A method for preparing diarrhetic shellfish toxin standard substance from marine microalgae

Abstract

This invention discloses a method for preparing diarrhetic shellfish toxin standards using marine microalgae, specifically including the following steps: disrupting toxin-producing algal cells with NaOH solution and hydrolyzing esterified toxins, followed by liquid-liquid extraction to extract the toxins from the algal culture medium. The crude toxin extract is concentrated and purified using dextran gel chromatography, then further purified by semi-preparative liquid chromatography, and finally purified by solid-phase extraction to obtain OA and DTX1 toxin standards. Qualitative analysis was performed using high-resolution secondary mass spectrometry, and purity analysis was conducted using liquid chromatography-diode array detection (LC-DAD). The results showed that the purity of both OA and DTX1 standards was above 99%. This invention features a simple process, high toxin extraction rate, and good purification effect, and has significant application value for the large-scale preparation of toxin standards.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product separation, and in particular relates to a method for separating and preparing high-purity diarrheal shellfish toxin from marine dinoflagellates. Background Art

[0002] Diarrhetic shellfish toxins (DSTs) are a class of marine algae toxins composed of Okadaic acid (OA), Dinophysistoxins (DTXs) ​​and their derivatives. Figure 1 As shown in the figure, it is insoluble in water but soluble in organic reagents such as methanol, acetonitrile, and chloroform. This type of toxin is often enriched in biological tissues such as shellfish, threatening the safety of shellfish products, causing diarrheal poisoning in consumers, and inducing the development of tumors, potentially threatening human health and marine ecological safety.

[0003] Whether it is the development of new drugs for marine toxins or the research on monitoring methods for toxic red tides, algal toxin standards are needed. In particular, the routine testing of the sanitary quality of shellfish products by regulatory authorities also requires marine toxin standards for qualitative and quantitative analysis. As more and more marine toxins are discovered, the development of corresponding toxin standards is significantly lagging behind. Therefore, there is a huge market demand for marine algal toxin standards. At present, algal toxin standards are expensive and have low concentrations. They can only meet the quantitative needs of toxins for daily monitoring, but are difficult to meet the needs of algal toxin toxicology research and rapid detection method development. Due to the complex chemical structures of OA and DTXs, they cannot be produced through chemical synthesis. They are mainly isolated and prepared from toxin-producing algae or poisoned shellfish tissues. Therefore, it is of great significance to establish and develop a preparation process for obtaining OA and DTX1 toxin standard substances from toxin-producing microalgae. Summary of the invention

[0004] In view of the current situation that domestic diarrhetic shellfish poisoning standards are seriously lacking, the present invention provides a method for separating and purifying high-purity okanamic acid (OA) and dimorphotoxin-1 (DTX1) from a large-volume toxin-producing algae culture system to make up for the deficiencies of the prior art.

[0005] The method provided by the present invention comprises the following steps:

[0006] 1) adding alkaline solution to a culture system of Prorocentrum lima containing diarrhetic shellfish toxin and standing to break algal cells, then adjusting the pH value of the solution to 2.0-5.0, adding dichloromethane for extraction, and collecting the lower organic phase to obtain a crude toxin extract;

[0007] The alkali solution, as specifically described in the embodiment, is a NaOH solution;

[0008] Preferably, the dichloromethane is added in an amount of 7% (v / v);

[0009] Preferably, the pH value of the solution is adjusted to 3.0;

[0010] 2) The crude toxin extract collected by liquid-liquid extraction is concentrated, re-dissolved with methanol and added to an activated dextran gel chromatography column, eluted with a methanol solution, and the eluate is collected in sections; the elution volume of the toxin is determined based on the results of liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection of the toxin composition in the separated components, and the components containing OA and DTX1 toxins are selected, combined and concentrated, and further purified by semi-preparative liquid chromatography;

[0011] 3) using semi-preparative liquid chromatography to further separate and purify the OA and DTX1 components, analyzing them by LC-MS / MS, combining the toxin components, and further purifying them using a solid phase extraction column;

[0012] The chromatographic column is a C18 semi-preparative column, the mobile phase A is ultrapure water containing 0.1% formic acid, the mobile phase B is a 100% methanol solution, and the mobile phase A and B are used in a ratio of 2:8 for isocratic elution;

[0013] 4) The OA and DTX1 toxin samples obtained by semi-preparative liquid chromatography separation and purification were added to an activated solid phase extraction (SPE) column, eluted with 10-20% methanol to remove the matrix in the sample, and then eluted with a methanol solution to obtain OA and DTX1 standard samples.

[0014] The present invention directly utilizes NaOH solution to break up algae cells, thereby hydrolyzing the esterified forms of OA and DTX1 toxins in the cells into free toxins, and then adopts a liquid-liquid extraction method to collect the toxins in the culture medium; the method of the present invention effectively reduces the operation processes such as collection, breaking, and hydrolysis of algae cells, and is time-saving and has a high recovery rate; at the same time, dextran gel chromatography and semi-preparative liquid chromatography techniques are used for separation and purification, the process is simple, the purification effect is good, and the purity of the prepared OA and DTX1 standard substances is above 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Chemical structures of diarrhetic shellfish toxins OA and DTX1;

[0016] Figure 2 The technical flow chart of the present invention for preparing OA and DTX1 toxin standard substances from Prorocentrum lima;

[0017] Figure 3Comparison of liquid-liquid extraction recoveries of OA and DTX1 toxins under different pH conditions;

[0018] Figure 4 Effect of dichloromethane dosage on the extraction recovery rate of Prorocentrum lima. Different letters indicate significant differences among the groups (p<0.05).

[0019] Figure 5 Chromatograms of samples before purification under high performance liquid chromatography ultraviolet (HPLC-UV) detection under different ratios of mobile phase;

[0020] Figure 6 Full scan mass spectra (m / z 200-1500) of samples at each stage of separation and purification of the present invention;

[0021] Figure 7 High-resolution secondary mass spectra of OA (A) and DTX1 (B) toxins obtained by semi-preparative liquid chromatography purification of the present invention. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. However, the protection scope of the present invention is not limited to the scope covered by the embodiments.

[0023] Example 1: Extraction of toxins from toxic algae

[0024] 10% (v / v) 2.5 mol / L NaOH was added to the culture medium of Prorocentrum lima in the stable growth phase and allowed to stand for 24 hours to break up the algal cells and hydrolyze the esterified toxins. The results showed that after NaOH treatment, the total amount of OA increased by 4.5 times, the total amount of DTX1 increased by 2.2 times, and the OA and DTX1 toxins were basically completely released into the culture medium.

[0025] At 2.5 mol L -1 The pH of the culture medium after the disruption was adjusted to 3.0 by HCl, and 7% (v / v) dichloromethane was added thereto. After magnetic stirring for 5 minutes, it was poured into a separatory funnel and allowed to stand for 20 minutes. The lower organic phase was collected and centrifuged at 4800×g for 10 minutes. The clear liquid in the lower layer was collected, which was the crude extract of OA and DTX1 toxins. The results showed that the extraction recovery rates of OA and DTX1 were 90.9±3.7% and 65.1±2.3%, respectively.

[0026] Example 2: Further separation and purification of diarrhoeal shellfish poison

[0027] (1) Purification by dextran gel chromatography

[0028] Weigh 15g of Sephadex LH-20 filler and soak it in chromatographic grade methanol overnight to fully swell and activate the filler and remove some fat-soluble impurities. After soaking, the column was filled with wet column packing, and the final filling specification was 16mm×700mm.

[0029] The crude extracts of OA and DTX1 collected from Prorocentrum lima cells were concentrated, dried by nitrogen blowing, and then redissolved in 3-5 mL of methanol. The mobile phase used for column chromatography was methanol solution, and the flow rate was set at 1-3 mL min -1 . The concentrated toxin sample was loaded onto the column, the first 30 minutes of eluate was discarded, and then the sample was collected in sections, with 2 to 4 mL collected in each tube. According to the results of high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS), the elution volume of OA and DTX1 was 75-85 mL. The components containing the toxin were combined and concentrated, and further purified by semi-preparative liquid chromatography.

[0030] (2) Semi-preparative liquid chromatography purification

[0031] The sample purified by the dextran gel chromatography column was further purified by semi-preparative liquid chromatography (HITACHI) to separate OA and DTX1 toxins. The chromatographic column used was a Waters X-Bridge C18 OBD semi-preparative column (250×10 mm, 5 μm), the mobile phase A was ultrapure water containing 0.1% formic acid, the mobile phase B was 100% methanol solution, and the elution was performed by isocratic elution (80% B) at a flow rate of 4.0 mL min -1 The detector was a diode array detector (DAD) with a detection wavelength of 210 nm. The results showed that the peak time of OA was 6.87 min and the peak time of DTX1 was 9.78 min. The OA and DTX1 toxin components were collected separately and further purified by solid phase extraction column.

[0032] (3) Solid phase extraction purification

[0033] The specific operation is as follows: Waters Oasis-HLB SPE column (200 mg, 6 cc) was used for purification. First, 5 mL of methanol and 5 mL of 20% methanol aqueous solution (v / v) were used to activate and equilibrate the SPE column (about 1 mL min -1 ); Then, the purified toxin was prepared into a loading solution containing 20% ​​methanol and added to the SPE column (flow rate of about 0.5 mL min -1 ); then, rinse with 5 mL of 20% methanol aqueous solution (about 1 mL min -1 ); finally, elute with 1 mL of methanol (approximately 1 mL min -1). The eluate was collected and used as the standard substance of OA and DTX1 toxins.

[0034] The technical process for preparing OA and DTX1 toxin standard substances from Prorocentrum lima is as follows: Figure 2 shown.

[0035] Example 3: Process Optimization Study

[0036] (1) Toxin extraction process: NaOH was used to simultaneously disrupt algal cells and hydrolyze esterified toxins, and dichloromethane was used to extract free OA and DTX1 toxins in the culture medium. The extraction pH conditions ( Figure 3 ) and the amount of extractant ( Figure 4 The results showed that the best extraction effect was achieved when the pH of the culture system of Prorocentrum lima was adjusted to 3.0 and the addition ratio of dichloromethane was 7% (v / v).

[0037] (2) Preparation and purification process: The present invention compares the separation of toxin samples under different mobile phase ratios. The LC-UV chromatogram is shown in FIG. Figure 5 As shown in the figure, with the increase of the proportion of organic phase, the elution time of toxin is greatly shortened, and the peak width is narrowed. After optimization, isocratic elution (80% methanol) is used in the preparation, and the separation and purification effect of OA and DTX1 is better, and the preparation can be completed within 15 minutes.

[0038] Example 4: Analysis of purification results

[0039] In order to compare the impurity content in samples at each stage and analyze the removal effect of impurities in each step, the full scan mass spectra of samples at each stage were collected in the range of m / z 200-1500 using LC-MS Q1 full scan mode ( Figure 6 ). The results showed that after purification by dextran gel column chromatography, the impurity response of the sample was reduced by nearly 5 times, and the impurity peaks were significantly reduced, but the chromatography column purification could not separate the OA and DTX1 toxins; after purification by semi-preparative liquid chromatography, the toxins were completely separated, and the impurities in the sample were basically completely removed. The purity of the toxin products in the wavelength range of 200-400nm was analyzed by LC-UV, and the purity of OA was measured to be 99.66%, and the purity of DTX1 was 99.97%; the purpose of purification by solid phase extraction (SPE) column is to remove the aqueous phase in the sample, and further concentrate the toxins, elute the toxins in the solid phase extraction filler with methanol, and finally keep the toxins in a neutral environment.

[0040] The semi-preparative purified OA and DTX1 toxins were analyzed by LC-HR / MS. TMC18 chromatographic column (150×3mm id, 5μm), HRMS system in negative ion mode for full scan analysis of samples, the scanning range of m / z 100-1300; set the monitoring parent ion m / z 803.4587 and 817.4743, MS analysis of samples within the specified retention time range 2 The fragmentation voltage was 155.0 V and the collision energy was -60 eV. The measured masses of the adduct ions and fragment ions of the purified product are shown in Table 1. The high-resolution secondary mass spectrum is shown in Figure 7 shown.

[0041] Table 1 Measured masses and accurate masses of [MH]- and fragment ions of OA and DTX1 products purified by semi-preparative HPLC

[0042]

[0043] The results showed that the fragment ions (m / z 563, 255, 113, etc.) in the sample secondary mass spectra were consistent with the characteristic fragment ions of OA and DTX1 toxins, and the measured mass of the sample fragment ions and the accurate mass error of the characteristic fragment ions were within ±1ppm. It can be determined that the prepared products are OA and DTX1.

Claims

1. A method for preparing a diarrhetic shellfish toxin standard substance from marine microalgae, characterized in that: The method comprises the following steps 1) Adding a 10% 2.5 mol / L NaOH solution to a solution of Prorocentrum lima containing diarrhetic shellfish toxins and leaving it to stand for 24 hours to break up the algal cells and hydrolyze the esterified toxins, thereby reducing the operation process of collecting, breaking up, and hydrolyzing the algal cells. Subsequently, adjusting the pH value of the solution to 3.0, adding dichloromethane for extraction, and collecting the lower organic phase to obtain a crude toxin extract; 2) After the toxin crude extract is concentrated, it is re-dissolved in methanol and added to an activated dextran gel chromatography column, eluted with a methanol solution, and the eluate is collected in sections; the elution volume of the toxin is determined based on the results of LC-MS / MS detection of the toxin composition in the separated components, and the components containing OA and DTX1 toxins are selected for merging and concentration, and further purification is performed by semi-preparative liquid chromatography; 3) Semi-preparative liquid chromatography was used to further separate and purify the OA and DTX1 components. After LC-MS / MS analysis, the toxin components were combined and further purified using a C18 semi-preparative solid phase extraction column; the mobile phase A was ultrapure water containing 0.1% formic acid, the mobile phase B was 100% methanol solution, and 80% mobile phase B was used for isocratic elution; 4) The OA and DTX1 toxin samples obtained by semi-preparative liquid chromatography separation and purification were added to an activated solid phase extraction column, eluted with 10-20% methanol to remove the matrix in the sample, and then eluted with a methanol solution to obtain OA and DTX1 standard samples.

2. The method according to claim 1, characterized in that The added proportion of dichloromethane in the above 1) is 7% by volume.

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