CN113655147A - Method for detecting microcystin in water - Google Patents
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Abstract
The invention relates to a method for detecting microcystin in water, which comprises the following steps: filtering a water sample through a 0.22 mu m MCE filter membrane, and then carrying out online solid-phase extraction and analytical determination on the filtered water sample through an online solid-phase extraction ultra-high performance liquid chromatography tandem mass spectrometry; c8 solid phase extraction column is adopted for on-line solid phase extraction; the conditions of the ultra-high performance liquid chromatography are as follows: mobile phase: the solution A is 0.1% formic acid acetonitrile solution, and the solution B is 0.1% formic acid aqueous solution; gradient elution procedure: 0-4.6min, 98% of solution B; 4.6-10.0min, 98% B liquid-25% B liquid, 10.0-11.0min, 25% B liquid-100% B liquid; the flow rate is 0.4 mL/min; column temperature 35 ℃, sample injection volume: 2 mL. The method provided by the invention has the advantages of high sensitivity, high accuracy, good recovery rate and reproducibility, and simple and rapid operation, and can be used for simultaneously measuring 8 MCs in the water body.
Description
Technical Field
The invention belongs to the technical field of water environment monitoring, relates to a method for detecting microcystins in water, and particularly relates to a method for detecting microcystins in water by an online solid-phase extraction ultra-high performance liquid chromatography tandem mass spectrometry.
Background
With the development of industrial and agricultural production, a large amount of nitrogen and phosphorus are discharged into water, so that the eutrophication phenomenon of the water is increasingly serious. The eutrophication of water body can cause the appearance of cyanobacterial bloom, and the cyanobacterial bloom can release secondary metabolites of biotoxins, such as Microcystins (MCs), and the like, to cause pollution of various derivatives and harm the safety of human beings and other organisms. Epidemiological research finds that the high incidence of primary liver cancer in certain areas (such as Jiangsu Thaixing, Haimen and the like) in south China is also related to the pollution of drinking water by MCs. At least 279 MC isomers have been reported so far, wherein MC-LR is the most toxic and widely distributed configuration, and the limit standard of MC-LR in drinking water recommended by the Australian scholars of the World Health Organization (WHO) and the national Standard of Water environmental quality (GB3838-2002) is 1.0 mu g/L. MC-LR, MC-RR, MC-LA and MC-YR are mainly focused by the United States Environmental Protection Agency (USEPA), and the monitoring and evaluation of MC-LR only can greatly underestimate the pollution level of MCs in source water and can not meet the requirement of people on the safety of drinking water. In order not to endanger human safety and health, it is particularly important to develop a plurality of rapid and efficient detection methods for MCs.
At present, the methods for detecting MCs in water reported in the literature are roughly classified into the following four types: chemical analysis, biological analysis, biochemical analysis, and molecular bioinformation detection. The method mainly adopts a pretreatment mode of manual or automatic solid phase extraction to enrich and concentrate a large amount of water samples and then detect the water samples, can identify the types of the detected toxins by comparing the retention time of the detected toxins with the retention time of standard toxins, can quantify the detected toxins by comparing the peak areas of the detected toxins and the standard toxins, has the detection limit generally reaching the level of mu g/L or ng/L, and can better meet the detection requirement of trace MCs in water. There are studies on simultaneous determination of 12 MC concentrations using high performance liquid chromatography-triple quadrupole mass spectrometry (see: ZUXuXUO, Caixin, ZUXIANYI, Li-Ruifen. direct injection-ultra high performance liquid chromatography-triple quadrupole mass spectrometry for simultaneous rapid determination of 12 microcystins and 1 nodulotoxin [ J ] in water [ 2017,35(12):1286-1293 ]). Compared with other methods, the method has the advantages of high pressure, high speed, high efficiency, high sensitivity and wide application range, is suitable for separating, identifying and quantitatively detecting MCs and various isomers thereof, but has the defects of more complex sample pretreatment steps, time consumption, poorer sample repeatability and the like, and the solid phase extraction column is generally expensive in price and relatively higher in analysis cost. The liquid chromatography-mass spectrometry (LC-MS) analysis method optimizes pretreatment and detection conditions, and establishes an LC-MS/MS analysis method for synchronously extracting and simultaneously determining MCs. MCs are separated through liquid chromatography, mass spectrum is used as a detector for detection, acetonitrile-water-formic acid is generally used as a mobile phase, the type and content of toxins are determined according to accurate molecular weight and structure information, and the method has higher selectivity and sensitivity compared with other chromatographic detection technologies. LC-MS has good distinguishing effect on the type of MCs due to the fact that a mass spectrometer is connected in series, and can identify a plurality of algal toxins in a mixture at the same time, but a liquid chromatography-mass spectrometer is expensive and has high operation cost. The U.S. Environmental Protection Agency (US EPA, 2015) recommends direct injection-liquid chromatography tandem mass spectrometry (LC-MS/MS) detection of 6 microcystins (MC-LA, MC-LF, MC-LR, MC-LY, MC-RR, etc.), but this approach suffers from interference from complex matrices (see: US Environmental Protection Agency (US EPA) Method 544.). Zhangming and the like adopt a solid phase extraction-ultra-high performance liquid chromatography-electrospray tandem mass spectrometry method to simultaneously determine 9 microcystins in surface water (see: Zhang ming, Tang Dynasty, Chen Feng and the like. solid phase extraction-ultra-high performance liquid chromatography-electrospray tandem mass spectrometry method to simultaneously determine 9 microcystins [ J ] in surface water, 2012,30(01): 51-55.); li Dong gang et al detected 8 trace microcystins in water by three-channel automatic solid-phase extraction-high performance liquid chromatography quadrupole tandem mass spectrometry (see: Li Dong gang, Li Fang, Jawen Jing et al. three-channel automatic solid-phase extraction-high performance liquid chromatography quadrupole tandem mass spectrometry detected 8 trace microcystins in water). However, the methods of Zhangming et al require manual solid phase extraction for sample pretreatment, have complicated steps and take a long time, and are easily interfered by the surrounding environment in the sample pretreatment process; the detection limit of the method of Li's Boehringer et al is as high as 0.01-0.6 ug/L, and the sensitivity is not sufficient.
Therefore, the method which has the advantages of high sensitivity, high accuracy, good recovery rate and reproducibility, simple and convenient operation and capability of simultaneously measuring 8 MCs in the water body is established, and the method has important significance for monitoring, managing and timely managing the water body pollution condition.
Disclosure of Invention
In order to solve one or more problems in the prior art, the invention provides a method for detecting microcystins in water; the method provided by the invention has the advantages of high sensitivity, high accuracy, good recovery rate and reproducibility, and simple and rapid operation, and can be used for simultaneously measuring 8 MCs in the water body.
The invention provides a method for detecting microcystin in water, which comprises the following steps:
(1) filtering a water sample through a 0.22 mu m MCE filter membrane to obtain a pretreated water sample;
(2) performing online solid-phase extraction and analysis determination on the pretreated water sample by using an online solid-phase extraction ultra-performance liquid chromatography tandem mass spectrometry;
wherein, the online solid-phase extraction adopts a C8 solid-phase extraction column;
when the analysis and determination are carried out, the conditions of the ultra-high performance liquid chromatography are as follows: elution was performed in a gradient with mobile phase:
mobile phase: the solution A is 0.1% formic acid acetonitrile solution, and the solution B is 0.1% formic acid aqueous solution; gradient elution procedure: 0-4.6min, 2% of solution A and 98% of solution B; 4.6-10.0min, 2% of solution A-75% of solution A, 98% of solution B-25% of solution B, 10.0-11.0min, 75% of solution A-0% of solution A, and 25% of solution B-100% of solution B; the flow rate is 0.4 mL/min; column temperature 35 ℃, sample injection volume: 2 mL.
Preferably, the C8 solid phase extraction column used has a particle size of 10 μm, an inner diameter of 2.1mm and a column length of 30mm
Direct Connect HP solid phase extraction column.
Preferably, when the analytical determination is made, the ultra high performance liquid chromatography uses an ACQUITY UPLC BEH C18 column having a particle size of 1.6 μm, an inner diameter of 2.1mm and a column length of 50 mm.
Preferably, in performing the analytical determination, the mass spectrometric conditions are: a triple quadrupole mass spectrometer is used, and an electrospray ion source, a positive ion scanning mode and a multi-reaction monitoring mode are adopted; capillary voltage: 3.70 kV; ion source temperature: 150 ℃; desolventizing temperature: 500 ℃; desolventizing agent gas flow: 1000L/h; taper hole gas flow: 50L/h; taper hole air pressure: 30V; collision gas flow rate: 0.06 mL/min.
Preferably, the method is capable of detecting 8 microcystins in water simultaneously; the 8 kinds of microcystins are MC-LR, MC-YR, MC-RR, MC-WR, MC-LA, MC-LF, MC-LY and MC-LW.
Preferably, the mass spectrometric parameters of the method for detecting 8 microcystins are: MC-LR, parent ion 995.4, qualitative ion 135, quantitative ion 213, cone aperture voltage 85V, and collision energy 75 eV; MC-YR, parent ion 1045.6, qualitative ion 135, quantitative ion 213, cone hole voltage 85V, and collision energy 85 eV; MC-RR, parent ion 519.7, qualitative ion 135, quantitative ion 440.4, cone hole voltage 42V, collision energy 32 eV; MC-WR, parent ion 1068.5, qualitative ion 135, quantitative ion 213, taper hole voltage 60V, and collision energy 95 eV; MC-LA, parent ion 910, qualitative ion 135, quantitative ion 776, cone hole voltage 40V, and collision energy 80 eV; MC-LF, parent ion 986.3, qualitative ion 135, quantitative ion 213, cone hole voltage 42V, collision energy 68 eV; MC-LY, parent ion 1002.6, qualitative ion 135, quantitative ion 985, cone voltage 45V, collision energy 80 eV; MC-LW, parent ion 1025.5, qualitative ion 135, quantitative ion 213, cone-hole voltage 45V, and collision energy 65 eV.
Preferably, before the step (2), the method further comprises the step of adding a methanol solution of leucine enkephalin with the concentration of 1 mu g/L to the pretreated water sample obtained in the step (1) as an internal standard solution.
Preferably, before step (2), the method further comprises the step of performing online solid-phase extraction and analytical determination on the mixed standard solution containing 8 microcystins by online solid-phase extraction ultra-performance liquid chromatography-tandem mass spectrometry to draw a standard curve.
Preferably, 8 single standard solutions of microcystins are adopted to prepare 7 mixed standard solutions containing 8 microcystins with different concentrations of 10ng/L, 20ng/L, 50ng/L, 100ng/L, 200ng/L, 500ng/L and 1000ng/L respectively, and the mixed standard solutions are used for drawing a standard curve.
Preferably, the method has a linear relation in the range of 10ng/L-1000ng/L when detecting the microcystin in water, and the correlation coefficients are all more than 0.99; the detection limit of the method for detecting the microcystins in the water is 0.02-0.37ng/L, and the quantification limit is 0.07-1.24 ng/L.
Compared with the prior art, the method for detecting 8 microcystins in water by using the on-line solid-phase extraction ultra-high performance liquid chromatography-tandem mass spectrometry has the following beneficial effects:
1. compared with the existing sample pretreatment means, the method disclosed by the invention does not need a pretreatment step of manually carrying out solid-phase extraction on a water sample, namely a complex pretreatment step, so that the detection is faster and more efficient, the on-machine detection time of one sample is 11 minutes, the method is economic, fast and efficient, and the requirement of fast analysis can be better met.
2. The method has high recovery rate and good repeatability, 8 microcystins have good linear relation in the range of 10ng/L-1000ng/L, the correlation coefficients are all more than 0.99, the detection Limit (LOD) of the 8 microcystins is 0.02-0.37ng/L, the quantitative Limit (LOQ) is 0.07-1.24ng/L and is far lower than the limit standard of the microcystins in the water body in China, and trace MCs in a water environment sample can be detected; compared with the prior art, the method has the advantages of good chromatographic peak separation effect, low detection limit, low quantitative limit, high sensitivity, high accuracy and the like.
3. The method overcomes the defects of complex pretreatment steps, time consumption, poor sample repeatability and high analysis cost of a manual solid phase extraction method, effectively reduces the interference of the surrounding environment in a direct sample injection method, and provides a scientific analysis method for comprehensively understanding the pollution conditions of various MCs in eutrophicated water in China.
Drawings
FIG. 1 is a chromatogram for the detection of 8 microcystins by the method of the present invention.
FIG. 2 is a graph showing the recovery of 8 microcystins from 3 different membranes of the present invention. In the figure, PES filter membrane, PTFE filter membrane and MCE filter membrane are adopted from left to right in the corresponding recovery rate column diagram of each microcystin.
FIG. 3 is a chromatogram of 8 microcystins from 3 different filters according to the invention.
FIG. 4 is a chromatogram of 8 microcystins in the case of using the C8 and C18 solid phase extraction columns of the present invention.
FIG. 5 is a chromatogram of 8 microcystins in 6 different mobile phase systems of the present invention. In the figure, (a) mobile phase system: methanol + water; (b) mobile phase system: acetonitrile and water; (c) mobile phase system: 0.1% methanoic acid in methanol + 0.1% aqueous formic acid; (d) mobile phase system: 0.1% formic acid acetonitrile solution + 0.1% formic acid aqueous solution; (e) mobile phase system: 0.25% formic acid acetonitrile solution + 0.1% formic acid aqueous solution; (f) mobile phase system: 0.5% formic acid in acetonitrile + 0.1% formic acid in water.
FIG. 6 is a chromatogram of 8 microcystins from four different gradient elution procedures according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The invention provides a method for detecting microcystin in water, which comprises the following steps:
(1) filtering a water sample through an MCE filter membrane (mixed cellulose ester filter membrane) with the particle size of 0.22 mu m to obtain a pretreated water sample; in the invention, the water sample is filtered by the MCE filter membrane with the diameter of 0.22 mu m before being loaded on the machine, so that impurities in the water can be effectively removed, and the phenomenon that the impurities in the water sample block the chromatographic column is prevented, thereby shortening the service life of the chromatographic column; the invention discovers that under the condition of using a 0.22 mu m MCE filter membrane, the recovery rate range of 8 Microcystins (MCs) is 79-108%, the MCE filter membrane keeps the quantity of the MCs less than that of a PES filter membrane (polyether sulfone filter membrane) and a PTFE filter membrane (polytetrafluoroethylene filter membrane), so that the recovery rate of the MCs in a water sample is increased, meanwhile, the MCE filter membrane has similar recovery rates to the 8 microcystins, and the MCE filter membrane has the most stable influence on microcystin homologues, therefore, in the invention, the 0.22 mu m MCE filter membrane is selected to filter the water sample to obtain a pretreated water sample;
(2) performing online solid-phase extraction and analysis determination on the pretreated water sample by using an online solid-phase extraction ultra-performance liquid chromatography tandem mass spectrometry; in the invention, after the pretreated water sample is subjected to on-line solid-phase extraction by adopting a solid-phase extraction column, the analysis and the determination can be directly carried out by ultra-high performance liquid chromatography tandem mass spectrometry; wherein, the online solid-phase extraction adopts a C8 solid-phase extraction column; compared with a C18 solid-phase extraction column, the method can not elute MC-LA, MC-LY, MC-LW and MC-LF (4/8), and on the contrary, the method has an obviously better effect on separating 8 MCs by adopting the C8 solid-phase extraction column; when the analysis and determination are carried out, the conditions of the ultra-high performance liquid chromatography are as follows: elution was performed in a gradient with mobile phase: mobile phase: the solution A is 0.1% formic acid acetonitrile solution, and the solution B is 0.1% formic acid aqueous solution; gradient elution procedure: 0-4.6min, 2% of solution A and 98% of solution B; 4.6-10.0min, 2% of solution A-75% of solution A, 98% of solution B-25% of solution B, 10.0-11.0min, 75% of solution A-0% of solution A, and 25% of solution B-100% of solution B; the flow rate is 0.4 mL/min; column temperature 35 ℃, sample injection volume: 2 mL; in the present invention, a flow rate of 0.4mL/min means that the flow rates of both mobile phase A and mobile phase B are 0.4 mL/min; the invention discovers that the selection of the mobile phase system and the gradient elution program in the invention can simultaneously ensure that 8 MCs can be well separated, the response value of each substance is similar, the base line is stable during analysis and test, and the accuracy is high. In particular, the percentage contents involved in the mobile phase and the gradient elution procedure of the invention are volume percentage contents; in the present invention, the 0.1% formic acid acetonitrile solution (formic acid to acetonitrile volume ratio: 0.1:99.9) refers to an acetonitrile solution containing 0.1% by volume fraction formic acid, and the 0.1% formic acid aqueous solution (formic acid to pure water volume ratio: 0.1:99.9) refers to an aqueous solution containing 0.1% by volume fraction formic acid.
The method can simultaneously detect 8 microcystins in the water body by an online solid-phase extraction ultra-high performance liquid chromatography tandem mass spectrometry, selects a Mixed Cellulose (MCE) filter membrane to carry out filtration pretreatment on a water sample, adopts an online solid-phase extraction method to carry out online enrichment, elution and concentration treatment on the water sample, and simultaneously selects a C8 solid-phase extraction column and an optimized mobile phase and mobile phase gradient elution condition to carry out rapid and high-sensitivity detection on the 8 microcystins in the water body; the method provided by the invention has the advantages of good chromatographic peak separation effect, low detection limit, low quantitative limit, high sensitivity, high accuracy, good recovery rate and reproducibility, and simple and rapid operation, and can be used for simultaneously measuring 8 MCs in the water body. Compared with the existing sample pretreatment means, the method disclosed by the invention does not need a pretreatment step of manually carrying out solid-phase extraction on a water sample, namely a complex pretreatment step, so that the detection is quicker and more efficient, the on-machine detection time of one sample is 11 minutes, the method is economic, quick and efficient, and the requirement of quick analysis can be better met; the method has high recovery rate and good repeatability, 8 microcystins have good linear relation in the range of 10ng/L-1000ng/L, the correlation coefficients are all more than 0.99, the detection Limit (LOD) of the 8 microcystins is 0.02-0.37ng/L, the quantitative Limit (LOQ) is 0.07-1.24ng/L and is far lower than the limit standard of the microcystins in the water body in China, and trace MCs in a water environment sample can be detected; the method overcomes the defects of complex pretreatment steps, time consumption, poor sample repeatability and high analysis cost of a manual solid phase extraction method, effectively reduces the interference of the surrounding environment in a direct sample injection method, and provides a scientific analysis method for comprehensively understanding the pollution conditions of various MCs in eutrophicated water in China.
According to some preferred embodiments, a C8 solid phase extraction column with a particle size of 10 μm, an internal diameter of 2.1mm and a column length of 30mm is used
Direct Connect HP solid phase extraction column (also abbreviated as
column)。
According to some preferred embodiments, the ultra performance liquid chromatography uses an ACQUITY UPLC BEH C18 column with a particle size of 1.6 μm, an inner diameter of 2.1mm and a column length of 50mm when performing analytical measurements.
According to some preferred embodiments, in performing the analytical determination, the mass spectrometry conditions are: a triple quadrupole mass spectrometer is used, and an electrospray ion source, a positive ion scanning mode and a multi-reaction monitoring mode are adopted; capillary voltage: 3.70 kV; ion source temperature: 150 ℃; desolventizing temperature: 500 ℃; desolventizing agent gas flow: 1000L/h; taper hole gas flow: 50L/h; taper hole air pressure: 30V; collision gas flow rate: 0.06 mL/min.
According to some preferred embodiments, the method is capable of detecting 8 microcystins in water simultaneously; the 8 kinds of microcystins are MC-LR (microcystin-LR), MC-YR (microcystin-YR), MC-RR (microcystin-RR), MC-WR (microcystin-WR), MC-LA (microcystin-LA), MC-LF (microcystin-LF), MC-LY (microcystin-LY) and MC-LW (microcystin-LW).
According to some preferred embodiments, the mass spectrometric parameters of the method for detecting 8 microcystins are: MC-LR, parent ion 995.4, qualitative ion 135, quantitative ion 213, cone aperture voltage 85V, and collision energy 75 eV; MC-YR, parent ion 1045.6, qualitative ion 135, quantitative ion 213, cone hole voltage 85V, and collision energy 85 eV; MC-RR, parent ion 519.7, qualitative ion 135, quantitative ion 440.4, cone hole voltage 42V, collision energy 32 eV; MC-WR, parent ion 1068.5, qualitative ion 135, quantitative ion 213, taper hole voltage 60V, and collision energy 95 eV; MC-LA, parent ion 910, qualitative ion 135, quantitative ion 776, cone hole voltage 40V, and collision energy 80 eV; MC-LF, parent ion 986.3, qualitative ion 135, quantitative ion 213, cone hole voltage 42V, collision energy 68 eV; MC-LY, parent ion 1002.6, qualitative ion 135, quantitative ion 985, cone voltage 45V, collision energy 80 eV; MC-LW, parent ion 1025.5, qualitative ion 135, quantitative ion 213, cone-hole voltage 45V, collision energy 65eV, see Table 1.
Table 1: mass spectrometric parameters of 8 microcystins.
According to some preferred embodiments, before step (2), the method further comprises the step of adding a 1 μ g/L leucine enkephalin methanol solution (i.e. leucine enkephalin internal standard solution) as an internal standard solution into the pretreated water sample obtained in step (1); the leucine enkephalin methanol solution adopts chromatographically pure methanol as a solvent.
According to some preferred embodiments, before step (2), the method further comprises the step of performing online solid phase extraction and analytical determination on the mixed standard solution containing 8 microcystins simultaneously by online solid phase extraction ultra performance liquid chromatography-tandem mass spectrometry to draw a standard curve.
According to some preferred embodiments, 8 single standard solutions of microcystin are used to prepare 7 mixed standard solutions with different concentrations of 10ng/L, 20ng/L, 50ng/L, 100ng/L, 200ng/L, 500ng/L and 1000ng/L and simultaneously containing 8 microcystins for drawing a standard curve.
In the present invention, the standard curve is obtained by:
preparation of standard stock solutions: preparing 8 methanol single standard stock solutions (namely 8 microcystin single standard solutions) with the concentration of 1.0mg/L (MC-RR, MC-LR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY and MC-LW), storing at-20 deg.C in the dark, and taking; when 8 kinds of microcystin single-standard solution is prepared, a proper amount of 8 kinds of microcystin standard substances are diluted by methanol (chromatographic pure methanol), and are stored in the dark at the temperature of minus 20 ℃; when 8 single standard solutions of the microcystin are used, the single standard solutions are diluted to required mass concentration by using chromatographic pure methanol.
8 single-standard solutions of microcystin are prepared into mixed standard solutions with the concentrations of 10ng/L, 20ng/L, 50ng/L, 100ng/L, 200ng/L, 500ng/L and 1000ng/L by using chromatographic pure methanol, and the mixed standard solutions simultaneously contain 7 mixed standard solutions of 8 microcystin with different concentrations, are used for drawing a standard curve and are stored in a dark place at the temperature of minus 20 ℃, and the concentration of each microcystin contained in the mixed standard solution with each concentration is the same.
After the instrument is stabilized, carrying out online solid-phase extraction and analytical determination on 7 mixed standard solutions with different concentrations and containing 8 microcystins by using an online solid-phase extraction ultra-high performance liquid chromatography tandem mass spectrometry to obtain mass spectrograms under each concentration, and drawing a standard curve by using a chromatographic peak area (Y) as a vertical coordinate and a concentration (X) as a horizontal coordinate; the linear relation, the correlation coefficient (R) and the correlation coefficient (R) of the standard curve of 8 kinds of microcystins determined by the invention2) As shown in table 2.
Table 2: linear relation of standard curve of standard solution of 8 kinds of microcystin, correlation coefficient (R) and correlation coefficient (R)2)。
According to some preferred embodiments, the method has a linear relationship in the range of 10ng/L to 1000ng/L with correlation coefficients of > 0.99 when detecting microcystins in water; the detection limit of the method for detecting the microcystins in the water is 0.02-0.37ng/L, and the quantification limit is 0.07-1.24 ng/L.
The invention will be further illustrated by way of example, but the scope of protection is not limited to these examples.
Example 1
Simultaneously detecting 8 microcystins in a water sample by an Online solid-phase extraction ultra-high performance liquid chromatography tandem mass spectrometry (Online-SPE UPLC-MS/MS).
Preparing a standard stock solution: preparing 1.0 mg/L8 kinds of methanol single standard stock solutions (MC-RR, MC-LR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, MC-LW), storing at 20 deg.C in dark place, and removing. The right amount of 8 microcystin standard substances is diluted by chromatographic pure methanol, and when in use, the standard substances are diluted by the chromatographic pure methanol to the required mass concentration. Preparing leucine enkephalin internal standard solution with the concentration of 50.0 mu g/L by using chromatographic pure methanol, storing the internal standard solution at the temperature of below 20 ℃ below zero for use, and diluting the internal standard solution to the required mass concentration by using the chromatographic pure methanol when the internal standard solution is used.
Preparing mixed standard solutions with the concentrations of 10ng/L, 20ng/L, 50ng/L, 100ng/L, 200ng/L, 500ng/L and 1000ng/L and containing 7 different concentrations of 8 microcystins by using 8 single standard solutions of the microcystins and using chromatographic pure methanol for drawing a standard curve, storing the standard solutions at minus 20 ℃ in a dark place, wherein the concentration of each microcystin contained in each mixed standard solution with each concentration is the same; and when the mixed standard solution is subjected to online solid-phase extraction and analysis and measurement, leucine enkephalin internal standard solution is also added, and the concentration of the added leucine enkephalin internal standard solution is 1 mu g/L.
Ultra-high performance liquid phase by on-line solid phase extractionPerforming online solid phase extraction and analytical determination on 7 mixed standard solutions with different concentrations and containing 8 microcystins by using a chromatographic tandem mass spectrometry to obtain mass spectrograms under each concentration, and drawing a standard curve by using a chromatographic peak area (Y) as a vertical coordinate and a concentration (X) as a horizontal coordinate; the linear relation, the correlation coefficient (R) and the correlation coefficient (R) of the standard curve of 8 kinds of microcystins determined by the invention2) As shown in table 2.
Secondly, pretreatment of the water sample: taking 20 ml of water sample, filtering the water sample through an MCE filter membrane of 0.22 mu m, adding 1 mu g/L leucine enkephalin internal standard solution, and waiting for feeding the sample.
③ the on-line solid phase extraction column is
Direct Connect HP (10 μm, 2.1 mm. times.30 mm) (abbreviated as
A solid phase extraction column); the conditions of the ultra-high performance liquid chromatography are as follows: microcystins in the sample were separated by an ACQUITY UPLC BEH C18(1.6 μm, 2.1 mm. times.50 mm) chromatography column, elution being carried out in a gradient fashion with a binary pump mobile phase: the solution A is 0.1% formic acid acetonitrile solution, and the solution B is 0.1% formic acid aqueous solution; gradient elution procedure: 0-4.6min, 2% of solution A and 98% of solution B; 4.6-10.0min, 2% of solution A-75% of solution A, 98% of solution B-25% of solution B, 10.0-11.0min, 75% of solution A-0% of solution A, and 25% of solution B-100% of solution B; the flow rate is 0.4 mL/min; column temperature 35 ℃, sample injection volume: 2 mL; the gradient elution procedure in this example is shown in table 3.
The tandem mass spectrum conditions are as follows: using a triple quadrupole mass spectrometer, electrospray ion source (ESI), positive ion scanning, Multiple Reaction Monitoring (MRM) mode; capillary voltage: 3.70 kV; ion source temperature: 150 ℃; desolventizing temperature: 500 ℃; desolventizing agent gas flow: 1000L/h; taper hole gas flow: 50L/h; taper hole air pressure: 30V; collision gas flow rate: 0.06 mL/min; the mass spectrometric parameters of the 8 microcystins are shown in table 1.
Table 3: gradient elution procedure of example 1.
| Time (min) | Solution A (%) | Solution B (%) |
| 0 | 2％ | 98％ |
| 4.6 | 2％ | 98％ |
| 10.0 | 75％ | 25％ |
| 11.0 | 0％ | 100％ |
The chromatogram for detecting 8 microcystins in a water sample in this example is shown in fig. 1, and it can be seen from fig. 1 that the method of the present invention can separate 8 MCs well.
This example shows the retention times (min) and correlation coefficients (R) obtained by detecting 8 Microcystins (MCs)2) Linear range (ng/L), signal to noise ratio (S/N), limit of detection (LOD), limit of quantitation (LOQ), accuracy (%) and precision (%), as shown in Table 4; wherein, the detection limit and the quantification limit of the detection of the present embodiment are calculated by 3 times and 10 times of signal-to-noise ratio (S/N).
Table 4: this example measures the retention times obtained by 8 Microcystins (MCs)(min), correlation coefficient (R)2) Linear range (ng/L), signal to noise ratio (S/N), limit of detection (LOD), limit of quantitation (LOQ), accuracy (%), and precision (%).
From the results in Table 4, it can be seen that 8 kinds of microcystins have a good linear relationship in the range of 10ng/L-1000ng/L when detected by the method of the present invention, and the correlation coefficients are all greater than 0.99. The detection Limit (LOD) of 8 kinds of microcystins calculated by 3 times and 10 times of signal-to-noise ratio (S/N) is 0.02-0.37ng/L, and the quantification Limit (LOQ) is 0.07-1.24ng/L which are all lower than the standard value 1 mu g/L specified in GB 3838-2002; where the accuracy and precision in table 4 were obtained by a spiking recovery experiment.
Example 2
Respectively measuring 20 milliliters of 24 parts of ultrapure water samples, wherein 6 parts of ultrapure water samples are used as sample blanks, adding a microcystin mixed standard solution into the other 18 parts of samples, respectively making 3 adding levels, respectively measuring the total concentration of 8 added microcystins as 50ng/L, 100ng/L and 500ng/L, and parallelly measuring each adding level for 6 times; wherein the microcystin mixed standard solution is a mixed standard solution containing 8 MCs at the same time, and during preparation, 1.0mg/L methanol single standard stock solution (MC-RR, MC-LR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, MC-LW) is prepared, and is stored and removed in dark at the temperature of 20 ℃. Mixing 100 μ L of 1.0mg/L single standard stock solution and 200 μ L of methanol to obtain 100 μ g/L mixed standard solution, and diluting the rest of each solution with 50ng/L, 100ng/L and 500ng/L methanol (chromatographic pure methanol) to desired mass concentration.
Filtering through MCE filter membrane of 0.22 mu m, adding leucine enkephalin internal standard liquid of 1 mu g/L, and waiting for the determination of Online-SPE UPLC-MS/MS.
③ the on-line solid phase extraction column is
Direct Connect HP (10 μm, 2.1 mm. times.30 mm); the conditions of the ultra-high performance liquid chromatography are as follows: by ACQUITY UPLC BEH C18(1.6 μm, 2.1 mm. times.50 mm)Separating microcystin in the sample by a chromatographic column, eluting by a mobile phase in a gradient mode, and mixing the mobile phase with a binary pump: the solution A is 0.1% formic acid acetonitrile solution, and the solution B is 0.1% formic acid aqueous solution; gradient elution procedure: 0-4.6min, 2% of solution A and 98% of solution B; 4.6-10.0min, 2% of solution A-75% of solution A, 98% of solution B-25% of solution B, 10.0-11.0min, 75% of solution A-0% of solution A, and 25% of solution B-100% of solution B; the flow rate is 0.4 mL/min; column temperature 35 ℃, sample injection volume: 2 mL; the gradient elution procedure in this example is shown in table 3.
The tandem mass spectrum conditions are as follows: using a triple quadrupole mass spectrometer, electrospray ion source (ESI), positive ion scanning, Multiple Reaction Monitoring (MRM) mode; capillary voltage: 3.70 kV; ion source temperature: 150 ℃; desolventizing temperature: 500 ℃; desolventizing agent gas flow: 1000L/h; taper hole gas flow: 50L/h; taper hole air pressure: 30V; collision gas flow rate: 0.06 mL/min; the mass spectrometric parameters of the 8 microcystins are shown in table 1.
In this example, after the detection of each sample according to the above-described steps (i) to (iv), the average recovery (%) and the relative standard deviation (RSD,%) were calculated as shown in table 5.
Table 5: example 2 the blank sample was normalized to recover the experimental results (n ═ 6).
Example 3
Example 3 is essentially the same as example 1, except that:
in the pretreatment of a water sample, three groups of experiments are carried out by adopting three filter membranes, and other experimental conditions of each group of experiments are the same as those of the example 1; the three filters are respectively a 0.22 μm polyethersulfone filter (PES filter), a 0.22 μm polytetrafluoroethylene filter (PTFE) filter and a 0.22 μm mixed cellulose ester filter (MCE filter).
In this example, the recovery rates of 8 microcystins under 3 different filter membrane use conditions are shown in FIG. 2, and the chromatograms of 8 microcystins under 3 different filter membrane use conditions are shown in FIG. 3.
As can be seen from FIG. 2, the recovery rate of 8 MCs with the MCE filter membrane is in the range of 79-108%, which meets the EPA standard of 70-130%, and from the recovery rate results in FIG. 2, the MCE filter membrane retains a smaller amount of MCs than the PES filter membrane and the PTFE filter membrane, and the similar recovery rates of 8 microcystins indicate that the MCE filter membrane has the most stable effect on the homologues of microcystins; the chromatogram of fig. 3 shows that the separation of 8 MCs was more effective when the MCE filter was used than when PES filter and PTFE filter were used.
Example 4
Example 4 is essentially the same as example 1, except that:
two different online solid phase extraction columns are adopted for solid phase extraction, and other detection conditions are the same as those in the example 1; the two solid phase extraction columns are respectively columns
Direct Connect HP (abbreviated as
A solid-phase extraction column),
C18 solid phase extraction column.
This example compares the peak separation effect of the substances using two solid phase extraction columns, as shown in FIG. 4; as can be seen from the view of figure 4,
the C18 solid phase extraction column was unable to elute MC-LA, LY, LW and LF (4/8), but instead,
the solid phase extraction column has good effect of separating 8 MCs.
According to the degree of separationFormula (I) calculation yields
The separation degree of each chromatographic peak after solid-phase extraction of the solid-phase extraction column is determined by analysis and measurement as follows: 2.47, 0.73, 1.13, 5.43, 1.11, 4.36, 0.83. The two peaks of MC-YR and MC-LR, MC-LF and MC-LW are slightly overlapped, and the separation degree between the peaks of other adjacent substances>1, description of the invention
The solid phase extraction column has good separation effect.
t2: retention time of the latter of two adjacent peaks;
t1: retention time of the previous peak in two adjacent peaks;
w1，w2: the width of two adjacent peaks.
Example 5
This example compares methanol (chromatographically pure methanol) and acetonitrile as the organic phases of the mobile phase and performs 6-fold comparative experiments with formic acid as the buffer solutions in amounts of 0.1%, 0.25%, 0.5% by volume, respectively:
experimental group (a): the mobile phase is selected from: the liquid A is methanol, and the liquid B is water; experimental group (b): the mobile phase is selected from: the liquid A is acetonitrile, and the liquid B is water; experimental group (c): the mobile phase is selected from: the solution A is 0.1% formic acid methanol solution (methanol solution containing 0.1% formic acid by volume fraction), and the solution B is 0.1% formic acid aqueous solution; experimental group (d): the mobile phase is selected from: the solution A is 0.1% formic acid acetonitrile solution, and the solution B is 0.1% formic acid aqueous solution; experimental group (e): the mobile phase is selected from: the solution A is 0.25% formic acid acetonitrile solution, and the solution B is 0.1% formic acid aqueous solution; experimental group (f): the mobile phase is selected from: the solution A is 0.5% formic acid acetonitrile solution, and the solution B is 0.1% formic acid aqueous solution. Other detection conditions of the 6-group comparison experiment were the same as those of example 1.
The chromatogram of 8 microcystins in this example under 6 different mobile phase systems is shown in FIG. 5. In the figure, (a) to (f) correspond to the results of the experimental groups (a) to (f), respectively.
From the results of FIG. 5, it is shown that under the conditions of the two mobile phases (c) and (d), 8 MCs can obtain better separation effect and the response value of each substance is similar, but when (c) methanol is used as the organic phase of the mobile phase, the peak time is later than that when acetonitrile is used as the organic phase, and the base line is unstable. Therefore, the optimized mobile phase system of the invention is as follows: 0.1% formic acid acetonitrile solution and 0.1% formic acid water solution are used as a mobile phase system.
Example 6
This example compares the peak appearance of 8 microcystins under 4 gradient elution procedures, and four comparative experiments were performed with four gradient elution procedures as follows:
experimental group (a): the gradient elution procedure was: 0-4.1min, 0% of solution A, and 100% of solution B; 4.1-7.0min, 0% solution A-100% solution A, 100% solution B-0% solution B; 7.0-11.0min, 100% A liquid-0% A liquid, 0% B liquid-100% B liquid;
experimental group (b): the gradient elution procedure was: 0-4.6min, 2% of solution A and 98% of solution B; 4.6-10.0min, 2% of solution A-75% of solution A, 98% of solution B-25% of solution B, 10.0-11.0min, 75% of solution A-0% of solution A, and 25% of solution B-100% of solution B;
experimental group (c): the gradient elution procedure was: 0-4.1min, 5% of solution A and 95% of solution B; 4.1-6.0min, 5% of solution A-40% of solution A, 95% of solution B-60% of solution B; 6.0-9.0min, 40% of solution A-95% of solution A, and 60% of solution B-5% of solution B; 9.0-11.0min, 95% solution A-5% solution A, 5% solution B-95% solution B;
experimental group (d): the gradient elution procedure was: 0-6.0min, 5% of solution A and 95% of solution B; 6.0-9.0min, 5% of solution A-95% of solution A, and 95% of solution B-5% of solution B; 9.0-11.0min, 95% of solution A-5% of solution A, and 5% of solution B-95% of solution B. The other test conditions for the four sets of comparative experiments were the same as in example 1.
The chromatogram of 8 microcystins from this example under four different gradient elution procedures is shown in FIG. 6; in the figure, (a) to (d) correspond to the results of the experimental groups (a) to (d), respectively.
As can be seen from the results in FIG. 6, the 8 MCs under the condition of the experimental group (a) do not give full peaks and are not completely separated; under the condition of the experimental group (b), 8 MCs have complete peaks and good separation degree; in both the experimental group (c) and the experimental group (d), only 4 MCs showed peaks, and the other 4 MCs showed no peaks or were not separated. The invention discovers that the optimized gradient elution program of the invention is as follows: 0-4.6min, 2% of solution A and 98% of solution B; 4.6-10.0min, 2% of solution A-75% of solution A, 98% of solution B-25% of solution B, 10.0-11.0min, 75% of solution A-0% of solution A, and 25% of solution B-100% of solution B.
The invention has not been described in detail and is in part known to those of skill in the art.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for detecting microcystin in water, said method comprising the steps of:
(1) filtering a water sample through a 0.22 mu m MCE filter membrane to obtain a pretreated water sample;
(2) performing online solid-phase extraction and analysis determination on the pretreated water sample by using an online solid-phase extraction ultra-performance liquid chromatography tandem mass spectrometry;
wherein, the online solid-phase extraction adopts a C8 solid-phase extraction column;
when the analysis and determination are carried out, the conditions of the ultra-high performance liquid chromatography are as follows: elution was performed in a gradient with mobile phase:
mobile phase: the solution A is 0.1% formic acid acetonitrile solution, and the solution B is 0.1% formic acid aqueous solution; gradient elution procedure: 0-4.6min, 2% of solution A and 98% of solution B; 4.6-10.0min, 2% of solution A-75% of solution A, 98% of solution B-25% of solution B, 10.0-11.0min, 75% of solution A-0% of solution A, and 25% of solution B-100% of solution B; the flow rate is 0.4 mL/min; column temperature 35 ℃, sample injection volume: 2 mL.
2. The method of claim 1, wherein:
the adopted C8 solid phase extraction column has particle diameter of 10 μm, inner diameter of 2.1mm, and column length of 30mm
C8 Direct Connect HP solid phase extraction column.
3. The method of claim 1, wherein:
for analytical determination, the column used for the ultra high performance liquid chromatography was an ACQUITY UPLC BEH C18 column having a particle size of 1.6 μm, an inner diameter of 2.1mm and a column length of 50 mm.
4. The method of claim 1, wherein:
in performing the analytical determination, the mass spectrometry conditions were: a triple quadrupole mass spectrometer is used, and an electrospray ion source, a positive ion scanning mode and a multi-reaction monitoring mode are adopted; capillary voltage: 3.70 kV; ion source temperature: 150 ℃; desolventizing temperature: 500 ℃; desolventizing agent gas flow: 1000L/h; taper hole gas flow: 50L/h; taper hole air pressure: 30V; collision gas flow rate: 0.06 mL/min.
5. The method of claim 1, wherein:
the method can simultaneously detect 8 kinds of microcystins in water;
the 8 kinds of microcystins are MC-LR, MC-YR, MC-RR, MC-WR, MC-LA, MC-LF, MC-LY and MC-LW.
6. The method of claim 5, wherein:
the mass spectrometric parameters of the method for detecting 8 microcystins are as follows:
MC-LR, parent ion 995.4, qualitative ion 135, quantitative ion 213, cone aperture voltage 85V, and collision energy 75 eV;
MC-YR, parent ion 1045.6, qualitative ion 135, quantitative ion 213, cone hole voltage 85V, and collision energy 85 eV;
MC-RR, parent ion 519.7, qualitative ion 135, quantitative ion 440.4, cone hole voltage 42V, collision energy 32 eV;
MC-WR, parent ion 1068.5, qualitative ion 135, quantitative ion 213, taper hole voltage 60V, and collision energy 95 eV;
MC-LA, parent ion 910, qualitative ion 135, quantitative ion 776, cone hole voltage 40V, and collision energy 80 eV;
MC-LF, parent ion 986.3, qualitative ion 135, quantitative ion 213, cone hole voltage 42V, collision energy 68 eV;
MC-LY, parent ion 1002.6, qualitative ion 135, quantitative ion 985, cone voltage 45V, collision energy 80 eV;
MC-LW, parent ion 1025.5, qualitative ion 135, quantitative ion 213, cone-hole voltage 45V, and collision energy 65 eV.
7. The method of claim 1, wherein:
before the step (2), the method also comprises the step of adding a leucine enkephalin methanol solution with the concentration of 1 mu g/L into the pretreated water sample obtained in the step (1) as an internal standard solution.
8. The method of claim 5, wherein:
before the step (2), the method also comprises the step of carrying out online solid-phase extraction and analysis measurement on the mixed standard solution containing 8 microcystins by using online solid-phase extraction ultra-performance liquid chromatography tandem mass spectrometry to draw a standard curve.
9. The method of claim 8, wherein:
8 kinds of microcystin single standard solutions are adopted to prepare 7 mixed standard solutions containing 8 kinds of microcystin with different concentrations, wherein the concentrations of the mixed standard solutions are respectively 10ng/L, 20ng/L, 50ng/L, 100ng/L, 200ng/L, 500ng/L and 1000ng/L, and the mixed standard solutions are used for drawing a standard curve.
10. The method according to any one of claims 1 to 9, characterized in that:
the method has a linear relation in the range of 10ng/L-1000ng/L when detecting the microcystins in water, and the correlation coefficients are all more than 0.99;
the detection limit of the method for detecting the microcystins in the water is 0.02-0.37ng/L, and the quantification limit is 0.07-1.24 ng/L.
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