Preparation method for detecting OA in seawater by liquid chromatography tandem mass spectrometry

Abstract

The application discloses a pretreatment method for detecting OA in seawater by using liquid chromatography tandem mass spectrometry, and is characterized by comprising the following steps: (1) taking seawater samples in a centrifugal tube, adding SBA-15-C 18 materials, oscillating, centrifuging, discarding seawater, and reserving SBA-15-C 18 materials; (2) adding formic acid methanol solution in the SBA-15-C 18 materials, oscillating, ultrasonicating, centrifuging, transferring supernatant to another centrifugal tube, reserving SBA-15-C 18 materials, adding formic acid methanol solution in the SBA-15-C 18 materials, repeating the previous steps, and combining the supernatant; (3) nitrogen blowing the supernatant to near dryness, using methanol-2mmol ammonium acetate solution to constant volume, mixing, filtering to a sample bottle, and instrument analysis. The application can enrich OA toxin in seawater by oscillating after adsorbing OA in seawater by using SBA-15-C 18 materials, and can effectively remove the influence of matrix effect on seawater purification, has the advantages of fast speed, simple steps, good purification effect, and greatly improved efficiency of the traditional seawater pretreatment process.

Description

TECHNICAL FIELD

[0001] The present application relates to the OA detection technical field, especially to a pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry. BACKGROUND

[0002] Red tide is a phenomenon caused by the sudden proliferation of some bacteria, protozoa and phytoplankton in water under certain environmental conditions. Single-cell microalgae is the main organism causing red tide. These algae can produce toxins, and the toxins are filtered by shellfish in seawater, and the shellfish will produce shellfish toxins. Diarrhetic shellfish toxins (DSP) belong to one of the eight major marine shellfish toxins, and are widely distributed in global coastal waters, including free and esterified toxins such as dinophysistoxins and the like. The content of OA in seawater matrix is low, which leads to inaccurate results in the process of detecting OA in seawater, and the toxins are difficult to enrich.

[0003] Liquid chromatography tandem mass spectrometry (HPLC-MS / MS) is a main method for detecting OA in seawater. In this method, the pretreatment method of seawater is mainly based on SPE method. However, the traditional SPE treatment method needs activation, elution and elution steps, which is time-consuming and complicated, and affects the stability of the results and the test efficiency. Therefore, in the process of detecting OA in seawater by liquid chromatography tandem mass spectrometry, how to optimize the pretreatment of seawater is particularly important. SUMMARY

[0004] The present application provides a pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry, which is simple to operate and has high purification efficiency.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: the pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry of the present application comprises the following steps:

[0006] (1) Take seawater sample and place it in a centrifugal tube, add SBA-15-C 18 material, oscillate and centrifuge, discard the seawater and reserve the SBA-15-C 18 material. The SBA-15-C 18 material has good adsorption effect on OA, and the impurities and OA toxins in seawater are adsorbed by the SBA-15-C18 material, which has high specificity, and can be reused after desorption. In addition, the SBA-15-C 18 material is used as an adsorbent to purify the seawater sample, which is very simple to operate and has high purification efficiency.

[0007] (2) In the SBA-15-C18 The material is added with methanol solution of formic acid, oscillated, ultrasonically treated, centrifuged, and the supernatant is transferred to another centrifuge tube, and the SBA-15-C 18 The material is added with methanol solution of formic acid, oscillated, ultrasonically treated, centrifuged, and the supernatant is transferred to another centrifuge tube, and the SBA-15-C 18 The material is added with methanol solution of formic acid, and the previous steps are repeated, and the supernatants are combined. In the present application, the methanol solution of formic acid is used as a desorption reagent, and the machine results show that the desorption process only releases the adsorbed OA toxin from the material, and does not release impurities, and the reuse of the SBA-15-C18 material can be achieved.

[0008] (3) The supernatant is nitrogen blown to near dryness, and is diluted with methanol-2mmol ammonium acetate solution, mixed, and then filtered into a sample bottle for instrument analysis.

[0009] As preferred, in step (1), 5mL of seawater sample is taken, 15mg of SBA-15-C 18 The material is oscillated for 20-40s, centrifuged at 7000-9000r / min for 3-7min. When the OA concentration is high, the SBA-15-C 18 The material is added in an amount to complete the adsorption process.

[0010] As preferred, the SBA-15-C 18 The material is prepared by the following method:

[0011] (a) The polyethylene glycol-polypropylene glycol-polyethylene glycol triblock polymer (P123) is stirred and dissolved in hydrochloric acid at room temperature, and then tetraethyl orthosilicate (TEOS) is added, and pre-hydrolysis is carried out under stirring at 40±2℃ to obtain a pre-hydrolysis solution;

[0012] (b) Dimethyloctadecylchlorosilane is slowly added to the pre-hydrolysis solution, and the obtained mixture is stirred at 40±2℃ and then transferred to a reaction kettle for hydrothermal reaction. After reaction, the product is filtered, and the filtrate is washed with anhydrous ethanol and water, and dried to obtain a dry product;

[0013] (c) The dry product is placed in a muffle furnace for temperature rising calcination to obtain the SBA-15-C 18 material. The SBA-15-C 18 The material is prepared by one-pot synthesis method, and the SBA-15 material is first prepared, and then C 18 is modified on the SBA-15 material to obtain the SBA-15-C 18 material. The prepared SBA-15-C 18 material can achieve the goal of efficient purification of sample matrix and reduction of matrix effect.

[0014] As preferred, in step (a), 3g of polyethylene glycol-polypropylene glycol-polyethylene glycol triblock polymer is dissolved in hydrochloric acid prepared by mixing 97.5ml of water and 15ml of hydrogen chloride, 6mL of ethyl silicate is added, and pre-hydrolysis is carried out by stirring at 40±2℃ for 1-2h.

[0015] As preferred, in step (b), 1.305g of dimethyl octadecyl chlorosilane is slowly added to the pre-hydrolysis solution, and after stirring at 40±2℃ for 8-12h, it is transferred to a reaction kettle, and hydrothermal reaction is carried out in a static state, the reaction temperature is 80-100℃, the reaction time is 18-36h, and the drying temperature is 60-80℃. The reaction temperature cannot be too high, and a temperature that is too high can damage the SBA-15-C 18 The order of the material greatly affects its use effect.

[0016] As preferred, in step (c), the temperature rising calcination program of the muffle furnace is as follows: the temperature rising speed is 1-2℃ per minute, and after rising to 500-600℃, it is kept for 4-6h.

[0017] As preferred, in step (2), 3mL of 1% formic acid methanol solution by volume percentage is added, the oscillation time is 20-40s, the ultrasonic time is 3-5min, the centrifugal speed is 7000-9000r / min, and the centrifugal time is 3-7min. Even if the amount of adsorbed OA toxin increases, the OA toxin can still be desorbed from the material by 1% formic acid methanol, and the material can be reused.

[0018] As preferred, in step (3), in the methanol-2mmol ammonium acetate solution, the volume ratio of methanol to 2mmol ammonium acetate solution is 7:3, the volume is fixed to 1mL, and after mixing, it is filtered through a 0.22μm filter membrane into a sample bottle.

[0019] Therefore, the present application has the following beneficial effects: SBA-15-C 18 The material adsorbs OA in seawater, and the enrichment of OA toxin in seawater can be completed by oscillation, and the influence of matrix effect on seawater purification can be effectively removed, the speed is fast, the steps are simple, the purification effect is good, and the efficiency of the traditional seawater pretreatment process is greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the SEM image of SBA-15-C 18 The material.

[0021] Figure 2 is the TEM image of SBA-15-C 18 The material. DETAILED DESCRIPTION

[0022] The present application will be further described below in combination with the drawings and specific embodiments.

[0023] I. SBA-15-C 18 Material Preparation

[0024] SBA-15-C 18 The material was prepared by the following method:

[0025] (a) 3 g of polyethylene-polypropylene glycol-polyethylene triblock polymer was stirred and dissolved in hydrochloric acid (hydrochloric acid was prepared by mixing 97.5 ml of water and 15 ml of hydrogen chloride) at room temperature, and then 6 mL of ethyl silicate was added. Pre-hydrolysis was carried out by stirring at 40°C for 1.5 h to obtain a pre-hydrolysis solution;

[0026] (b) 1.305 g of dimethyloctadecylchlorosilane was slowly added to the pre-hydrolysis solution. After the resulting mixture was stirred at 40°C for 10 h, it was transferred to a reaction kettle and allowed to react statically at 100°C for 24 h. After the reaction, the product was filtered, and the filtrate was washed with anhydrous ethanol and water, and dried at 75°C for 12 h to obtain a dry product;

[0027] (c) The dry product was placed in a muffle furnace and calcined by increasing the temperature to obtain SBA-15-C 18 The muffle furnace temperature increase calcination procedure was as follows: the temperature was increased at a rate of 1°C per minute, and then maintained at 550°C for 4 h.

[0028] 1. SEM characterization

[0029] The SBA-15-C 18 material was scanned by electron microscopy (SEM) as shown in Figure 1 It can be clearly observed that the SBA-15-C 18 material has a two-dimensional pore structure, and the SBA-15-C 18 material has uniform particle size, is in the form of a curved cylinder, the pore surface morphology is in the form of a uniform rod structure, the particles are aggregated into bundles, and have a relatively regular length, indicating that the SBA-15-C 18 mesoporous molecular sieve has structural integrity, and even after octadecyl functionalization, the complete structural framework is still maintained.

[0030] 2. TEM characterization

[0031] The SBA-15-C 18 material was scanned by transmission electron microscopy (TEM) as shown in Figure 2 It can be seen that in the direction parallel to the axis, the material presents a honeycomb structure, and in the direction perpendicular to the axis, it has a regular pore structure, presenting long-range ordered and regular pore arrangement, further proving that the synthesized SBA-15-C 18The mesostructure belongs to two-dimensional hexagonal structure. It can be observed from the picture that the pore diameter of the sample is about 1 μm-2 μm, and the pore wall thickness is about 1 μm, which is the characteristic of typical SBA-15 mesoporous molecular sieve. It is proved that the SBA-15-C 18 The material still maintains the original morphology and structure of SBA-15.

[0032] II. SBA-15-C 18 Selection of material use environment

[0033] 5 mL of the filtered seawater was taken, 10 μL of OA standard solution with a concentration of 1000 ng / ml was added, and 15 mg of SBA-15-C 18 material was put in. 4 mL of methanol, 25% methanol, 50% methanol, 75% methanol and pure water were added to the seawater respectively, and vortexed for 30 s. Then, the mixture was centrifuged at a speed of 8000 r / min for 5 min. 1 mL of supernatant was taken, filtered through a 0.22 μm filter membrane, and injected into the machine (LCMS-8060NX liquid chromatograph-mass spectrometer, Shimadzu Enterprise Management Co., Ltd., Japan). It was found through detection that the adsorption process did not need to involve organic reagents, and SBA-15-C 18 material can directly adsorb OA toxin, so 15 mg of SBA-15-C 18 was directly put into seawater to complete the adsorption.

[0034] The chromatographic conditions are as follows: mobile phase A: 5 mmol / L ammonium acetate solution containing 0.1% formic acid; mobile phase B: acetonitrile; chromatographic column: 1.9 μm C 18 -AQ column (2.1 x 100 mm, HSS), column temperature 40 ℃; flow rate: 0.5 mL / min; injection volume: 10 μL. The liquid chromatography gradient elution conditions are shown in Table 1.

[0035] Table 1 Gradient elution conditions of liquid chromatography

[0036] Time (min) Flow rate (mL / min) 5 mmol ammonium acetate solution with 0.1% formic acid (%) Acetonitrile (%) 0.00 0.5 70 30 3.00 0.5 45 55 4.00 0.5 10 90 5.00 0.5 0 100 6.00 0.5 70 30 8.00 0.5 70 30

[0037] The mass spectrometry conditions are as follows: using electrospray ion source (ESI) negative ion mode scanning; selecting multiple reaction monitoring mode (MRM) for quantitative detection; interface voltage: 1.0 kV; interface current: 0.2 μA; interface temperature: 400 ℃; drying gas flow rate: 3.0 L·min -1 . The mass spectrometry parameters are shown in Table 2, in which "*" represents the quantitative ion.

[0038] Table 2 OA multiple reaction monitoring mode mass spectrometry parameters

[0039]

[0040] III. Selection of desorption solution

[0041] To compare the effect of different desorption solutions on the desorption of the material, SBA-15-C 18 The material was subjected to adsorption-desorption cycle experiments. The total volume of the desorption solution was temporarily set to 3 mL. The OA toxin is a fat-soluble compound and is insoluble in water but easily soluble in organic solvents such as methanol and dichloromethane. It usually disappears after alkaline hydrolysis, and appropriate acidic conditions are beneficial to the elution of the OA toxin. Therefore, dichloromethane and 1% formic acid methanol were selected as the desorption solutions, and the specific steps were as follows: 2 mL of 1% formic acid methanol solution was added to the SBA-15-C 18 material (as above) after the completion of the adsorption process, vortexed and shaken for 30 s, ultrasonicated for 5 min, centrifuged at 8000 r / min for 5 min, and the supernatant was transferred to a 15 mL centrifuge tube and blown to dryness with nitrogen. 1 mL of methanol-2 mmol ammonium acetate solution (volume ratio 7:3) was used for re-dissolution, and a disposable needle was used to take the re-dissolved solution, which was filtered through a 0.22 μm needle polytetrafluoroethylene filter membrane into a sample injection vial for liquid chromatography-mass spectrometry analysis. The experimental results are shown in Table 3. Therefore, 1% formic acid methanol was finally determined as the desorption solution.

[0042] Table 3 Effect of different desorption solutions on desorption

[0043]

[0044] Four, specificity and adsorption effect test

[0045] To verify the specificity and effectiveness of the adsorption effect of SBA-15-C 18 on OA, 100 μL of OA standard solution with a concentration of 1000 ng / ml was added to 5 mL of seawater, and then 15 mg of SBA-15-C 18 , C 18 , SBA15, and PSA material were added to adsorb OA. The results showed that no OA was detected in the seawater with SBA-15-C18, and the content of OA in the seawater with other materials did not change, indicating that SBA-15-C 18 can effectively adsorb OA, while C 18 , SBA15, and PSA material cannot adsorb OA and cannot be applied to the detection of OA in seawater.

[0046] Example 1

[0047] (1) 5 mL of seawater sample was taken in a 15 mL centrifuge tube, 15 mg of SBA-15-C 18 material was added, and after shaking for 30 s, it was centrifuged at 8000 r / min for 5 min, and the seawater was discarded, and the SBA-15-C 18 material was retained;

[0048] (2) In SBA-15-C 18 The 3 mL volume percentage 1% formic acid methanol solution was added to the material, oscillated for 30 s, and ultrasonically treated for 5 min. The material was centrifuged at 8000 r / min for 5 min, and the supernatant was transferred to another 15 mL centrifuge tube. The SBA-15-C 18 material was reserved, and the above steps were repeated. The supernatants were combined. 18 The 3 mL volume percentage 1% formic acid methanol solution was added to the material, and the above steps were repeated. The supernatants were combined.

[0049] The above-described embodiments are only a preferred scheme of the present application, and do not limit the present application in any form. Other variants and modifications can be made without departing from the technical scheme recited in the claims.

Claims

1. A pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry, characterized in that, The method comprises the following steps: (1) Take 5 mL seawater sample into a centrifugal tube, add 15 mg SBA-15-C 18 material, shake and centrifuge, shaking time 20-40 s, centrifugal speed 7000-9000 r / min, centrifugal time 3-7 min, discard the seawater and reserve the SBA-15-C 18 material; (2) 3 mL of 1% formic acid methanol solution was added to the SBA-15-C 18 material, oscillated, ultrasonically treated, centrifuged, oscillation time was 20-40 s, ultrasonic treatment time was 3-5 min, centrifugal speed was 7000-9000 r / min, centrifugal time was 3-7 min, the supernatant was transferred to another centrifuge tube, and the SBA-15-C 18 material was reserved. 18 The formic acid methanol solution was added to the SBA-15-C 18 material, and the previous steps were repeated, and the supernatants were combined. (3) The supernatant is blown to near dryness with nitrogen, and then diluted with methanol-2mmol ammonium acetate solution, the volume ratio of methanol to 2mmol ammonium acetate solution being 7:3, and the total volume being 1ml; after mixing, the mixture is filtered through a 0.22μm filter membrane into a sample bottle for instrument analysis.

2. The pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, The SBA-15-C 18 The material was prepared by the following method: (a) stirring and dissolving the polyethylene glycol-polypropylene glycol-polyethylene glycol triblock polymer in hydrochloric acid at room temperature, then adding ethyl silicate, and stirring for pre-hydrolysis at 40±2℃ to obtain a pre-hydrolysis solution; (b) slowly adding dimethyloctadecylchlorosilane into the pre-hydrolysis solution, stirring the obtained mixture at 40±2℃, and then transferring the mixture into a reaction kettle for hydrothermal reaction, filtering the product after reaction, washing the filter with anhydrous ethanol and water, and drying to obtain a dry product; (c) placing the dried material in a muffle furnace to heat and calcine, to obtain SBA-15-C 18 Materials.

3. The pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry according to claim 2, characterized in that, In step (a), 3g of the polyethylene glycol-polypropylene glycol-polyethylene glycol triblock polymer is dissolved in hydrochloric acid, the hydrochloric acid is prepared by mixing 97.5ml of water and 15ml of hydrogen chloride, 6ml of ethyl silicate is added, and pre-hydrolysis is carried out by stirring at 40±2℃ for 1-2h.

4. The pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry according to claim 2, characterized in that, In step (b), 1.305g of dimethyloctadecylchlorosilane is slowly added into the pre-hydrolysis solution, the mixture is stirred at 40±2℃ for 8-12h, and then transferred into a reaction kettle for hydrothermal reaction, the reaction is carried out in a static state, the reaction temperature is 80-100℃, the reaction time is 18-36h, and the drying temperature is 60-80℃.

5. The pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry according to claim 2, characterized in that, In step (c), the temperature rising and calcination program of the muffle furnace is as follows: the temperature rising speed is 1-2℃ per minute, the temperature is raised to 500-600℃, and then kept for 4-6h.

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