CN101788538A - Detection method of microcystic toxins and metabolites thereof in organisms - Google Patents
Detection method of microcystic toxins and metabolites thereof in organisms Download PDFInfo
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Abstract
The invention discloses a detection method of microcystic toxins and metabolites thereof in organisms, which comprises the following steps: 1. finely grinding freeze drying biological tissue samples, and then, adding extraction liquid into ingredients for extracting materials to be detected; 2. enriching and eluting the extraction liquid obtained in the first step through a strong cation exchange SPE cartridge, and then, processing eluting liquid into a water solution used for analysis and detection; 3. using HPLC-MS for determining the nature of the materials to be detected; and 4. using an external reference method for defining the quantity of the materials to be detected. The method of the invention can simultaneously determine the nature and define the quantity for detecting MC-RR, MC-RR-GSH and MC-RR-Cys in the biological samples, the detection results are reliable, the reproduction quality is good, the sensitivity is high, and when being used, the method detects the existence of MC-RR, MC-RR-GSH and MC-RR-Cys in acute toxicity big-headed fish bodies for the first time.
Description
Technical field
The present invention relates to the qualitative and quantitative detecting method of Microcystin in a kind of biosome and metabolic product thereof, relate in particular in a kind of biosome Microcystin MC-RR and in metabolic process with the bond MC-RR-GSH of reduced glutathione GSH and halfcystine Cys and the qualitative and quantitative detecting method of MC-RR-Cys.The inventive method is applicable to the qualitative and detection by quantitative of Microcystin MC-RR in the biosome and metabolic product MC-RR-GSH and MC-RR-Cys, and be applicable to the research Microcystin in biosome accumulation, transmission and metabolism detoxifcation and the security of aquatic products assessed.
Background technology
Along with the significantly raising of human society yield-power and the continuous increase of population pressure, body eutrophication causes that blue-green alga bloom takes place frequently, and the harmful secondary metabolites of release, Microcystin (microcystins wherein, be called for short MCs) be that the class that produces of blue-green algae is to the bigger hepatotoxin of biotic influence in environment and the fresh water water body, liver is its main target organ, simultaneously MCs also have renal toxicity, genetoxic, embryo and development toxicity (thank flat. historical development of Taihu Lake blue-green algae and wawter bloom disaster. Beijing: Science Press, 2008).MCs can pass through potable water, approach such as food accumulation enter human body, thereby human health is threatened, Yu Shunzhang etc. find to contain in the potable water hepatitis in area of MCs and onset of liver cancer rate apparently higher than the phreatic water crowd (Yu Shunzhang that drinks no toxin by epidemiology survey, Zhao Ning, money woods dawn etc. Chinese tumour magazine, 2001 (23): 96-99).Microcystin directly causes human dead incident to occur in Brazilian Caruaru dialysis center in 1996, because this center has used the water that is subjected to the Microcystin pollution as the kidney water for dialysis, the result has caused 116 people to poison, the serious consequence of 52 people's death (Jochimsen, E.M., Carmichael, W.W., An J., New Eng.J.Med., 1998 (338): 873-878).
Glutathione is a kind of small-molecule peptide material, extensively be present in the biosome, be made up of glutamic acid, halfcystine and 3 amino acid of glycocoll, glutathione has reduced form (GSH) and two kinds of forms of oxidized form (GSSG), accounts for the overwhelming majority with reduced glutathione GSH under physiological condition.Halfcystine (Cys) is a common amino acid in a kind of biosome, can be transformed by the methionine in the body, can transform mutually with cystine.MC-RR is a kind of Microcystin the most general in the lake, subtropics, when having MC-RR to exist in the biosome, metabolism by biosome self, MC-RR can form the MC-RR-GSH bond with reduced glutathione GSH, and the one-step hydrolysis of going forward side by side becomes water-soluble stronger MC-RR-Cys bond (Kondo, F., Ikai, Y., Oka, H., et al., Chem.Res.Toxicol.1992 (5): 591, Pflugmacher S, Wiegand C, BeattieKA,, et al., Environmental Toxicology and Chemistry.2001 (20): 846-852).
At present for MCs, the research of its metabolic product and detoxification processes thereof focuses mostly in experiment in vitro, also be confined to simultaneously qualitative detection, people such as Kondo have synthesized Microcystin-glutathione and halfcystine in conjunction with the product standard items by chemical method preparation first in 1992, studied its toxicity, and by LC-FAB-MS in big mouse liver first qualitative detection arrived Microcystin-glutathione and halfcystine in conjunction with product (Kondo, F., Ikai, Y., Oka, H., et al., Chem.Res.Toxicol.1992 (5): 591-596, Kondo, F., Matsumoto, H., Yamada, S., et al., Chem.Res.Toxicol.1996 (9): 1355-1359), Pflugmacher etc. are by liquid chromatography and the qualitative Microcystin glutathione bond (Pflugmacher, the S. that form by enzymatic in the experiment in vitro of having identified of substance assistant laser desorpted ionized time-of-flight mass spectrometry, Wiegand, C., Oberemm, A., et al., Acta.1998 (1425): 527-533).For the then rare report of the vivo and vitro quantitative examination of Microcystin and metabolic product thereof, because MC-RR and metabolic product MC-RR-GSH thereof and the concentration of MC-RR-Cys in biological sample are very low usually, interfering material is many, at present without any method energy while these three kinds of materials of qualitative and quantitative detection, thereby need set up reliable, stable quantitative analysis method, and, lay the foundation for further studying MC-RR metabolism and detoxification processes thereof in vivo by effective enrichment, separation and purge process realization high sensitivity, high commissioner's attributive analysis.
Summary of the invention
The object of the present invention is to provide the detection method of interior Microcystin of a kind of biosome and metabolic product thereof, this inventive method can be qualitative simultaneously and the detection by quantitative biological sample in Microcystin MC-RR and metabolic product MC-RR-GSH and MC-RR-Cys, testing result is reliable, favorable reproducibility, highly sensitive.
In order to achieve the above object, the present invention by the following technical solutions, this scheme is carried out according to following steps:
1, the extraction of Microcystin MC-RR and metabolic product MC-RR-GSH and MC-RR-Cys: after biological tissue's organ samples freeze drying, place the stone roller alms bowl levigate, then in acetate-disodium ethylene diamine tetraacetate (EDTA) aqueous solution, make histoorgan clasmatosis with Ultrasonic Cell Disruptor, discharge determinand, centrifugal 10min under 12000r/min pipettes supernatant and keeps in Dark Place to clean container then.Repeat to extract twice according to above-mentioned steps, merge three times supernatant;
2, enrichment, isolation and purification: step 1 gained supernatant is realized enrichment and separated with strong cation exchange solid-phase extraction column (specification is 3cc/60mg), eluent evaporate to dryness on Rotary Evaporators of collecting, methyl alcohol with 100v/v% divides three sample dissolution and changes centrifuge tube over to again, evaporate to dryness on Rotary Evaporators, the solid residue that obtains is used for assay determination with deionized water dissolving and constant volume;
3, qualitative analysis: the HPLC-MS coupling, according to MC-RR, the retention time of MC-RR-GSH and MC-RR-Cys standard items and the mass-to-charge ratio m/z of feature fragment ion peak carry out qualitative;
Chromatographic condition: moving phase: A is the formic acid of 0.05v/v%, and B is the formic acid acetonitrile solution of 0.05v/v%; Flow velocity: 0～20.0min is 0.2mL/min, and 20.01～25.0min is 0.3mL/min; Chromatographic column: C18,100mm * 2.1mm ID, 3.5 μ m; Column temperature: 40.0 ℃; Sample disc temperature: 10.0 ℃; Sampling volume is 10 μ L; Elution program: 0min:95v/v%A, 5%v/v%B, 1.0min:65v/v%A, 35v/v%B, 17.0min:55v/v%A, 45v/v%B, 17.5min:30v/v%A, 70v/v%B, 18.0min:5v/v%A, 95v/v%B, 20.0min:5v/v%A, 95v/v%B, 20.01min:95v/v%A, 5v/v%B, 25.0min:95v/v%A, 5v/v%B;
Mass spectrum condition: electro-spray ionization (ESI); Positive ion mode; Nozzle needle voltage is 4.5KV; Lens voltage: MC-RR is 55.50V, and MC-RR-GSH and MC-RR-Cys are 45.50V; Automatic gain control is opened; The sheath gas velocity is 20 units, and this unit is a U.S. Finnigan LCQ system definition, from 0-100; Auxiliary air to close closes; Capillary temperature is 250 ℃;
4, quantitative test: use external standard method, quantitative with chromatographic peak area, preparation MC-RR, MC-RR-GSH and MC-RR-Cys mixing standard specimen series aqueous solution, MC-RR, MC-RR-GSH and MC-RR-Cys concentration range are 0.01～2.5 μ g/mL, with the same chromatographic condition of determinand under direct injected, respectively with MC-RR, the concentration of MC-RR-GSH and MC-RR-Cys is horizontal ordinate, corresponding chromatographic peak area is an ordinate, and the drawing standard curve is carried out quantitatively testing sample by its regression equation.
All freeze drying conditions are at-65 ℃ in the inventive method, and vacuum tightness is dry 48h in the vacuum freeze drier of 0.1mbar.
The inventive method compared with prior art has following advantage and effect:
1, MC-RR, MC-RR-GSH and the MC-RR-Cys in the simultaneously qualitative and detection by quantitative biological sample;
2, to the qualitative of MC-RR, MC-RR-GSH and MC-RR-Cys and detection by quantitative reliable results, favorable reproducibility, highly sensitive;
3, detect the existence of acute contamination bighead body interior MC-RR, MC-RR-GSH and MC-RR-Cys first.
Description of drawings
Fig. 1 and Fig. 2 are respectively the extract behind the blank fish liver mark-on and the total ions chromatogram of aqueous solution mark-on direct injected.Be followed successively by the one-level total ions chromatogram among Fig. 1 from top to bottom, MC-RR-GSH, the secondary total ions chromatogram of MC-RR-Cys and MC-RR is followed successively by the one-level total ions chromatogram from top to bottom among Fig. 2, MC-RR-GSH, the secondary total ions chromatogram of MC-RR-Cys and MC-RR.
Fig. 3 is injected into behind the MC-RR in the 48h content situation of MC-RR, MC-RR-GSH and MC-RR-Cys in its liver for acute contamination bighead.
By Fig. 1 and Fig. 2 as can be seen, the endogenous material in the fish liver is not disturbed the mensuration of MC-RR, MC-RR-GSH and MC-RR-Cys; As seen from Figure 3, injected interior metabolism spontaneous generation MC-RR-GSH of energy and the MC-RR-Cys of passing through of healthy bighead body of MC-RR.
Embodiment
Below in conjunction with specific embodiment the inventive method is described in further detail.
Embodiment 1: the detection method of Microcystin and metabolic product thereof in a kind of biosome the steps include:
1, the extraction of Microcystin MC-RR and metabolic product MC-RR-GSH and MC-RR-Cys: get cryodesiccated fish liver sample 50mg, levigate being placed in the centrifuge tube in grinding alms bowl, add acetate-EDTA aqueous solution of 5mL 5v/v%, wherein EDTA concentration is 0.01mol/L.Continue ultrasonic 3～5min with Ultrasonic Cell Disruptor under 0 ℃, centrifugal 10min under 12000r/min pipettes supernatant and keeps in Dark Place to clean container then.Repeat to extract twice again according to above-mentioned steps, merge above three supernatants and get solution I, it is pure that used glacial acetic acid and disodium ethylene diamine tetraacetate are analysis;
2, the enrichment of determinand, isolation and purification: carry out enrichment in the strong cation exchange solid-phase extraction column (specification 3cc/60mg) that the solution I adding that step 1 is obtained activates in advance, after treating that enrichment finishes, use the formic acid of 3mL 2v/v% successively, the methyl alcohol drip washing of 3mL90v/v% contains the methanol solution wash-out of 15v/v% strong aqua at last with 10mL.Eluent is collected in the 25mL round-bottomed flask, evaporate to dryness on Rotary Evaporators, divide three sample dissolution and change the 1.5mL centrifuge tube over to 1mL 100v/v% methyl alcohol again, evaporate to dryness once more, the solid residue that obtains is with deionized water dissolving and be settled to 100 μ L, be used for assay determination, it is pure that used formic acid, methyl alcohol and strong aqua are analysis;
3, qualitative analysis: the HPLC-MS coupling, according to MC-RR, the retention time of MC-RR-GSH and MC-RR-Cys standard items and the mass-to-charge ratio m/z of feature fragment ion peak carry out qualitative;
The used MC-RR of the inventive method is commercially available environmental analysis solid standard items, and MC-RR-GSH and MC-RR-Cys standard items reference literature method are synthesized (Kondo, F., Ikai, Y., Oka, H., et al., Chem.Res.Toxicol.1992 (5): 592).Preparation MC-RR, MC-RR-GSH and MC-RR-Cys concentration are the mixing standard specimen aqueous solution of 1.0 μ g/mL and as standard reference material testing sample are carried out qualitative analysis.The electro-spray ionization second order ms cracking characteristic ion of MC-RR and metabolin MC-RR-GSH and MC-RR-Cys sees Table 1, MC-RR, and the parent ion of MC-RR-GSH and MC-RR-Cys is respectively 520.06,673.61 and 580.56.
The ESI second order ms cracking characteristic ion of table 1MC-RR and metabolin MC-RR-GSH and MC-RR-Cys
4, quantitative test: use external standard method, quantitative with chromatographic peak area, preparation MC-RR, MC-RR-GSH and MC-RR-Cys mixing standard specimen series aqueous solution, MC-RR, MC-RR-GSH and MC-RR-Cys concentration range are 0.01～2.5 μ g/mL, with the same chromatographic condition of determinand under direct injected, respectively with MC-RR, the concentration of MC-RR-GSH and MC-RR-Cys is horizontal ordinate, corresponding chromatographic peak area is an ordinate, and the drawing standard curve is carried out quantitatively testing sample by its regression equation.
Embodiment 2: the selectivity of the inventive method, accuracy, precision and sensitivity are investigated:
1, method selectivity: the cryodesiccated sample 50mg of blank fish liver that the HPLC-MS that learns from else's experience detects, with 50 μ LMC-RR, MC-RR-GSH and MC-RR-Cys concentration are 1 μ gmL
-1Aqueous solution be added in this blank sample, operate according to the method step of embodiment 1, obtain the MC-RR with same concentrations, the essentially identical secondary total ions chromatogram of MC-RR-GSH and MC-RR-Cys mixed aqueous solution direct injected.This experimental result shows that the endogenous material in the fish liver sample is not disturbed the mensuration of determinand, sees Fig. 1 and Fig. 2;
2, method calibration curve: the cryodesiccated sample 50mg of blank fish liver that the HPLC-MS that learns from else's experience detects, press step 1 and the operation of step 2 method among the embodiment 1, obtain the matrix concentrate sample of a series of extraction and cleanings.The MC-RR that adds variable concentrations in above sample successively, MC-RR-GSH and MC-RR-Cys are mixed with that to be equivalent to the concentration of each determinand in the fish liver be 0.02,0.1,0.2,1.0,2.0,5.0 μ g.g
-1Serial mixed aqueous solution, set up calibration curve.With MC-RR, MC-RR-GSH and MC-RR-Cys concentration are horizontal ordinate respectively, and chromatographic peak area is that ordinate obtains calibration curve.MC-RR, MC-RR-GSH and MC-RR-Cys the results are shown in Table 2 at the calibration curve of liver:
Calibration curve (the X: concentration μ gg of table 2MC-RR, MC-RR-GSH and MC-RR-Cys
-1, Y: peak area)
3, recovery of standard addition: the cryodesiccated sample 50mg of blank fish liver that the HPLC-MS that learns from else's experience detects adds basic, normal, high three concentration respectively and (is respectively 0.2,1.0,5.0 μ gg
-1) three kinds of determinands, each concentration is carried out 3 sample analyses.By comparing, the ratio of the standard solution direct injected gained peak area that above processed group chromatographic peak area is identical with containing the determinand amount calculates the averaging method recovery of each concentration point, the results are shown in Table 3.
Table 3MC-RR, MC-RR-GSH and the MC-RR-Cys recovery of standard addition in fish liver sample
4, the precision of method: prepare MC-RR respectively, MC-RR-GSH and MC-RR-Cys are basic, normal, high, and three concentration (are respectively 0.2,1.0,5.0 μ gg
-1) fish liver quality control sample (QC), prepare two groups of samples simultaneously, one group of each concentration analysis of sample 5 times, another group sample METHOD FOR CONTINUOUS DETERMINATION 3 days, every day 1 time.Thereby try to achieve the precision of this assay method, represent with the coefficient of variation.The results are shown in Table 4:
MC-RR in the table 4 fish liver, the precision that MC-RR-GSH and MC-RR-Cys measure
5, method detectability and quantitative limit: mark-on in the blank fish liver that detects through HPLC-MS, measure the blank mark-on of low concentration of 7 repetitions, mark-on concentration is generally 1～5 times of expection method detectability (MDL) value, and handle and measure according to the overall process of method, calculate the standard deviation SD that measures concentration for 7 times again, mark-on concentration is 0.02 μ gg
-1, computing method are as follows:
Method detectability=t
(n-1, α=0.01)* standard deviation SD
Quantitative limit (LOQ)=2.5 * method detectability
Degree of confidence t wherein
(n-1, α=0.01)Be 99%, the t value when degree of freedom is n-1 is tabled look-up and can be got, and is 2.896, and n is the sample number of replicate analysis, the results are shown in Table 5:
MC-RR in the table 5. mark-on liver sample, the detectability of MC-RR-GSH and MC-RR-Cys and quantitative limit
Embodiment 3: the acute exposure experiment of healthy bighead:
Go into the MC-RR aqueous solution from lumbar injection behind a collection of healthy bighead fasting 48h, the injected dose of MC-RR is 50 μ g/kg body weight, collects the bighead liver sample of injection back 1,3,12,24 and 48h respectively.The sample of collecting is preserved in-20 ℃ of lower seals, and is standby with postlyophilization.The qualitative and quantitative analysis method of setting up according to the inventive method is measured the MC-RR in the bighead liver of the acute contamination of different time respectively, and MC-RR-GSH and MC-RR-Cys the results are shown in Figure 3.
Claims (1)
1. the detection method of Microcystin and metabolic product thereof in the biosome the steps include:
1., take by weighing cryodesiccated biological tissue samples, levigate being placed in the centrifuge tube in grinding alms bowl, acetate-EDTA the aqueous solution that adds 5v/v% according to the ratio of 0.1mL/mg, wherein EDTA concentration is 0.01mol/L, in 0 ℃ of following ultrasonication 3～5min, centrifugal under 12000r/min then, pipette supernatant and to clean container, keep in Dark Place, repeat to extract twice again, merge solution I;
2., carry out enrichment in the strong cation exchange solid-phase extraction column with solution I adding activation in advance, after treating that enrichment finishes, use the formic acid of 2v/v% successively, the methyl alcohol drip washing of 90v/v%, at last with the methanol solution wash-out that contains the 15v/v% strong aqua, eluent is collected in the round-bottomed flask, evaporate to dryness on Rotary Evaporators, methyl alcohol with 100v/v% divides three sample dissolution and changes centrifuge tube over to again, evaporate to dryness on Rotary Evaporators, the solid residue that obtains is used for assay determination with deionized water dissolving and constant volume;
3., qualitative analysis: the HPLC-MS coupling, according to MC-RR, the retention time of MC-RR-GSH and MC-RR-Cys standard items and the mass-to-charge ratio m/z of feature fragment ion peak carry out qualitative;
Chromatographic condition: moving phase: A is the formic acid of 0.05v/v%, and B is the formic acid acetonitrile solution of 0.05v/v%; Flow velocity: 0～20.0min is 0.2mL/min, and 20.01～25.0min is 0.3mL/min; Chromatographic column: C18,100mm * 2.1mm ID, 3.5 μ m; Column temperature: 40.0 ℃; Sample disc temperature: 10.0 ℃; Sampling volume is 10 μ L; Elution program: 0min:95v/v%A, 5%v/v%B, 1.0min:65v/v%A, 35v/v%B, 17.0min:55v/v%A, 45v/v%B, 17.5min:30v/v%A, 70v/v%B, 18.0min:5v/v%A, 95v/v%B, 20.0min:5v/v%A, 95v/v%B, 20.01min:95v/v%A, 5v/v%B, 25.0min:95v/v%A, 5v/v%B;
Mass spectrum condition: electro-spray ionization; Positive ion mode; Nozzle needle voltage is 4.5KV; Lens voltage: MC-RR is 55.50V, and MC-RR-GSH and MC-RR-Cys are 45.50V; Automatic gain control is opened; The sheath gas velocity is 20 units; Auxiliary air to close closes; Capillary temperature is 250 ℃;
4., quantitative test: use external standard method, quantitative with chromatographic peak area, preparation MC-RR, MC-RR-GSH and MC-RR-Cys mixing standard specimen series aqueous solution, MC-RR, MC-RR-GSH and MC-RR-Cys concentration range are 0.01～2.5 μ g/mL, with the same chromatographic condition of determinand under direct injected, respectively with MC-RR, the concentration of MC-RR-GSH and MC-RR-Cys is horizontal ordinate, corresponding chromatographic peak area is an ordinate, and the drawing standard curve is carried out quantitatively testing sample by its regression equation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197021A (en) * | 2013-04-09 | 2013-07-10 | 上海海洋大学 | Method for detecting microcystic toxins MC-LR in organism |
| CN107389825A (en) * | 2017-08-11 | 2017-11-24 | 浙江省食品药品检验研究院 | The method that algae toxin in water is determined based on full-automatic on-line solid phase extraction ultra performance liquid chromatography linear ion hydrazine tandem mass spectrum |
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2010
- 2010-01-11 CN CN201010028952A patent/CN101788538A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197021A (en) * | 2013-04-09 | 2013-07-10 | 上海海洋大学 | Method for detecting microcystic toxins MC-LR in organism |
| CN107389825A (en) * | 2017-08-11 | 2017-11-24 | 浙江省食品药品检验研究院 | The method that algae toxin in water is determined based on full-automatic on-line solid phase extraction ultra performance liquid chromatography linear ion hydrazine tandem mass spectrum |
| CN107389825B (en) * | 2017-08-11 | 2020-07-07 | 浙江省食品药品检验研究院 | Method for determining algae toxins in water based on full-automatic online solid-phase extraction-ultra-high performance liquid chromatography-linear ion trap tandem mass spectrometry |
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| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20100728 |