Pretreatment method for detecting OA in seawater by using liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a pretreatment method for detecting OA in seawater by liquid chromatography-tandem mass spectrometry, which is characterized by comprising the following steps: (1) putting a seawater sample into a centrifuge tube, adding an SBA-15-C18 material, oscillating, centrifuging, discarding seawater, and reserving the SBA-15-C18 material; (2) adding a formic acid methanol solution into the SBA-15-C18 material, oscillating, performing ultrasonic treatment, centrifuging, transferring supernate into another centrifugal tube, retaining the SBA-15-C18 material, adding the formic acid methanol solution into the SBA-15-C18 material, repeating the previous steps, and combining the supernate; and (3) nitrogen-blowing the supernate to be nearly dry, fixing the volume by using methanol-2mmol ammonium acetate solution, uniformly mixing, filtering, and feeding into a sample injection bottle for instrument analysis. According to the method, the SBA-15-C18 material is used for adsorbing OA in the seawater, enrichment of OA toxin in the seawater can be completed through oscillation, the influence of the matrix effect on seawater purification can be effectively removed, the speed is high, the steps are simple, the purification effect is good, and the efficiency of the traditional seawater pretreatment process is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of OA detection, and in particular to a pretreatment method for detecting OA in seawater by utilizing liquid chromatography tandem mass spectrometry. Background Art

[0002] Red tide is a phenomenon in which some bacteria, protozoa and phytoplankton in the water suddenly proliferate and aggregate under specific environmental conditions, causing the water to change color. Single-celled microalgae are the main organisms that cause red tides. These algae produce toxins, which are filtered and eaten by shellfish in the seawater, and the shellfish will produce shellfish toxins. Diarrheal shellfish toxin (DSP) is one of the eight major marine shellfish toxins and is widely distributed in coastal waters around the world. It includes free and esterified toxins such as okanamic acid (OA) and its derivatives (Dinophysistoxins). The low OA content in the seawater matrix leads to inaccurate results in the detection of OA in seawater, and the toxin is difficult to enrich.

[0003] Liquid chromatography tandem mass spectrometry (HPLC-MS / MS) is a major method for detecting OA in seawater. In this method, the pretreatment method for seawater is mainly based on SPE. However, the traditional SPE treatment method requires activation, elution, and other steps, which is time-consuming and cumbersome, affecting the stability of the results and the efficiency of the test. Therefore, in the process of using liquid chromatography tandem mass spectrometry to detect OA in seawater, how to optimize the pretreatment of seawater is particularly important. Summary of the invention

[0004] The present invention aims to solve the technical problems existing in the existing solid phase extraction (SPE) treatment method and provides a pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry, which has simple operation and high purification efficiency.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme: The pretreatment method of the present invention for detecting OA in seawater by liquid chromatography tandem mass spectrometry comprises the following steps: (1) Take a seawater sample and place it in a centrifuge tube. Add SBA-15-C 18 Materials, shake and centrifuge, discard seawater, retain SBA-15-C 18 Material: SBA-15-C 18 The material has a good adsorption effect on OA. Impurities and OA toxins in seawater are adsorbed by SBA-15-C18 material with strong specificity. It can be reused after desorption. 18 The material is used as an adsorbent to purify seawater samples. The operation is very simple and the purification efficiency is high.

[0006] (2) In SBA-15-C 18Add formic acid methanol solution to the material, shake, sonicate, centrifuge, transfer the supernatant to another centrifuge tube, and keep SBA-15-C 18 Materials, in SBA-15-C 18 A formic acid methanol solution is added to the material, the previous steps are repeated, and the supernatant is combined. In the present invention, a formic acid methanol solution is used as a desorption reagent. The results of the machine show that the desorption process only releases the adsorbed OA toxin from the material, and does not release impurities, so that the SBA-15-C18 material can be reused.

[0007] (3) The supernatant liquid was blown to nearly dryness with nitrogen, and the volume was made up with methanol-2mmol ammonium acetate solution. After mixing, it was filtered into a sample injection bottle for instrument analysis.

[0008] Preferably, in step (1), 5 mL of seawater sample is taken and 15 mg of SBA-15-C is added. 18 materials, oscillation time 20-40s, centrifugal speed 7000-9000r / min, centrifugal time 3-7min. When the OA concentration is high, it can be increased by increasing the SBA-15-C 18 The material input amount completes the adsorption process.

[0009] Preferably, the SBA-15-C 18 The material was prepared by the following method: (a) dissolving a polyethylene glycol-polypropylene glycol-polyethylene glycol triblock polymer (P123) in hydrochloric acid under stirring at room temperature, adding tetraethyl orthosilicate (TEOS), and stirring at 40±2° C. for pre-hydrolysis to obtain a pre-hydrolyzed solution; (b) slowly adding dimethyloctadecylsilyl chloride to the pre-hydrolyzed solution, stirring the obtained mixture at 40±2° C. and transferring it to a reactor for hydrothermal reaction, filtering the product after the reaction, washing the filtered product with anhydrous ethanol and water, and drying to obtain a dried product; (c) The dried product is placed in a muffle furnace and calcined at elevated temperature to obtain SBA-15-C 18 Material: SBA-15-C 18 The material was prepared by a one-pot synthesis method. SBA-15 material was first prepared, and then C was modified on the SBA-15 material. 18 To obtain SBA-15-C 18 Materials, prepared SBA-15-C 18 The material can achieve the goal of efficient purification of sample matrix and reduction of matrix effect.

[0010] Preferably, in step (a), 3 g of polyethylene glycol-polypropylene glycol-polyethylene glycol triblock polymer is dissolved in hydrochloric acid, wherein the hydrochloric acid is prepared by mixing 97.5 ml of water and 15 ml of hydrogen chloride, and then 6 mL of ethyl silicate is added, and the mixture is stirred at 40±2° C. for 1 to 2 hours for pre-hydrolysis.

[0011] Preferably, in step (b), 1.305 g of dimethyloctadecylsilyl chloride is slowly added to the pre-hydrolyzed solution, stirred at 40±2°C for 8 to 12 hours, and then transferred to a reaction kettle, and the hydrothermal reaction is carried out in a static state, the reaction temperature is 80 to 100°C, the reaction time is 18 to 36 hours, and the drying temperature is 60 to 80°C. The reaction temperature cannot be too high, as it may damage SBA-15-C 18 The orderliness of the material greatly affects its use effect.

[0012] Preferably, in step (c), the muffle furnace heating calcination procedure is: the heating rate is 1-2°C per minute, and after heating to 500-600°C, it is maintained for 4-6 hours.

[0013] Preferably, in step (2), 3 mL of 1% by volume formic acid methanol solution is added, the shaking time is 20 to 40 seconds, the ultrasound is performed for 3 to 5 minutes, the centrifugal speed is 7000 to 9000 r / min, and the centrifugal time is 3 to 7 minutes. Even if the amount of adsorbed OA toxin increases, the OA toxin can still be desorbed from the material by 1% formic acid methanol, thereby realizing the reuse of the material.

[0014] Preferably, 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 an injection bottle.

[0015] Therefore, the present invention has the following beneficial effects: 18 The material adsorbs OA in seawater and can enrich OA toxins in seawater through oscillation. It can also effectively remove the influence of matrix effect on seawater purification. It has fast speed, simple steps and good purification effect, which greatly improves the efficiency of traditional seawater pretreatment process. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 SBA-15-C 18 SEM images of the materials.

[0017] Figure 2 SBA-15-C 18 TEM image of the material. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0019] 1. SBA-15-C 18 Material preparation SBA-15-C 18 The material was prepared by the following method: (a) 3 g of polyethylene glycol-polypropylene glycol-polyethylene glycol triblock polymer was 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, and the mixture was stirred at 40° C. for 1.5 h for pre-hydrolysis to obtain a pre-hydrolyzed solution; (b) 1.305 g of dimethyloctadecylsilyl chloride was slowly added to the pre-hydrolyzed solution, and the obtained mixture was stirred at 40° C. for 10 h and then transferred to a reaction kettle, and statically reacted at 100° C. for 24 h. After the reaction, the product was filtered, and the filtered product was washed with anhydrous ethanol and clean water, and dried at 75° C. for 12 h to obtain a dry product; (c) The dried product is placed in a muffle furnace and calcined at elevated temperature to obtain SBA-15-C 18 The material was calcined in a muffle furnace at a heating rate of 1 °C per minute until it reached 550 °C and then maintained for 4 h.

[0020] 1. SEM characterization Scanning electron microscopy of SBA-15-C 18 Materials (such as Figure 1 As shown in the figure, it can be clearly observed that SBA-15-C 18 The material has a two-dimensional through-hole structure, SBA-15-C 18 The particle size of the material is uniform, cylindrical and curved, the pore surface morphology is a uniform rod-like structure, the particles are clustered into bundles, and have a relatively regular length, indicating that SBA-15-C 18 The mesoporous molecular sieve has structural integrity and can maintain a complete structural framework even after being functionalized with octadecyl.

[0021] 2. TEM characterization Scanning SBA-15-C by transmission electron microscopy 18 Materials (such as Figure 2 As shown in the figure, it can be seen that the pores present a honeycomb structure in the direction parallel to the axis, and in the direction perpendicular to the axis, it has a regular pore structure, showing a long-range orderly and regular pore arrangement, which further proves that the synthesized SBA-15-C 18 The mesoscopic structure belongs to a two-dimensional hexagonal structure. From the picture, it can be observed that the pore size of the sample is about 1μm-2μm, and the pore wall thickness is about 1μm, which is a typical feature of SBA-15 mesoporous molecular sieves. This shows that the functionalized SBA-15-C 18The material still maintains the original morphology and structure of SBA-15.

[0022] 2. SBA-15-C 18 Selection of material use environment Take 5 mL of filtered seawater, add 10 μL of OA standard solution with a concentration of 1000 ng / ml, and add 15 mg of SBA-15-C 18 Materials, 4 mL of methanol, 25% methanol, 50% methanol, 75% methanol and pure water were added to seawater, vortexed for 30 seconds, centrifuged at 8000 r / min for 5 minutes, 1 mL of supernatant was taken, filtered through a 0.22 μm filter membrane, injected, and loaded on a machine (LCMS-8060NX liquid chromatography-mass spectrometer, Shimadzu Enterprise Management Co., Ltd., Japan). After testing, it was found that the adsorption process did not require the participation of organic reagents. SBA-15-C in seawater 18 The material can directly adsorb OA toxins, so 15 mg SBA-15-C was directly added during the determination process. 18 Adsorption can be completed by adding it to seawater.

[0023] Chromatographic conditions were 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 (2.1×100 mm, HSS), column temperature 40°C; flow rate: 0.5 mL / min; injection volume: 10 μL. Liquid chromatography gradient elution conditions are shown in Table 1.

[0024] Table 1 Gradient elution conditions of liquid chromatography Time (min) Flow rate (mL / min) 5mmol / ammonium acetate solution containing 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 The mass spectrometry conditions were as follows: negative ion mode scanning using electrospray ionization (ESI); multiple reaction monitoring (MRM) mode for quantitative detection; interface voltage: 1.0 kV; interface current: 0.2 μA; interface temperature: 400 °C; drying gas flow rate: 3.0 L min -1 , mass spectrometry parameters are shown in Table 2, where “*” represents quantitative ions.

[0025] Table 2 Mass spectrometry parameters in OA multiple reaction detection mode 3. Selection of desorption liquid In order to compare the effects of different desorption liquids on the desorption effect of materials, SBA-15-C 18The material was subjected to adsorption-desorption cycle experiment. The total volume of desorption liquid in the experimental process was temporarily set at 3 mL. OA toxins are all fat-soluble compounds, insoluble in water, and easily soluble in organic solvents such as methanol and dichloromethane. They usually disappear after alkaline hydrolysis. Proper acidic conditions are conducive to the elution of OA toxins. Therefore, dichloromethane and 1% formic acid methanol were selected as desorption liquids, respectively. The specific steps are as follows: Add 2% 4-(2-methyl-2-oxo-1-yl)-2-nitropropene to the SBA-15-C that has completed the adsorption process. 18 Add 2mL 1% formic acid methanol solution to the material (same as above) for desorption, vortex oscillation for 30s, ultrasonic for 5min, centrifuge at 8000r / min for 5min, transfer the supernatant to a 15mL centrifuge tube, blow nitrogen to dryness. Redissolve with 1ml methanol-2mmol ammonium acetate solution (volume ratio 7:3), use a disposable needle to absorb the reconstituted solution, pass through a 0.22μm needle-type polytetrafluoroethylene filter membrane in a sample vial, and provide 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.

[0026] Table 3 Effect of different desorption liquids on desorption effect 4. Specificity and adsorption effect test To verify SBA-15-C 18 To investigate the specificity of OA adsorption and the effectiveness of the adsorption effect, 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 materials adsorbed OA. The results showed that OA was not detected in the seawater added with SBA-15-C18, and the OA content in the seawater added with other materials did not change, indicating that SBA-15-C 18 It can effectively adsorb OA, while C 18 , SBA15, and PSA materials cannot adsorb OA, and cannot be used for OA detection in seawater.

[0027] Example 1 (1) Take 5 mL of seawater sample and place it in a 15 mL centrifuge tube. Add 15 mg of SBA-15-C 18 The material was shaken for 30 seconds and then centrifuged at 8000r / min for 5 minutes. The seawater was discarded and SBA-15-C was retained. 18 Material; (2) In SBA-15-C 18 Add 3 mL of 1% formic acid methanol solution to the material, shake for 30 seconds, ultrasonicate for 5 minutes, centrifuge at 8000 r / min for 5 minutes, transfer the supernatant to another 15 mL centrifuge tube, and keep SBA-15-C 18 Materials, in SBA-15-C18 Add 3 mL of 1% by volume formic acid methanol solution to the material, repeat the previous steps, and combine the supernatants; (3) blow the supernatant to near dryness with nitrogen, make up to volume with methanol-2 mmol ammonium acetate solution (volume ratio of 7:3), mix well, and filter into a sample injection bottle for instrument analysis.

[0028] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A pretreatment method for detecting OA in seawater using liquid chromatography tandem mass spectrometry, characterized in that: The following steps are involved: (1) Take a seawater sample and place it in a centrifuge tube, add SBA-15-C 18 Materials, shake and centrifuge, discard seawater, retain SBA-15-C 18 Material; (2) In SBA-15-C 18 Add formic acid methanol solution to the material, shake, sonicate, centrifuge, transfer the supernatant to another centrifuge tube, and keep SBA-15-C 18 Materials, in SBA-15-C 18 Add formic acid methanol solution to the material, repeat the previous steps, and combine the supernatants; (3) Blow the supernatant liquid with nitrogen until it is almost dry, make up to volume with methanol-2mmol ammonium acetate solution, mix well and filter into a sample injection 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: In step (1), take 5 mL of seawater sample and add 15 mg of SBA-15-C 18 Materials, oscillation time 20~40s, centrifugal speed 7000~9000r / min, centrifugal time 3~7min.

3. The pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry according to claim 1 or 2, characterized in that: The SBA-15-C 18 The material was prepared by the following method: (a) dissolving a polyethylene glycol-polypropylene glycol-polyethylene glycol triblock polymer in hydrochloric acid at room temperature with stirring, then adding ethyl silicate, and stirring at 40±2° C. for prehydrolysis to obtain a prehydrolyzed solution; (b) slowly adding dimethyloctadecylsilyl chloride to the pre-hydrolyzed solution, stirring the obtained mixture at 40±2° C. and transferring it to a reactor for hydrothermal reaction, filtering the product after the reaction, washing the filtered product with anhydrous ethanol and clean water, and drying to obtain a dried product; (c) The dried product is placed in a muffle furnace and calcined at elevated temperature to obtain SBA-15-C 18 Material.

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

5. The pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry according to claim 3, characterized in that: In step (b), 1.305 g of dimethyloctadecylsilyl chloride is slowly added to the pre-hydrolyzed solution, stirred at 40±2° C. for 8 to 12 hours, and then transferred to a reactor, and the hydrothermal reaction is carried out in a static state, the reaction temperature is 80 to 100° C., the reaction time is 18 to 36 hours; the drying temperature is 60 to 80° C.

6. The pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry according to claim 3, characterized in that: In step (c), the muffle furnace heating calcination procedure is: the heating rate is 1-2°C per minute, and after heating to 500-600°C, it is maintained for 4-6 hours.

7. The pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In step (2), 3 mL of 1% by volume formic acid methanol solution is added, the shaking time is 20-40 s, the ultrasound is applied for 3-5 min, the centrifugal speed is 7000-9000 r / min, and the centrifugal time is 3-7 min.

8. The pretreatment method for detecting OA in seawater by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In step (3), the volume ratio of methanol to 2 mmol ammonium acetate solution in methanol-2 mmol ammonium acetate solution is 7:3, the volume is fixed to 1 mL, and after mixing, it is filtered through a 0.22 μm filter membrane into a sample injection bottle.

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