CN103439445B - Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry the detection method of triphenylmethane and metabolin thereof in water body - Google Patents
Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry the detection method of triphenylmethane and metabolin thereof in water body Download PDFInfo
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Abstract
The invention belongs to environmental monitoring technology field, be specifically related to the detection method of triphenylmethane and metabolin Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry in a kind of water body.Yet there are no can detect MG, LMG, CV, LCV, BG five kinds of materials simultaneously and detect the detection method being limited to ng rank.The present invention adopts compound mass spectrum to be the novel mass spectrometer of a kind of advanced person, series connection quadrupole rod linear ion hydrazine technology, greatly improve MS/MS scan capability, series connection Ultra Performance Liquid Chromatography instrument simultaneously, a kind of simple, quick, sensitive method can be established, 5 kinds of triphenylmethanes and metabolin thereof in Simultaneously test water body, in further assay ecologic environment, pollution condition provides reliable detection method.
Description
Technical field
The invention belongs to environmental monitoring technology field, be specifically related to the detection method of triphenylmethane and metabolin Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry in a kind of water body.
Background technology
At present, domestic and foreign literature reports that the mensuration about triphenylmethane material adopts liquid phase chromatography (UV detect, fluoroscopic examination) or LC-MS technology more.Due to LMG, LCV in UV-visible region without absorption peak, and MG, CV unstressed configuration response, therefore adopt and often need by certain pre-treatment means during high-efficient liquid phase technique this few class material of Simultaneously test, as need through PbO when UV, visible light method measures
2post-column derivation, is oxidized to dominant material MG, CV by recessive material LMG, LCV, but derivative column packing proportioning is subject to the impact of mobile phase and sample substrate, there is the problems such as conversion ratio instability; And when using fluorescence detector to measure, by adding reductive agent in pretreatment process, MG, CV are reduced into LMG, LCV; For avoiding the loaded down with trivial details of pre-treatment oxide pillars or reductive agent, also utilize HLPC-UV-FLD series process, directly measure MG, CV and Metabolic residue thereof in aquaculture water, the method detects and is limited to 0.4 μ g/L.Yet there are no can detect MG, LMG, CV, LCV, BG five kinds of materials simultaneously and detect the detection method being limited to ng rank.
Summary of the invention
In order to solve above-mentioned technical matters, the invention provides the Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of triphenylmethane and metabolin thereof in a kind of water body.The present invention adopts compound mass spectrum to be the novel mass spectrometer of a kind of advanced person, series connection quadrupole rod linear ion hydrazine technology, greatly improve MS/MS scan capability, a kind of simple, quick, sensitive method can be established, 5 kinds of triphenylmethanes and metabolin thereof in Simultaneously test water body, in further assay ecologic environment, pollution condition provides reliable detection method.
The present invention is realized by following technical scheme:
Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of triphenylmethane and metabolin thereof in water body,
Equipment used by the method comprises Ultra Performance Liquid Chromatography instrument and triple quadrupole bar/compounded linear ion trap mass spectrometer,
Described Ultra Performance Liquid Chromatography instrument is ACQUITY UPLC, and chromatographic column is ACQUITY UPLCTM BEH C18, and chromatographic column specification is 2.1mm × 50mm, 1.7 μm; Column temperature is 50 DEG C; Flow velocity is 0.5mL/min, sampling volume 5 μ L; Mobile phase is: with 5mmol/L ammonium acetate solution for mobile phase A, and mobile phase A formic acid is adjusted to PH=4.5, and acetonitrile is Mobile phase B; Elution requirement is: adopt gradient elution mode, 0min, 20%B, keeps 0.5min; During 2.9min, linear increase, to 80%B, keeps 1min; Revert to 20%B after 4min, balance 1min terminates;
Described triple quadrupole bar/compounded linear ion trap mass spectrometer is AB SCIEX QTRAP 5500 mass spectrometer, the ionized voltage of described mass spectrometer is 5000V, ion source temperature is 600 DEG C, gas curtain gas is 40pis, and spraying gas is 55pis, and auxiliary heating gas is 60psi, impinger is High, ion gun is electric spray ion source, and scan mode is positive ion scan mode, " bar-trap scanning " mode;
The method comprises the interior preparation section of mark mixed liquor, the preparation section of sample solution and upper machine testing operation,
The preparation section of described interior mark mixed liquor is as follows: interior mark mixed liquor configuration: get MG-D
5, LMG-D
6, CV-D
6and LCV-D
6, add acetontrile and become concentration to be MG-D
5500 μ g/L, LMG-D
6100 μ g/L, CV-D
6100 μ g/L and LCV-D
6the mixed solution of 500 μ g/L, for subsequent use as interior mark mixed liquor;
The preparation section of described sample solution is as follows: get the solid-phase extraction column activated, measure 100mL water sample, add 2mL10% hydroxylamine hydrochloride solution, 1mL 1.0mol/L p-toluenesulfonic acid solution, add mark mixed solution in 10 μ L again, after shaking up, with the flow velocity of 1mL/min, water sample is passed through solid-phase extraction column, with the Synthesis of diaminodiphenyl in enrichment water sample, after loading, use 3mL acetone, 5mL acetonitrile, the removal of impurities of 5mL water wash solid-phase extraction column successively, purge solid-phase extraction column with nitrogen, remove the moisture in solid-phase extraction column; Finally use ammoniacal liquor-acetonitrile solution wash-out solid-phase extraction column, collect eluent; Eluent is placed in nitrogen and blows concentrating instrument, blow to dry to the greatest extent, be settled to 1.0mL with acetonitrile, vortex oscillation mixes, and miillpore filter filters, and gets filtrate, obtains sample solutions;
Described upper machine testing operation is as follows: sample solution is placed in sample injection bottle, and upper machine is detected by Ultra Performance Liquid Chromatography-mass spectrometer, measurement result, the detection of the method is limited to MG2.18ng/L, LMG1.19ng/L, CV1.07ng/L, LCV0.97ng/L, BG0.42ng/L, quantitative limit MG8.73ng/L, LMG4.75ng/L, CV4.26ng/L, LCV3.90ng/L, BG1.68ng/L.
In the Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of above-mentioned triphenylmethane and metabolin thereof, the water in mobile phase 5mmol/L ammonium acetate solution is Millipore ultrapure water.
In the Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of above-mentioned triphenylmethane and metabolin thereof, described solid-phase extraction column is Oasis MCX 150mg/6cc.
In the Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of above-mentioned triphenylmethane and metabolin thereof, the activation method of described MCX solid-phase extraction column is respectively with 5mL acetonitrile, 5mL 5% aqueous formic acid activation MCX solid-phase extraction column 30min.
Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear the ion trap mass spectrometry of above-mentioned triphenylmethane and metabolin thereof detects
In method, described nitrogen purge time is 20min.
In the Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of above-mentioned triphenylmethane and metabolin thereof, the volume ratio of described ammoniacal liquor-acetonitrile is 5:95.
In the Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of above-mentioned triphenylmethane and metabolin thereof, described ammoniacal liquor-acetonitrile solution wash-out consumption is 10ml.
In the Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of above-mentioned triphenylmethane and metabolin thereof, described miillpore filter is 0.22 μm of PVDF filter membrane.
The present invention has carried out choice experiment to Mass Spectrometry Conditions, according to the chemical property of MG, LMG, CV, LCV, BG and corresponding Isotopic Internal Standard thing thereof, adopts " bar-trap scanning " (MRM-IDA-EPI) pattern, selects [M+H]
-for parent ion, then with suitable collision energy, second order ms scanning is carried out to it, find daughter fragment ion.Quasi-molecular ions responds the strongest daughter ion as quota ion, responds relatively strong daughter ion as qualitative ion.Detail parameters is in table 1.
Triple quadrupole bar/compounded linear ion trap mass spectrometry, had both retained the series connection better selectivity of quadrupole rod and sensitivity, also can make linear ion hydrazine and strengthen secondary fragment ion qualitative function." bar-trap scanning " (MRM-IDA-EPI) pattern, single injected sampling can obtain for quantitative MRM chromatogram and for MS qualitatively
2mass spectrogram, compared with common triple quadrupole bar scan pattern, enhances the scanning of secondary fragment daughter ion, greatly improves the scan capability of MS/MS, be more conducive to microanalysis, increase the qualitative ability of target compound in complex dielectrics.MG illustrates, and compares EPI scan pattern and MS/MS daughter ion scan pattern contrast, and at the MG of same concentrations 10 μ g/L under EPI scan pattern, fragmention responds nearly 100 times of enhancing.
Because Synthesis of diaminodiphenyl belongs to polar molecule, select ESI ion gun compared to APCI ion gun, Ionization Efficiency is higher.MG, CV, BG have ammonium group, and LMG, LCV have dimethylamino, and therefore ionization adopts positive ion ESI source.
The present invention has carried out choice experiment target compound triphenylmethane material to chromatographic condition mobile phase and has belonged to alkalescent material, in Ultra Performance Liquid Chromatography-mass spectrometry, for positive ionization electrospray scan pattern, mobile phase adds appropriate ammonium acetate under weak acid environment condition can improve determinand Ionization Efficiency.When adding ammonium acetate and formic acid in aqueous phase simultaneously, be conducive to the molecular ionization of target analytes, the chromatographic peak peak shape of target compound is greatly improved.This experiment has investigated mobile phase acetonitrile-5mmol/L ammonium acetate (respectively containing 0.01%, 0.02%, 0.05%, 0.10% and 0.20% formic acid) to the impact of flowing relative target compound.Result shows, when formic acid content is higher, the silanol base of target compound and C18 post reacts, and peak shape is wider and with hangover, degree of separation is also very poor.PH value is lower, especially larger on the impact of the peak shape of LCV, chromatographic peak respond weaken, peak shape broadens hangover.When acid concentration lower than 0.02% time, on peak shape without impact.Consider according to composite factors such as peak shape, response intensity, degree of separation, finally select acetonitrile-5mmol/L ammonium acetate (0.02% formic acid) aqueous solution to make mobile phase.
The present invention has carried out choice experiment to internal standard compound, this experiment finds, triphenylmethane material is very strict to the requirement of internal standard compound, due to some characteristics such as cracking in the instability of triphenylmethane material itself and source, select different interior marks, remarkable to the linear effect of each object typical curve, list in table 1 under the identical range of linearity, select the linearly dependent coefficient that different internal standard compound obtains.Result is visible, selects and self structure and the similar as far as possible interior mark of character, can well correct the error problem because of cracking generation in unstable and source.
Table 1 compound selects the linearly dependent coefficient (n=5) of different internal standard compound
| MG-D 5 | LMG-D 6 | CV-D 6 | LCV-D 6 | |
| MG | 0.9993 | 0.9693 | 0.9857 | 0.9700 |
| LMG | 0.9915 | 0.9986 | 0.9287 | 0.9856 |
| CV | 0.9807 | 0.9674 | 0.9991 | 0.9882 |
| LCV | 0.9850 | 0.9821 | 0.9644 | 0.9991 |
| BG | 0.9988 | 0.9632 | 0.9965 | 0.9640 |
The condition of the present invention to Solid-Phase Extraction pre-treatment is optimized selection,
The test findings of the selection of Solid-Phase Extraction is as follows: compare C18, HLB post and MCX post to the extraction efficiency of triphenylmethane material, result shows that MCX has good extraction efficiency to triphenylmethane material, and when adopting C18, HLB post to extract, the recovery of five kinds of materials is between 10%-50%, relatively low.Because triphenylmethane material belongs to alkali ion type compound, the enrichment that cation exchange column is applicable to alkali ion type compound be separated, this and experimental result match.
The test findings of the activation of solid-phase extraction column is as follows: triphenylmethane material easily forms kation in acid condition.According to cationic solid phases extraction column loading condition, sample solution pH value is less than its pKa Liang Ge unit, ensures that load solution is positively charged.In experiment, for solid-phase extraction column activated solution, whether acidifying is investigated, and after acetonitrile activation, compares pure water activation and sour water activation.If select acetonitrile, pure water activates, and for dominant material MG, CV, BG recovery about 65%, and recessive material LMG, LCV recovery is lower than 50%.And the activated solution of solid-phase extraction column is with after acid solution acidifying, extraction efficiency is clearly better, and the recovery reaches more than 80%.So cation exchange column adopts acid solution activation, loading water sample regulates pH to be acid, is conducive to the enrichment of cation exchange column to target compound.Triphenylmethane material polarity is comparatively large, water sample ion-pairing agent p-toluenesulfonic acid acidifying, is conducive to triphenylmethane material and forms ion pair, increase extraction efficiency.MG, LMG, CV, LCV, BG are to light, heat, oxygen sensitive simultaneously, easily methylate, be oxidized, so, add reductive agent oxammonium hydrochloride when sample extraction, be conducive to the degraded preventing determinand.
The Optimum Experiment result of eluting solvent is as follows:
For the electric charge of neutralization analysis thing, the elute soln pH value of cation exchange column is greater than its pKa Liang Ge unit, so eluting solvent adopts alkaline solution wash-out.On the basis of some documents, this experiment compares the elute effect (see Fig. 1) of ammoniacal liquor-acetonitrile (5:95), ammonia water-methanol (5:95), ammoniacal liquor-methanol-ethyl acetate (5:45:50) 3 kinds of eluents.Ammoniacal liquor-acetonitrile (5:95) solution recovery is fine; Ammonia water-methanol (5:95) to most of substance recovery lower than 40%; Ammoniacal liquor-methanol-ethyl acetate (5:45:50) is comparatively large to BG loss, the recovery only 30%.Final eluting solvent selects ammoniacal liquor-acetonitrile (5:95).The Optimum Experiment of eluting solvent the results are shown in Figure 1.
In water body of the present invention, the typical curve of the Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of triphenylmethane and metabolin thereof is as follows:
With the peak area of each component (Y) to concentration (X, μ g/L) drawing standard curve, MG, LCV, BG range of linearity 0.02-20.0 μ g/L, LMG, CV range of linearity 0.02-5.0 μ g/L, mass concentration is good linear relationship with peak area in the range of linearity, and r is greater than more than 0.998.The linear relationship of triphenylmethane material, related coefficient is listed in table 2.
Linear relationship, the related coefficient of table 2 triphenylmethane material
In water body of the present invention the detection limit of the Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of triphenylmethane and metabolin thereof and quantitative limit as follows: the detection limit assay method not detecting target substance according to National Environmental standard blank assay, the concentration that this experiment measures using the water sample concentration of 5.0ng/L as detection limit, prepare 7 parts of parallel water samples, volume of water sample is 100mL.Through whole process analysis, calculate the standard deviation S of its concentration.Take 3.14S as detection limit (LOD), 4 times detect and are limited to lower limit of quantitation (LOQ), detecting of 5 kinds of materials is limited to 0.32 ~ 2.18ng/L, and (see table 3) method has higher sensitivity, and the analysis meeting triphenylmethane in water body completely measures.
Table 3 method detection limit result of calculation (n=7)
In water body of the present invention the precision of the Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of triphenylmethane and metabolin thereof and the recovery as follows:
Method adopts blank water sample mark-on mode, and the blank mark-on water sample being respectively 5.0ng/L, 10.0ng/L, 20.0ng/L at concentration level carries out determination of recovery rates, and each mark-on water sample configured in parallel 6 increment product, its recovery of standard addition and precision the results are shown in Table 4.
Triphenylmethane recovery of standard addition and relative standard deviation (n=6) in the blank water sample of table 4
Beneficial effect of the present invention is:
(1) the present invention adopts compound mass spectrum to be the novel mass spectrometer of a kind of advanced person, series connection quadrupole rod linear ion hydrazine technology, greatly improve MS/MS scan capability, series connection Ultra Performance Liquid Chromatography instrument simultaneously, a kind of simple, quick, sensitive method can be established, 5 kinds of triphenylmethanes and metabolin thereof in Simultaneously test water body, in further assay ecologic environment, pollution condition provides reliable detection method.
(2) triple quadrupole bar/compounded linear ion trap mass spectrometry, had both retained the series connection better selectivity of quadrupole rod and sensitivity, also can make linear ion hydrazine and strengthen secondary fragment ion qualitative function." bar-trap scanning " (MRM-IDA-EPI) pattern, single injected sampling can obtain for quantitative MRM chromatogram and for MS qualitatively
2mass spectrogram, compared with common triple quadrupole bar scan pattern, enhances the scanning of secondary fragment daughter ion, greatly improves the scan capability of MS/MS, be more conducive to microanalysis, increase the qualitative ability of target compound in complex dielectrics.
(3) in EU member country Ireland regulation cultivation discharge water, MG content must not more than 100 μ g/L, must not more than 1.0 μ g/L in potable water, and detecting of this method is limited to 0.42 ~ 2.18ng/L, far below the restriction required, meet environment supervision demand completely.
Accompanying drawing explanation
The recovery of the different eluting solvent of Fig. 1.
Embodiment
Below in conjunction with specific embodiment, the present invention is further described, so that those skilled in the art more understands the present invention, but does not therefore limit the present invention.
Embodiment 1
Instrument and equipment used by the present embodiment has:
Ultra Performance Liquid Chromatography ACQUITY UPLC (Waters, US); AB SCIEX QTRAP 5500 mass spectrometer (American AB company); Solid-phase extracting instrument (Supelco company of the U.S.); Solid-phase extraction column Oasis MCX 150mg/6cc (Waters, US), nitrogen blows concentrating instrument (Zymark company of the U.S.); 0.22 μm of polyvinylidene fluoride (PVDF) film syringe-driven filter (Millipore company of the U.S.); Milli-Q Superpure water machine (Millipore company of the U.S.); Turbine mixer (Japanese LMS company).
Reagent used by the present embodiment has:
Standard items malachite green (MG, purity 98.0%), recessive peacock stone (LMG, purity 97.0%), crystal violet (CV, purity 92.5%), Recessive Crystal Violet (LCV, purity 99.0%), BG (BG, purity 96.0%), deuterated recessive peacock stone (LMG-D6, purity 98.5%), deuterated crystal violet (CV-D6, purity 99.0%) is German Dr.Ehrenstorfer company reagent; Deuterated malachite green (MG-D5, purity 99.7%) and deuterated Recessive Crystal Violet (LCV-D6, purity 99.8%) are German Witega company reagent.
Acetonitrile, methyl alcohol, formic acid, acetic acid, isopropyl alcohol, ammonium acetate, isopropyl alcohol, ammonium acetate (chromatographically pure, German Merck reagent); Oxammonium hydrochloride, p-toluenesulfonic acid (purity 99%+, Adamas company of Switzerland); Experimental water Millipore ultrapure water (TOC:<1ppb).
Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of triphenylmethane and metabolin thereof in water body,
Equipment used by the method comprises Ultra Performance Liquid Chromatography instrument and triple quadrupole bar/compounded linear ion trap mass spectrometer,
Described Ultra Performance Liquid Chromatography instrument is ACQUITY UPLC, and chromatographic column is ACQUITY UPLCTM BEH C18, and chromatographic column specification is 2.1mm × 50mm, 1.7 μm; Column temperature is 50 DEG C; Flow velocity is 0.5mL/min, sampling volume 5 μ L; Mobile phase is: with 5mmol/L ammonium acetate solution for mobile phase A, and mobile phase A formic acid is adjusted to PH=4.5, and acetonitrile is Mobile phase B; Elution requirement is: adopt gradient elution mode, 0min, 20%B, keeps 0.5min; During 2.9min, linear increase, to 80%B, keeps 1min; Revert to 20%B after 4min, balance 1min terminates;
Described triple quadrupole bar/compounded linear ion trap mass spectrometer is AB SCIEX QTRAP 5500 mass spectrometer, the ionized voltage of described mass spectrometer is 5000V, ion source temperature is 600 DEG C, gas curtain gas is 40pis, and spraying gas is 55pis, and auxiliary heating gas is 60psi, impinger is High, ion gun is electric spray ion source, and scan mode is positive ion scan mode, " bar-trap scanning " mode;
The method comprises the interior preparation section of mark mixed liquor, the preparation section of sample solution and upper machine testing operation,
The preparation section of described interior mark mixed liquor is as follows: interior mark mixed liquor configuration: get MG-D
550mg, LMG-D
610mg, CV-D
610mg and LCV-D
650mg, puts in 100ml volumetric flask, add acetonitrile dissolve and constant volume to be mixed with concentration be MG-D
5500 μ g/L, LMG-D
6100 μ g/L, CV-D
6100 μ g/L and LCV-D
6the mixed solution of 500 μ g/L, for subsequent use as interior mark mixed liquor;
The preparation section of described sample solution is as follows: get the solid-phase extraction column activated, measure 100mL water sample, add 2mL10% hydroxylamine hydrochloride solution, 1mL 1.0mol/L p-toluenesulfonic acid solution, add mark mixed solution in 10 μ L again, after shaking up, with the flow velocity of 1mL/min, water sample is passed through solid-phase extraction column, with the Synthesis of diaminodiphenyl in enrichment water sample, after loading, use 3mL acetone, 5mL acetonitrile, the removal of impurities of 5mL water wash solid-phase extraction column successively, purge solid-phase extraction column 20min with nitrogen, remove the moisture in solid-phase extraction column; Finally use 10ml ammoniacal liquor-acetonitrile (5:95) eluant solution solid-phase extraction column, collect eluent; Eluent is placed in nitrogen and blows concentrating instrument, blow to dry to the greatest extent, be settled to 1.0mL with acetonitrile, vortex oscillation mixes, and 0.22 μm of PVDF filter membrane miillpore filter filters, and gets filtrate, obtains sample solutions;
Described upper machine testing operation is as follows: sample solution is placed in sample injection bottle, and upper machine is detected by Ultra Performance Liquid Chromatography-mass spectrometer, measurement result.Triple quadrupole bar/compounded linear ion trap mass spectrometer measures with reference to Mass Spectrometry Conditions in table 5.
The polyion reaction monitoring of table 5 target compound and mass spectrometry parameters
* quota ion
The detection of the method is limited to MG2.18ng/L, LMG1.19ng/L, CV1.07ng/L, LCV0.97ng/L, BG0.42ng/L, quantitative limit MG8.73ng/L, LMG4.75ng/L, CV4.26ng/L, LCV3.90ng/L, BG1.68ng/L.
In the Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of above-mentioned triphenylmethane and metabolin thereof, the activation method of described MCX solid-phase extraction column is respectively with 5mL acetonitrile, 5mL 5% aqueous formic acid activation MCX solid-phase extraction column 30min.
Carry out analysis to the environmental water sample of 30 some positions, In Nanjing to measure.Result shows, the concentration near certain pond, chemical plant water sample MG is 3.64ng/L, and the concentration of certain the Changjiang river, chemical plant waste water discharge opening CV is that each material of other water head site of 2.11ng/L detects all lower than detection limit.By analyzing, the water sample of potable water source district does not all detect target compound, but the water sample near pollution source, affects by chemical plant etc., can detect wherein Cucumber.In EU member country's Ireland regulation cultivation discharge water, MG content must not more than 100 μ g/L, must not more than 1.0 μ g/L in potable water, and detecting of this method is limited to 0.42 ~ 2.18ng/L, far below the restriction required, meets environment supervision demand completely.In the situation of a large amount of sample analysis or aquaculture water too high levels, water sample, can direct injected after pH value regulates and filters, and detects and is limited to 26.7-40.8ng/L, also far below control limit.
Although the present invention with embodiment openly as above; but it is also not used to limit protection scope of the present invention; any technician being familiar with this technology, not departing from the change and retouching done in the spirit and scope of the present invention, all should belong to protection scope of the present invention.
Claims (1)
1. Ultra Performance Liquid Chromatography-triple quadrupole bar/compounded linear ion trap mass spectrometry detection method of water body Malachite Green, concealed malachite green, crystal violet, Recessive Crystal Violet and BG, is characterized in that,
Equipment used by the method comprises Ultra Performance Liquid Chromatography instrument and triple quadrupole bar/compounded linear ion trap mass spectrometer,
Described Ultra Performance Liquid Chromatography instrument is ACQUITY UPLC, and chromatographic column is ACQUITY UPLCTM BEH C18, and chromatographic column specification is 2.1 mm × 50 mm, 1.7 μm; Column temperature is 50 DEG C; Flow velocity is 0.5 mL/ min, sampling volume 5 μ L; Mobile phase is: with 5 mmol/L ammonium acetate solutions for mobile phase A, mobile phase A formic acid is adjusted to PH=4.5, and acetonitrile is Mobile phase B; Elution requirement is: adopt gradient elution mode, 0 min, 20% B, keep 0.5 min; During 2.9 min, linear increase to 80% B, keeps 1 min; Revert to 20% B after 4 min, balance 1 min and terminate;
Described triple quadrupole bar/compounded linear ion trap mass spectrometer is AB SCIEX QTRAP 5500 mass spectrometer, the ionized voltage of described mass spectrometer is 5000 V, ion source temperature is 600 DEG C, gas curtain gas is 40 pis, and spraying gas is 55 pis, and auxiliary heating gas is 60 psi, impinger is High, ion gun is electric spray ion source, and scan mode is positive ion scan mode, " bar-trap scanning " mode;
The method comprises the interior preparation section of mark mixed liquor, the preparation section of sample solution and upper machine testing operation,
The preparation section of described interior mark mixed liquor is as follows: interior mark mixed liquor configuration: get MG-D
5,lMG-D
6,cV-D
6and LCV-D
6, add acetontrile and become concentration to be MG-D
5500 μ g/L, LMG-D
6100 μ g/L, CV-D
6100 μ g/L and LCV-D
6the mixed solution of 500 μ g/L, for subsequent use as interior mark mixed liquor;
The preparation section of described sample solution is as follows: get the solid-phase extraction column activated, measure 100 mL water samples, add 2 mL 10% hydroxylamine hydrochloride solution, 1 mL 1.0 mol/L p-toluenesulfonic acid solution, add mark mixed solution in 10 μ L again, after shaking up, with the flow velocity of 1 mL/min, water sample is passed through solid-phase extraction column, with the Synthesis of diaminodiphenyl in enrichment water sample, after loading, use 3 mL acetone, 5 mL acetonitriles, 5 mL water wash solid-phase extraction column removal of impurities successively, purge solid-phase extraction column with nitrogen, remove the moisture in solid-phase extraction column; Finally use ammoniacal liquor-acetonitrile solution wash-out solid-phase extraction column, collect eluent; Eluent is placed in nitrogen and blows concentrating instrument, blow to dry to the greatest extent, be settled to 1.0 mL with acetonitrile, vortex oscillation mixes, and miillpore filter filters, and gets filtrate, obtains sample solutions;
Described upper machine testing operation is as follows: sample solution is placed in sample injection bottle, and upper machine is detected by Ultra Performance Liquid Chromatography-mass spectrometer, measurement result, the detection of the method is limited to MG2.18 ng/L, LMG1.19 ng/L, CV1.07 ng/L, LCV0.97 ng/L, BG0.42 ng/L, quantitative limit MG8.73 ng/L, LMG4.75ng/L, CV4.26 ng/L, LCV3.90 ng/L, BG1.68 ng/L;
Water in described mobile phase 5 mmol/L ammonium acetate solution is Millipore ultrapure water;
Described solid-phase extraction column is Oasis MCX 150 mg/ 6 cc;
The activation method of described MCX solid-phase extraction column is respectively with 5 mL acetonitriles, 5 mL 5% aqueous formic acid activation MCX solid-phase extraction column 30min;
Described nitrogen purge time is 20min;
The volume ratio of described ammoniacal liquor-acetonitrile is 5:95;
Described ammoniacal liquor-acetonitrile solution wash-out consumption is 10ml;
Described miillpore filter is 0.22 μm of PVDF filter membrane.
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|---|---|---|---|---|
| CN106198811B (en) * | 2016-08-16 | 2018-09-28 | 甘肃省商业科技研究所有限公司 | Quickly measure the kit and method of water and aquatic products Malachite Green and crystal violet |
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