0-4℃ high polarity acetonitrile → 2-8℃ weak polarity acetonitrile-methanol), even at a trace spiking level of 0.2 ng / g, the sample recovery rate still reached 94.7%, and the relative standard deviation (RSD) was as low as 2.9%, significantly better than the single precipitation process in Example 1 (Table 3). This confirms that the stepwise gradient precipitation method of the present invention achieves high recovery rate and high precision simultaneously by accurately removing matrix interference, with the lower limit of quantification exceeding 0.1 μg / L, and the sensitivity fully meeting the requirements for trace toxin detection. As shown in Table 4, using a stepwise protein precipitant can fully precipitate proteins, further improving the sample recovery rate, making it more suitable for the detection and identification of trace Bacillus cereus vomitoxin.

[0063] After effective protein precipitation, this invention introduces Anavo's highly efficient split 96-well plate purification tube, which achieves a dual breakthrough through its built-in HMR-Lipid packing material: on the one hand, it directionally adsorbs residual phospholipids in serum, achieving an ultra-high removal rate of 99.3% for lecithin and 99.9% for phosphatidylglycerol, completely eliminating the matrix effect and significantly improving detection accuracy; on the other hand, only 50 μL of serum sample is needed to simultaneously complete protein precipitation and sample purification, simplifying the multiple steps required by traditional solid-phase extraction, such as activation, rinsing, elution, and nitrogen blowing concentration, into a single-column operation, improving pretreatment efficiency by more than 60%, and gaining a critical time window for the emergency diagnosis of food poisoning patients.

[0064] Example 3 A method for detecting Bacillus cereus vomitoxin in serum samples, comprising the following steps: (1) Proteins in serum samples were precipitated using an extraction solvent to obtain a purified solution: 50 µl of serum sample was placed in a purification tube (Anavo Super-Efficient 96-well plate, HMR-Lipid packing material), 50 µl of Bacillus cereus vomitoxin internal standard solution (100 µg / L) was added, 150 µl of pure acetonitrile was added at 4℃, and the protein was allowed to stand for 5 min for the first precipitation. Then, at 8℃, 50 µl of mixed solvent 95% acetonitrile-5% methanol was added to the supernatant, and the protein was allowed to stand for 5 min until the protein precipitation was complete. The purified solution was obtained by high-speed centrifugation at 10000 r / min.

[0065] Steps (2)-(3) are the same as in Example 1.

[0066] The recovery rate and relative deviation of Bacillus cereus vomitoxin in serum samples at different spiking levels in this embodiment are shown in Table 5.

[0067] Table 5. Mean recovery rate and relative standard deviation of Bacillus cereus vomitoxin at different spiking levels in Example 3 .

[0068] Table 5 clearly shows that when the volume of the mixed solvent (95% acetonitrile - 5% methanol) exceeds twice the volume of the serum sample (i.e., volume ratio > 1:2), although secondary protein precipitation can be achieved, the average recovery rate is significantly reduced and the relative standard deviation (RSD) increases. This is because excessive mixed solvent may disrupt the polarity balance of the system, causing the precipitated hydrophobic lipoproteins to re-dissolve in locally supersaturated regions, re-encapsulating the target toxin molecules and resulting in loss; a high solvent ratio dilutes the internal standard concentration, weakening the isotope correction effectiveness, while the increase in residual free phospholipids exacerbates mass spectrometry ion suppression. Therefore, a mixed solvent volume ratio of 1:1-2 is the optimal range, which not only ensures complete lipoprotein precipitation with the lowest possible solvent strength but also avoids the risk of re-dissolution.

[0069] Example 4 A method for detecting Bacillus cereus vomitoxin in serum samples, comprising the following steps: (1) Proteins in serum samples were precipitated using an extraction solvent to obtain a purified solution: 50 µl of serum sample was placed in a purification tube (Anavo Super-Efficient 96-well plate, HMR-Lipid packing material), 50 µl of Bacillus cereus vomitoxin internal standard solution (100 µg / L) was added, 150 µl of pure acetonitrile was added at 4℃, and the mixture was allowed to stand for 5 min for the first protein precipitation. Then, at 4℃, 50 µl of mixed solvent 95% acetonitrile-5% methanol was added to the supernatant, and the mixture was allowed to stand for 5 min until the protein precipitation was complete. The purified solution was obtained by high-speed centrifugation at 10000 r / min.

[0070] Steps (2)-(3) are the same as in Example 1.

[0071] The recovery rate and relative deviation of Bacillus cereus vomitoxin in serum samples at different spiking levels in this embodiment are shown in Table 6.

[0072] Table 6. Mean recovery rate and relative standard deviation of Bacillus cereus vomitoxin at different spiking levels in Example 4 .

[0073] In Example 4, both precipitation processes were performed at a constant temperature of 4°C. The average recovery rate decreased by approximately 2-4% compared to Example 2 (stepwise temperature control at 4°C / 8°C), while the relative standard deviation (RSD) increased to 4%. This result clearly validates the advantages of the temperature gradient design: in the secondary precipitation step, the slightly higher temperature of 8°C (an increase from 4°C) can moderately enhance molecular thermal motion, promote the diffusion and aggregation of hydrophobic lipoproteins, and improve precipitation efficiency. However, the constant temperature of 4°C lacks this temperature difference, leading to a lower lipoprotein precipitation rate. Furthermore, it is more prone to forming solvent supersaturation zones due to uneven local mixing, causing re-dissolution of precipitated lipoproteins, resulting in entrainment loss of the target analyte and incomplete purification. The reduced hydrophobic protein precipitation efficiency directly leads to a decrease in residual phospholipid clearance, which in turn weakens mass spectrometry ionization efficiency, ultimately manifesting as a decrease in recovery rate and an increase in RSD.

[0074] Comparative Example 1 (1) Without using protein precipitation and solid-phase extraction column purification, the serum was directly diluted with water and injected. Step (2) is the same as step (3) in Example 1.

[0075] The recovery rate of Bacillus cereus vomitoxin was found to be below 50%. This is because the target substance competes with interfering substances in the serum for ionization, resulting in severe matrix inhibition and failing to achieve good experimental results, thus not meeting the detection requirements.

[0076] Comparative Example 2 No HMR-Lipid packing material was added to the purification tube in step (1), and all other operating steps were the same as in Example 1. The results of detecting the phospholipid interference peak using an evaporative light scattering detector are shown below. Figure 3 .

[0077] pass Figure 2 (Example 1) and Figure 3 (Comparative Example 2) shows that, Figure 3 The phospholipid interference peak at 7.374 minutes showed a significant interference peak on the evaporative light scattering detector, exceeding 20% ​​of the response value, indicating severe interference. However, after purification with HMR-Lipid packing material in Example 1, Figure 2 The phospholipid interference peak at 7.374 minutes was almost invisible, indicating that it was nearly completely removed, achieving a very good purification effect. This also reflects the good effect of using purification packing material on phospholipid removal.

[0078] Through multiple rounds of experimental condition optimization and result comparison studies, the applicant innovatively constructed a stepwise gradient precipitation technique for efficient extraction of proteins and toxins, synergistically combining it with the targeted purification effect of HMR-Lipid solid-phase extraction, successfully establishing a high-sensitivity LC-MS / MS detection system for Bacillus cereus vomitoxin in serum. This method optimizes the precipitation process in three dimensions—temperature, polarity, and volume ratio (0-4℃ high-polarity acetonitrile for hydrophilic proteins → 2-8℃ weak-polarity mixed solvent for targeting hydrophobic lipoproteins), combined with material-specific adsorption of residual phospholipids, eliminating over 90% of serum matrix interference; and employs environmentally friendly reagents for green pretreatment. Based on isotope internal standard (13C6-toxin) correction technology, the limit of quantitation was reduced to 0.1 μg / L, significantly superior to conventional methods. After multiple rounds of condition optimization and validation, this method combines the advantages of ease of operation, detection accuracy, and matrix tolerance, providing reliable technical support for the rapid clinical diagnosis of vomiting-related food poisoning.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting Bacillus cereus vomitoxin in serum samples, comprising the following steps: (1) Proteins in serum samples were precipitated using an extraction solvent to obtain a purified solution; (2) Concentrate and redissolve the purified solution; (3) The purified solution after reconstitution was separated by liquid chromatography column, and the content of Bacillus cereus vomitoxin was calculated by LC-MS / MS multiple reaction monitoring scanning mode.

2. The method according to claim 1, characterized in that, Step (1) involves stepwise protein precipitation: firstly, acetonitrile is used for primary precipitation, and then a mixed solvent is used for secondary precipitation; the mixed solvent is selected from a combination of two solvents among methanol, anhydrous ethanol, ethyl acetate, acetonitrile, and acetone.

3. The method according to claim 2, characterized in that, The mixed solvent is 95% acetonitrile-5% methanol; The volume ratio of the serum sample to acetonitrile was 1:(3-4). The volume ratio of the serum sample to the mixed solvent is 1:(1-2).

4. The method according to claim 2, characterized in that, The primary precipitation is carried out at a low temperature of 0-4℃, and the secondary precipitation is carried out at a temperature of 2-8℃, with the temperature of the secondary precipitation being higher than that of the primary precipitation.

5. The method according to any one of claims 1-4, characterized in that, In step (1), the serum sample is first placed in a purification tube, an internal standard solution is added, and then an extraction solvent is added. The volume ratio of the serum sample to the internal standard solution is 1:

1. The content of Bacillus cereus vomitoxin is quantitatively calculated using the internal standard method.

6. The method according to claim 1, characterized in that, In step (2), the purified liquid is first concentrated, and then redissolved with an organic solvent to obtain the redissolved purified liquid; the concentration temperature is 35-45℃; the organic solvent is methanol or acetonitrile.

7. The method according to claim 1, characterized in that, In step (1), the purification tube contains purification material, which is selected from one or more of C18, PSA, Carb, and HMR.

8. The method according to claim 7, characterized in that, The purification material is HMR, and the amount of HMR packing material used is 10-30 mg per 50 µl serum sample.

9. The method according to claim 1, characterized in that, In step (3), the liquid chromatography column is ACQUITY UPLCPeptide BEH C 18 ; The LC-MS / MS determination conditions are as follows: Column temperature 30-40℃, sample chamber temperature 2-4℃, injection volume 5μL; Mobile phase A is an acetonitrile solution containing 0.1-0.5 v% formic acid; mobile phase B is an aqueous solution containing 0.1-0.5 v% formic acid and 1-3 mmol / L ammonium acetate. The mass spectrometry conditions are as follows: Ion source mode: Electrospray positive ion mode (ESI+) ionization; Mass spectrometry scanning mode: multiple reaction monitoring mode, residence time 0.10s; capillary voltage 2.70-3.50kV, ion source temperature 130-150℃, desolvation temperature 480-500℃, desolvation gas flow rate 800-1000L / h, collision chamber pressure 0.3-0.4Pa, cone voltage 70-73.0V; The internal standard solution refers to the 13C6-Bacillus cereus vomitoxin internal standard solution.

10. The method according to any one of claims 1-9, used to determine food poisoning caused by Bacillus cereus vomitoxin.

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