CN103308639A - Detection method of content of blue algae toxin beta-methylamino-L-alanine in aquatic products - Google Patents
Detection method of content of blue algae toxin beta-methylamino-L-alanine in aquatic products Download PDFInfo
- Publication number
- CN103308639A CN103308639A CN201310186468XA CN201310186468A CN103308639A CN 103308639 A CN103308639 A CN 103308639A CN 201310186468X A CN201310186468X A CN 201310186468XA CN 201310186468 A CN201310186468 A CN 201310186468A CN 103308639 A CN103308639 A CN 103308639A
- Authority
- CN
- China
- Prior art keywords
- aquatic products
- methylamino
- alanine
- sample
- acid solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000003053 toxin Substances 0.000 title claims abstract description 29
- 231100000765 toxin Toxicity 0.000 title claims abstract description 29
- 238000001514 detection method Methods 0.000 title claims abstract description 16
- 241000195493 Cryptophyta Species 0.000 title abstract description 3
- UJVHVMNGOZXSOZ-UHFFFAOYSA-N 2-amino-3-(methylamino)propanoic acid Chemical compound CNCC(N)C(O)=O UJVHVMNGOZXSOZ-UHFFFAOYSA-N 0.000 title abstract 2
- 238000000034 method Methods 0.000 claims abstract description 29
- 238000001212 derivatisation Methods 0.000 claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 238000004885 tandem mass spectrometry Methods 0.000 claims abstract description 8
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 claims abstract description 7
- 150000002500 ions Chemical class 0.000 claims description 27
- 239000000047 product Substances 0.000 claims description 23
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 18
- 239000007789 gas Substances 0.000 claims description 18
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 14
- 239000007791 liquid phase Substances 0.000 claims description 12
- 239000007921 spray Substances 0.000 claims description 12
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 12
- 238000006460 hydrolysis reaction Methods 0.000 claims description 10
- 238000004811 liquid chromatography Methods 0.000 claims description 9
- 230000014759 maintenance of location Effects 0.000 claims description 9
- AJYLWDRNNNHBEV-VKHMYHEASA-N (2s)-2-hydrazinylbutanoic acid Chemical compound CC[C@H](NN)C(O)=O AJYLWDRNNNHBEV-VKHMYHEASA-N 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 8
- 230000007062 hydrolysis Effects 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- 238000004949 mass spectrometry Methods 0.000 claims description 6
- 238000001819 mass spectrum Methods 0.000 claims description 6
- 239000012071 phase Substances 0.000 claims description 6
- 238000001556 precipitation Methods 0.000 claims description 6
- 239000012141 concentrate Substances 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 238000005374 membrane filtration Methods 0.000 claims description 2
- UJVHVMNGOZXSOZ-VKHMYHEASA-N L-BMAA Chemical compound CNC[C@H](N)C(O)=O UJVHVMNGOZXSOZ-VKHMYHEASA-N 0.000 abstract description 44
- 238000011084 recovery Methods 0.000 abstract description 18
- 238000004458 analytical method Methods 0.000 abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000012472 biological sample Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 48
- 108700012359 toxins Proteins 0.000 description 24
- 239000012086 standard solution Substances 0.000 description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 241000237858 Gastropoda Species 0.000 description 11
- 238000005303 weighing Methods 0.000 description 10
- 239000012491 analyte Substances 0.000 description 8
- 239000012496 blank sample Substances 0.000 description 8
- 238000010812 external standard method Methods 0.000 description 8
- 241000251468 Actinopterygii Species 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 239000003643 water by type Substances 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 5
- 241000192700 Cyanobacteria Species 0.000 description 4
- 239000004809 Teflon Substances 0.000 description 4
- 229920006362 Teflon® Polymers 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 4
- 239000000413 hydrolysate Substances 0.000 description 4
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000003344 environmental pollutant Substances 0.000 description 3
- 231100000719 pollutant Toxicity 0.000 description 3
- SRUWWOSWHXIIIA-UKPGNTDSSA-N Cyanoginosin Chemical compound N1C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](C)[C@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C(=C)N(C)C(=O)CC[C@H](C(O)=O)N(C)C(=O)[C@@H](C)[C@@H]1\C=C\C(\C)=C\[C@H](C)[C@@H](O)CC1=CC=CC=C1 SRUWWOSWHXIIIA-UKPGNTDSSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 108010067094 microcystin Proteins 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 240000000163 Cycas revoluta Species 0.000 description 1
- 235000008601 Cycas revoluta Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000192710 Microcystis aeruginosa Species 0.000 description 1
- 239000005422 algal bloom Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000008239 natural water Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012502 risk assessment Methods 0.000 description 1
Images
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention provides a detection method of the content of blue algae toxin beta-methylamino-L-alanine in aquatic products. The method comprises the steps such as pretreatment, derivatization and liquid chromatography-Quadrupole-electrospray ionization mass spectrometry. The method is simple to operate and high in analysis speed; to solve the problems of complex biological sample basal body and technical difficulties in pretreatment in water environment, the pretreatment process used in the method can extract the free-state and binding-state BMAA (Beta-N-methylamino-L-alanine) from the aquatic products. The high performance liquid chromatography-Quadrupole tandem mass spectrometry associated detection method used in the method is low in detection limit, excellent in repeatability, high in sensitivity, higher in recovery rate and high in precision.
Description
Technical field
The invention belongs to the analytical chemistry field, relate to the detection method of pollutant in the aquatic products, more specifically relate to the detection method of cyanophycean toxin β-methylamino-ALANINE (BMAA) content in a kind of aquatic products.
Background technology
Blue-green alga bloom occurrence frequency and scale in world today's scope are all in rising trend, therefore the inland lake of a plurality of countries and coastal ocean water body all are in serious eutrophic state, carry out for the research of blue-green alga bloom pollutant and very urgent to human health risk assessment.The emphasis of research is on traditional pollutants such as Microcystin (Microcystin) at present, for other cyanophycean toxin researchs seldom, and most of blue-green algaes all can produce the phenomenon of β-methylamino-ALANINE BMAA toxin after 2003 are found, caused the great attention of international community, detection research has all been carried out in a plurality of areas.Even at present external research has shown in not depositing the ocean water environment of sago cycas, the BMAA(free state that two kinds of forms exist and in conjunction with attitude) still can move and amplify by hydrobiont food chain (blue-green algae-phytoplankton-animal plankton-fish).This achievement hints in China breakout of water bloom waters or blue-green algae grows on a large scale, and the waters also may exist similar toxin migration and amplification approach.China is breakout of cyanobacteria blooms one of the most serious zone in the world, and the investigation of carrying out blue-green algae BMAA toxin in the inland lake natural water environment research that distributes seems particularly important.
Owing to lacking at present the effective measures that prevent that algal bloom from occuring, therefore want the prevent and avoid cyanophycean toxin to people and animals' harm, monitoring and control cyanophycean toxin limiting the quantity of in all kinds of aquatic products are effective methods.Liquid chromatography mass coupling method has been applied to the detection of multiple algae toxin compound in water body and the biosome.Yet the detection research for BMAA is less.Existing BMAA adopts fluorescence HPLC method to detect usually, and sensitivity is not high, testing result is inaccurate.
Summary of the invention
Goal of the invention: the purpose of this invention is to provide the accurately detection method of cyanophycean toxin β-methylamino-ALANINE content in the aquatic products of a kind of highly sensitive, testing result.
Technical scheme: the detection method of cyanophycean toxin β-methylamino in a kind of aquatic products provided by the invention-ALANINE content may further comprise the steps:
(1) pre-service: cryodesiccated aquatic products are pulverized evenly, added trichloroacetic acid solution homogenate on ice bath, centrifugal, get supernatant; After precipitation added the hydrochloric acid solution hydrolysis, membrane filtration got filtrate; Merge supernatant and filtrate, it is concentrated that 55 ℃ of nitrogen blows, and gets concentrate;
(2) derivatization: concentrate is added dissolving with hydrochloric acid, get 20 μ L derivatizations, get liquid to be measured;
(3) liquid chromatography-level Four bar-Electrospray Ionization Mass Spectrometry: the liquid to be measured that adopts liquid chromatography-level Four bar-electrospray ionization mass spectrum detecting step (2), β-methylamino-ALANINE is determined with retention time and characteristic ion, the drawing standard curve, detect with the external standard standard measure, the chromatography-mass spectroscopy condition is respectively:
Liquid phase chromatogram condition: instrument model: Agilent1290/6460HPLC/MS/MS; Liquid-phase chromatographic column: the EclipseXDB-C18 post, 4.6 * 100mm * 5.0 μ m, column temperature is 45 ℃; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass spectrum condition: electric spray ion source: positive ion electrospray is from pattern ESI+; 320 ℃ of ion source temperatures of dry gas temperature; Gas flow rate 10Lmin
-1Air pressure 40psi; Capillary voltage 3500V; 320 ℃ of sheath temperature degree; Sheath gas velocity 11Lmin
-1Spray nozzle voltage 1000V; Select multiple-reaction monitoring pattern (MRM).
In the step (1), in the step (1), the amount ratio of aquatic products and trichloroacetic acid solution is (10-20) mg:2ml, preferred 15mg:2ml; The concentration of trichloroacetic acid solution is 0.05-0.2mol L
-1, preferred 0.1mol L
-1Ultrasonic homogenate is adopted in homogenate, and ultrasonic power is 600-800W, ultrasonic time 5-15min, preferred 10min; Centrifugal rotational speed is 8000-12000r/m, preferred 12000r/m, and centrifugation time is 6-15min, preferred 10min; The concentration of hydrochloric acid solution is 3-10molL
-1, preferred 6molL
-1Hydrolysis temperature is 110 ℃, and hydrolysis time is 18h-24h, preferred 24h; Filter membrane aperture 0.22 μ m or 0.45 μ m are preferably 0.22 μ m; Method for concentration is used in 45 ℃ of-70 ℃ of lower nitrogen and blows concentratedly, and preferred 55 ℃ of lower nitrogen blow concentrated.
In the step (2), the concentration of hydrochloric acid solution is 10mmolL
-1-20mmolL
-1, preferred 20mmolL
-1Derivatization method is AccQ-Tag Μ Ltra Derivatization Kit (Waters, USA) kit instructions method.
Beneficial effect: the detection method of cyanophycean toxin β-methylamino in the aquatic products provided by the invention-ALANINE content is simple to operate, analysis speed is fast, the problem that the method is complicated for biological specimen matrix in the water environment, the pretreatment technology difficulty is large, the pretreating process that adopts can extract free state in the aquatic products and in conjunction with the BMAA of attitude, the high performance liquid chromatography of employing-quadrupole rods tandem mass spectrometry method joint inspection survey method detectability is low, favorable reproducibility, highly sensitive, the recovery is better and degree of accuracy is high.
Description of drawings
Fig. 1 is that the BMAA standard items are (MRM; 459.1 289.1) chromatograms;
Fig. 2 is crucian flesh of fish sample (MRM; 459.1 289.1) chromatograms;
Fig. 3 is the mass spectrogram of BMAA standard items;
Fig. 4 is the mass spectrogram of crucian flesh of fish sample.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described concrete material proportion of embodiment, process conditions and result thereof only are used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
1. the extraction of sample
Cryodesiccated aquatic products crucian flesh of fish sample is pulverized evenly, taken by weighing 15mg dry powder and add 2mL0.1molL
-1Trichloroacetic acid solution is in homogenate sample on ice, and ultrasonic power is 600-800W, in the centrifugal 10min of 12000r/m, pipettes supernatant liquor behind the ultrasonic 10min, as BMAA free state to be measured.The precipitation part adds 6molL
-1HCL, and place abundant hydrolysis 24h under 110 ℃ of conditions, and be cooled to natural temperature, filter through 0.22 μ m teflon filter, the hydrolysate filtrate that obtains and free state BMAA two parts of collecting all under 55 ℃ of conditions nitrogen blow concentrated, to be analyzed in-20 ℃ of preservations.
2. analyte derivative
Before LC-MS analysis, the freezing sample that step 1 is preserved dissolves with an amount of 20mM HCl, and taking out 20 μ L for analyte derivative, derivatization method is AccQ-Tag Μ Ltra Derivatization Kit (Waters, USA) kit instructions method.Sample after derivative detected within a week.
3. liquid chromatography-level Four bar-Electrospray Ionization Mass Spectrometry condition
Liquid phase chromatogram condition: instrument model: Agilent1290/6460HPLC/MS/MS; Liquid-phase chromatographic column: the EclipseXDB-C18 post, 4.6 * 100mm * 5.0 μ m, column temperature is 45 ℃; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass spectrum condition: electric spray ion source: positive ion electrospray is from pattern ESI+; 320 ℃ of ion source temperatures of dry gas temperature; Gas flow rate 10Lmin
-1Air pressure 40psi; Capillary voltage 3500V; 320 ℃ of sheath temperature degree; Sheath gas velocity 11Lmin
-1Spray nozzle voltage 1000V.Select multiple-reaction monitoring pattern (MRM), target compound is definite with retention time and characteristic ion, and the drawing standard curve quantitatively detects with external standard method.
4. the drafting of typical curve is carried out quantitative measurement with external standard method
Accurately take by weighing BMAA toxin standard items, be dissolved in 50% acetonitrile solution, be mixed with the standard solution of a series of variable concentrations, take the concentration of BMAA toxin as horizontal ordinate, peak area after liquid phase-mass spectrometry analysis is measured is that ordinate carries out regretional analysis, get regression equation y=20533x-1609.3, related coefficient is 0.9957.Be used for the working sample object.Add the standard solution of variable concentrations in blank sample, through after the identical processing, the interpolation concentration that obtains 3 times of signal to noise ratio (S/N ratio)s is detectability; Detecting of the method is limited to 6ng/g, and peak area and sample concentration are good linear relationship in the scope of concentration 0.05 μ g/mL～1mg/mL.
Stratographic analysis retention time and the quota ion of BMAA see Table 1.
Table 1 is stratographic analysis retention time and the quota ion of BMAA
| Material | Retention time (min) | Quota ion |
| BMAA | 4.299min | 459.1>289.1 |
5. the mensuration of sample and the recovery
Gather aquatic products crucian flesh of fish sample to be measured, after effectively extracting by step 1 pair sample, carry out derivatization treatment by step 2, carrying out HPLC-MS by step 3 again detects, and with above-mentioned resulting typical curve relatively, finally obtain BMAA free state in the crucian flesh of fish sample and in conjunction with the content of attitude by calculating.
After adopting identical crucian flesh of fish sample to carry out freeze drying, take by weighing 15mg blank sample to be measured, add 2mL0.2molL on ice
-1Trichloroacetic acid, the addition according to 0.5 μ g/g adds standard items BMAA solution simultaneously.Carry out identical pre-service and measure the content of BMAA, and carry out the recovery according to following formula and calculate:
R=(C-C
0)/0.5×100%
R-recovery, %
BMAA content of toxins in the aquatic products sample behind C-interpolation standard solution, mg/g;
C
0-do not add the BMAA content of toxins of standard solution aquatic products sample, ng/g;
The mensuration of the recovery is all carried out 6 parallel experiments for blank sample and mark-on.The recovery of this method and precision, as shown in table 2:
The recovery of table 2 this method and precision
| Component to be measured (totalBMAA) | Crucian flesh of fish sample (μ g/g dry weight) | The recovery/% | RSD/% |
| BMAA | 0.25μg/g | 80.3% | 3.3% |
1. the extraction of sample
Cryodesiccated aquatic products river snail sample edible is partly pulverized evenly, taken by weighing 15mg dry powder and add 2mL0.1molL
-1Trichloroacetic acid is in homogenate sample on ice, and ultrasonic power is 600-800W, in the centrifugal 10min of 12000r/m, pipettes supernatant liquor behind the ultrasonic 10min, as BMAA free state to be measured.The precipitation part adds 6molL
-1HCL, and place abundant hydrolysis 24h under 110 ℃ of conditions, and be cooled to natural temperature, filter through 0.22 μ m teflon filter, the hydrolysate filtrate that obtains and free state BMAA two parts of collecting all under 55 ℃ of conditions nitrogen blow concentrated, to be analyzed in-20 ℃ of preservations.
2. analyte derivative
Before LC-MS analysis, the freezing sample that step 1 is preserved dissolves with an amount of 20mMHCL, and taking out 20 μ L for analyte derivative, derivatization method is AccQ-Tag Μ Ltra Derivatization Kit (Waters, USA) kit instructions method.Sample after derivative detected within a week.
3. liquid chromatography-level Four bar-Electrospray Ionization Mass Spectrometry condition
Liquid phase chromatogram condition: instrument model: Agilent1290/6460HPLC/MS/MS; Liquid-phase chromatographic column: the EclipseXDB-C18 post, 4.6 * 100mm * 5.0 μ m, column temperature is 45 ℃; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass spectrum condition: electric spray ion source: positive ion electrospray is from pattern ESI+; 320 ℃ of ion source temperatures of dry gas temperature; Gas flow rate 10Lmin
-1Air pressure 40psi; Capillary voltage 3500V; 320 ℃ of sheath temperature degree; Sheath gas velocity 11Lmin
-1Spray nozzle voltage 1000V.Select multiple-reaction monitoring pattern (MRM), target compound is definite with retention time and characteristic ion, and the drawing standard curve quantitatively detects with external standard method.
4. the drafting of typical curve is carried out quantitative measurement with external standard method
Accurately take by weighing BMAA toxin standard items, be dissolved in 50% acetonitrile solution, being mixed with a series of standard is dissolved in, take the concentration of BMAA toxin as horizontal ordinate, peak area after liquid phase-mass spectrometry analysis is measured is that ordinate carries out regretional analysis, regression equation is y=25608x-4231.5, and related coefficient is 0.9976.100 μ g/mLBMAA standard solutions are carried out replicate determination (n=6), and relative standard deviation RSD is 0.5%.Add the standard solution of variable concentrations in blank sample, through after the identical processing, the interpolation concentration that obtains 3 times of signal to noise ratio (S/N ratio)s is detectability (6ng/g).
5. the mensuration of sample and the recovery
Gather river snail sample edible part to be measured, after effectively extracting by step 1 pair sample, carry out derivatization treatment by step 2, carrying out HPLC-MS by step 3 again detects, and with above-mentioned resulting typical curve relatively, finally obtain BMAA free state in the river snail sample edible part by converting and in conjunction with the content of attitude.
After adopting identical river snail sample edible partly to carry out freeze drying, take by weighing 15mg blank sample to be measured, add 2mL0.1molL on ice
-1Trichloroacetic acid adds the BMAA standard solution by 0.5 μ g/g addition.Carry out identical pre-service and measure the content of BMAA, and carry out the recovery according to following formula and calculate:
R=(C-C
0)/0.5×100%
R-recovery, %
BMAA content of toxins in the aquatic products sample behind C-interpolation standard solution, mg/g;
C
0-do not add the BMAA content of toxins of standard solution aquatic products sample, ng/g;
The mensuration of the recovery is all carried out 6 parallel experiments for blank sample and mark-on.The recovery of this method and precision, as shown in table 3:
The recovery of table 3 this method and precision
| Component to be measured (totalBMAA) | River snail sample edible part (μ g/g dry weight) | The recovery/% | RSD/% |
| BMAA | 0.63μg/g | 78.6% | 5.7% |
1. the extraction of sample
Cryodesiccated aquatic products river snail sample edible is partly pulverized evenly, taken by weighing 10mg dry powder and add 2mL0.2molL
-1Trichloroacetic acid is in homogenate sample on ice, and ultrasonic power is 600W, in the centrifugal 15min of 8000r/m, pipettes supernatant liquor behind the ultrasonic 15min, as BMAA free state to be measured.The precipitation part adds 3molL
-1HCL, and place abundant hydrolysis 18h under 110 ℃ of conditions, and be cooled to natural temperature, filter through 0.22 μ m teflon filter, the hydrolysate filtrate that obtains and free state BMAA two parts of collecting all under 70 ℃ of conditions nitrogen blow concentrated, to be analyzed in-20 ℃ of preservations.
2. analyte derivative
Before LC-MS analysis, the freezing sample that step 1 is preserved dissolves with an amount of 10mM HCl, and taking out 20 μ L for analyte derivative, derivatization method is AccQ-Tag Μ Ltra Derivatization Kit (Waters, USA) kit instructions method.Sample after derivative detected within a week.
3. liquid chromatography-level Four bar-Electrospray Ionization Mass Spectrometry condition
Liquid phase chromatogram condition: instrument model: Agilent1290/6460HPLC/MS/MS; Liquid-phase chromatographic column: the EclipseXDB-C18 post, 4.6 * 100mm * 5.0 μ m, column temperature is 45 ℃; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass spectrum condition: electric spray ion source: positive ion electrospray is from pattern ESI+; 320 ℃ of ion source temperatures of dry gas temperature; Gas flow rate 10Lmin
-1Air pressure 40psi; Capillary voltage 3500V; 320 ℃ of sheath temperature degree; Sheath gas velocity 11Lmin
-1Spray nozzle voltage 1000V.Select multiple-reaction monitoring pattern (MRM), target compound is definite with retention time and characteristic ion, and the drawing standard curve quantitatively detects with external standard method.
4. the drafting of typical curve is carried out quantitative measurement with external standard method
Accurately take by weighing BMAA toxin standard items, be dissolved in 50% acetonitrile solution, being mixed with a series of standard is dissolved in, take the concentration of BMAA toxin as horizontal ordinate, peak area after liquid phase-mass spectrometry analysis is measured is that ordinate carries out regretional analysis, regression equation is y=22574x-4581.3, and related coefficient is 0.9981.100 μ g/mLBMAA standard solutions are carried out replicate determination (n=6), and relative standard deviation RSD is 0.5%.Add the standard solution of variable concentrations in blank sample, through after the identical processing, the interpolation concentration that obtains 3 times of signal to noise ratio (S/N ratio)s is detectability (6ng/g).
5. the mensuration of sample and the recovery
Gather river snail sample edible part to be measured, after effectively extracting by step 1 pair sample, carry out derivatization treatment by step 2, carrying out HPLC-MS by step 3 again detects, and with above-mentioned resulting typical curve relatively, finally obtain BMAA free state in the river snail sample edible part by converting and in conjunction with the content of attitude.
1. the extraction of sample
Cryodesiccated aquatic products river snail sample edible is partly pulverized evenly, taken by weighing 20mg dry powder and add 2mL0.05molL
-1Trichloroacetic acid is in homogenate sample on ice, and ultrasonic power is 800W, in the centrifugal 6min of 10000r/m, pipettes supernatant liquor behind the ultrasonic 5min, as BMAA free state to be measured.The precipitation part adds 10molL
-1HCL, and place abundant hydrolysis 20h under 110 ℃ of conditions, and be cooled to natural temperature, filter through 0.22 μ m teflon filter, the hydrolysate filtrate that obtains and free state BMAA two parts of collecting all under 40 ℃ of conditions nitrogen blow concentrated, to be analyzed in-20 ℃ of preservations.
2. analyte derivative
Before LC-MS analysis, the freezing sample that step 1 is preserved dissolves with an amount of 15mMHCL, and taking out 20 μ L for analyte derivative, derivatization method is AccQ-Tag Μ Ltra Derivatization Kit (Waters, USA) kit instructions method.Sample after derivative detected within a week.
3. liquid chromatography-level Four bar-Electrospray Ionization Mass Spectrometry condition
Liquid phase chromatogram condition: instrument model: Agilent1290/6460HPLC/MS/MS; Liquid-phase chromatographic column: the EclipseXDB-C18 post, 4.6 * 100mm * 5.0 μ m, column temperature is 45 ℃; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass spectrum condition: electric spray ion source: positive ion electrospray is from pattern ESI+; 320 ℃ of ion source temperatures of dry gas temperature; Gas flow rate 10Lmin
-1Air pressure 40psi; Capillary voltage 3500V; 320 ℃ of sheath temperature degree; Sheath gas velocity 11Lmin
-1Spray nozzle voltage 1000V.Select multiple-reaction monitoring pattern (MRM), target compound is definite with retention time and characteristic ion, and the drawing standard curve quantitatively detects with external standard method.
4. the drafting of typical curve is carried out quantitative measurement with external standard method
Accurately take by weighing BMAA toxin standard items, be dissolved in 50% acetonitrile solution, being mixed with a series of standard is dissolved in, take the concentration of BMAA toxin as horizontal ordinate, peak area after liquid phase-mass spectrometry analysis is measured is that ordinate carries out regretional analysis, regression equation is y=24821x-3815.5, and related coefficient is 0.9978.100 μ g/mLBMAA standard solutions are carried out replicate determination (n=6), and relative standard deviation RSD is 0.5%.Add the standard solution of variable concentrations in blank sample, through after the identical processing, the interpolation concentration that obtains 3 times of signal to noise ratio (S/N ratio)s is detectability (6ng/g).
5. the mensuration of sample and the recovery
Gather river snail sample edible part to be measured, after effectively extracting by step 1 pair sample, carry out derivatization treatment by step 2, carrying out HPLC-MS by step 3 again detects, and with above-mentioned resulting typical curve relatively, finally obtain BMAA free state in the river snail sample edible part by converting and in conjunction with the content of attitude.
Claims (3)
1. the detection method of cyanophycean toxin β-methylamino-ALANINE content in the aquatic products is characterized in that: may further comprise the steps:
(1) pre-service: cryodesiccated aquatic products are pulverized evenly, added trichloroacetic acid solution homogenate on ice bath, centrifugal, get supernatant; After precipitation added the hydrochloric acid solution hydrolysis, membrane filtration got filtrate; Merge supernatant and filtrate, concentrated, get concentrate;
(2) derivatization: concentrate is added dissolving with hydrochloric acid, get 20 μ L derivatizations, get liquid to be measured;
(3) liquid chromatography-level Four bar-Electrospray Ionization Mass Spectrometry: the liquid to be measured that adopts liquid chromatography-level Four bar-electrospray ionization mass spectrum detecting step (2), β-methylamino-ALANINE is determined with retention time and characteristic ion, the drawing standard curve, detect with the external standard standard measure, the chromatography-mass spectroscopy condition is respectively:
Liquid phase chromatogram condition: instrument model: Agilent1290/6460HPLC/MS/MS; Liquid-phase chromatographic column: the EclipseXDB-C18 post, 4.6 * 100mm * 5.0 μ m, column temperature is 45 ℃; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass spectrum condition: electric spray ion source: positive ion electrospray is from pattern ESI+; 320 ℃ of ion source temperatures of dry gas temperature; Gas flow rate 10Lmin
-1Air pressure 40psi; Capillary voltage 3500V; 320 ℃ of sheath temperature degree; Sheath gas velocity 11Lmin
-1Spray nozzle voltage 1000V; Select multiple-reaction monitoring pattern (MRM).
2. the detection method of cyanophycean toxin β-methylamino-ALANINE content in a kind of aquatic products according to claim 1, it is characterized in that: in the step (1), the amount ratio of aquatic products and trichloroacetic acid solution is (10-20) mg:2ml; The concentration of trichloroacetic acid solution is 0.05-0.2mol L
-1Ultrasonic homogenate is adopted in homogenate, and ultrasonic power is 600-800W, ultrasonic time 5-15min; Centrifugal rotational speed is 8000-12000r/m, and centrifugation time is 6-15min; The concentration of hydrochloric acid solution is 3-10molL
-1Hydrolysis temperature is 110 ℃, and hydrolysis time is 18h-24h; Filter membrane aperture 0.22 μ m or 0.45 μ m; Method for concentration is used in 45 ℃ of-70 ℃ of lower nitrogen and blows concentrated.
3. the detection method of cyanophycean toxin β-methylamino-ALANINE content in a kind of aquatic products according to claim 1, it is characterized in that: in the step (2), the concentration of hydrochloric acid solution is 10mmolL
-1-20mmolL
-1
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310186468.XA CN103308639B (en) | 2013-05-17 | 2013-05-17 | Detection method of content of blue algae toxin beta-methylamino-L-alanine in aquatic products |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310186468.XA CN103308639B (en) | 2013-05-17 | 2013-05-17 | Detection method of content of blue algae toxin beta-methylamino-L-alanine in aquatic products |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103308639A true CN103308639A (en) | 2013-09-18 |
| CN103308639B CN103308639B (en) | 2015-01-14 |
Family
ID=49134089
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310186468.XA Active CN103308639B (en) | 2013-05-17 | 2013-05-17 | Detection method of content of blue algae toxin beta-methylamino-L-alanine in aquatic products |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103308639B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107290452A (en) * | 2017-06-23 | 2017-10-24 | 江南大学 | A kind of method for detecting reference state heterocycle amine content |
| CN107389848A (en) * | 2017-07-25 | 2017-11-24 | 中国环境科学研究院 | The extraction of D types amino acid and assay method in a kind of poisons in freshwater |
| CN113466469A (en) * | 2021-07-07 | 2021-10-01 | 北京常友生物科技有限公司 | Biomarker for detecting autism, detection kit and detection system |
-
2013
- 2013-05-17 CN CN201310186468.XA patent/CN103308639B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| PAUL ALAN COX ET AL: "Cyanobacteria and BMAA exposure from desert dust:A possible link to sporadic ALS among Gulf War veterans", 《AMYOTROPHIC LATERAL SCLEROSIS》 * |
| SANDRA ANNE BANACK ET AL: "Production of the Neurotoxin BMAA by a Marine Cyanobacterium", 《MARINE DRUGS》 * |
| THOMAS KRUGER ET AL: "LC-MS/MS determination of the isomeric neurotoxins BMAA (β-N-methylamino-L-alanine) and DAB (2,4-diaminobutyric acid) in cyanobacteria and seeds of Cycas revoluta and Lathyrus latifoius", 《TOXICON》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107290452A (en) * | 2017-06-23 | 2017-10-24 | 江南大学 | A kind of method for detecting reference state heterocycle amine content |
| CN107290452B (en) * | 2017-06-23 | 2022-04-15 | 江南大学 | Method for detecting content of bound heterocyclic amine |
| CN107389848A (en) * | 2017-07-25 | 2017-11-24 | 中国环境科学研究院 | The extraction of D types amino acid and assay method in a kind of poisons in freshwater |
| CN113466469A (en) * | 2021-07-07 | 2021-10-01 | 北京常友生物科技有限公司 | Biomarker for detecting autism, detection kit and detection system |
| CN113466469B (en) * | 2021-07-07 | 2024-01-16 | 北京常友生物科技有限公司 | Biomarker, detection kit and detection system for autism detection |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103308639B (en) | 2015-01-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yan et al. | Determination of melamine in dairy products, fish feed, and fish by capillary zone electrophoresis with diode array detection | |
| Yang et al. | Capillary electrophoresis–chemiluminescence determination of norfloxacin and prulifloxacin | |
| CN107167539A (en) | The detection method of a variety of residues of veterinary drug in a kind of rapid screening flesh of fish | |
| CN104076109A (en) | Method for testing seven indicative PCBs in food with high fat content simultaneously | |
| CN103308639B (en) | Detection method of content of blue algae toxin beta-methylamino-L-alanine in aquatic products | |
| CN106124433B (en) | A method of it quickly detects pyrethroid pesticide remained | |
| CN106596790A (en) | Method for rapidly detecting content of tetrodotoxin in fish flesh through liquid chromatography-mass spectrometry | |
| Lan et al. | Dual-site mitochondria-targeted fluorescent probe for simultaneous distinguishing detection of hypochlorite and SO2 derivatives in real water samples and bioimaging | |
| CN113533565A (en) | Method for detecting concentrations of 8 flavonoid compounds in human urine by UPLC-MS/MS method | |
| Benkova et al. | Determination of plasma aminothiols by high performance liquid chromatography after precolumn derivatization with N-(2-acridonyl) maleimide | |
| Chen et al. | Determination of iodine and bromine compounds in foodstuffs by CE‐inductively coupled plasma MS | |
| Bai et al. | Study on the Methods of Separation and Detection of Chelates | |
| Hu et al. | Ratiometric pyrene-based fluorescent sensor for on-site monitoring of formaldehyde in foods and living cells | |
| CN104820032A (en) | Method for detecting ochratoxin A in vegetables and fruits | |
| CN106770871A (en) | A kind of method of quantitative determination containing alditol acid polysaccharide | |
| Tang et al. | Simultaneous determination of metal ions by capillary electrophoresis with contactless conductivity detection and insights into the effects of BGE component | |
| CN104391071B (en) | The analytical approach of 8-OhdG, 8-hydroxyguanosine and 8-Isoprostanes F2 α in a kind of human urine | |
| Wang et al. | Determination of aminoglycoside antibiotics by a colorimetric method based on the aggregation of gold nanoparticles | |
| Chang et al. | Simultaneous determination of eleven quinolones antibacterial residues in marine products and animal tissues by liquid chromatography with fluorescence detection | |
| CN106596765A (en) | Method for detecting addition amount of maltodextrin in food materials | |
| CN112051343B (en) | Method for determining antibiotic residues | |
| Ward et al. | Accelerated determination of selenomethionine in selenized yeast: validation of analytical method | |
| CN104215614A (en) | Method for detecting aloin A, aloin B and aloe-emodin in series through high performance liquid chromatography-diode array/fluorescent detector | |
| CN104020200A (en) | Electrochemical method for measuring melamine | |
| CN110618230B (en) | Method for detecting dodecyl paraben |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |