CN103308639B - 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
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- 239000003053 toxin Substances 0.000 title claims abstract description 28
- 231100000765 toxin Toxicity 0.000 title claims abstract description 28
- 238000001514 detection method Methods 0.000 title claims abstract description 18
- 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 32
- 238000001212 derivatisation Methods 0.000 claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 238000004885 tandem mass spectrometry Methods 0.000 claims abstract description 8
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 claims abstract description 7
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- 239000000047 product Substances 0.000 claims description 23
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- AJYLWDRNNNHBEV-VKHMYHEASA-N (2s)-2-hydrazinylbutanoic acid Chemical compound CC[C@H](NN)C(O)=O AJYLWDRNNNHBEV-VKHMYHEASA-N 0.000 claims description 10
- 238000010812 external standard method Methods 0.000 claims description 10
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- 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
- 230000007062 hydrolysis Effects 0.000 claims description 6
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- 238000005119 centrifugation Methods 0.000 claims description 2
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- 238000001556 precipitation 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
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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 analytical chemistry field, relate to the detection method of pollutant in 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 within the scope of the world today and scale all in rising trend, inland lake and the coastal ocean water body of multiple country are all in serious eutrophication state, therefore carry out for the research of blue-green alga bloom pollutant and very urgent to human health risk assessment.The emphasis of current research is on traditional pollutants such as Microcystin (Microcystin), for the research of other cyanophycean toxins seldom, and most of blue-green algae all can produce the phenomenon of β-methylamino-ALANINE BMAA toxin after 2003 are found, cause the great attention of international community, detect delay has been carried out in multiple area all.Even if research external at present has shown in the ocean water environment not depositing sago cycas, the BMAA(free state that two kinds of forms exist and in conjunction with state) still can pass through hydrobiont food chain (blue-green algae-phytoplankton-animal plankton-fish) and carry out moving and amplifying.Also similar toxin migration and amplification approach may be there is in this achievement hint in China's breakout of water bloom waters or blue-green algae grown on larger scale waters.China is one of most critical regions of breakout of cyanobacteria blooms in the world, and the investigation distribution research carrying out blue-green algae BMAA toxin in inland lake natural water environment seems particularly important.
Owing to lacking the effective measures preventing algal bloom from occurring at present, therefore will prevent and eliminate the harm of cyanophycean toxin to people and animals, the limitation of monitor and forecast cyanophycean toxin in all kinds of aquatic products is effective method.Liquid chromatography mass coupling method has been applied to the detection of multiple Algae toxins compound in water body and biosome.But it is less for the detect delay of BMAA.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 object of this invention is to provide a kind of highly sensitive, testing result detection method of cyanophycean toxin β-methylamino-ALANINE content in aquatic products accurately.
Technical scheme: the detection method of cyanophycean toxin β-methylamino-ALANINE content in a kind of aquatic products provided by the invention, comprises the following steps:
(1) pre-service: cryodesiccated aquatic products are pulverized evenly, adds trichloroacetic acid solution homogenate on ice bath, centrifugal, obtain supernatant; After precipitation adds hydrochloric acid solution hydrolysis, membrane filtration, obtains filtrate; Merge supernatant and filtrate, 55 DEG C of nitrogen blow concentrated, obtain concentrate;
(2) derivatization: concentrate is added dissolving with hydrochloric acid, gets 20 μ L derivatizations, obtains liquid to be measured;
(3) liquid chromatography-level Four bar-Electrospray Ionization Mass Spectrometry: the liquid to be measured adopting liquid chromatography-level Four bar-electrospray ionization mass spectrum detecting step (2), β-methylamino-ALANINE is determined with retention time and characteristic ion, drawing standard curve, detect with quantified by external standard method, chromatography-mass spectroscopy condition is respectively:
Liquid phase chromatogram condition: INSTRUMENT MODEL: Agilent1290/6460HPLC/MS/MS; Liquid-phase chromatographic column: EclipseXDB-C18 post, 4.6 × 100mm × 5.0 μm, column temperature is 45 DEG C; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass Spectrometry Conditions: electric spray ion source: positive ion electrospray is from pattern ESI+; Dry gas temperature 320 DEG C of ion source temperatures; Gas flow rate 10Lmin
-1; Air pressure 40psi; Capillary voltage 3500V; Sheath temperature degree 320 DEG C; Sheath gas velocity 11Lmin
-1; Spray nozzle voltage 1000V; Select multiple-reaction monitoring pattern (MRM).
In step (1), in 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
-1; Homogenate adopts ultrasonic 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
-1; Hydrolysis temperature is 110 DEG C, and hydrolysis time is 18h-24h, preferred 24h; Filter sizes 0.22 μm or 0.45 μm, be preferably 0.22 μm; At method for concentration is used in 45 DEG C-70 DEG C, nitrogen blows concentrated, and preferably nitrogen blows concentrated at 55 DEG C.
In step (2), the concentration of hydrochloric acid solution is 10mmolL
-1-20mmolL
-1, preferred 20mmolL
-1; Derivatization method is AccQ-Tag Μ Ltra Derivatization Kit (Waters, USA) kit instructions method.
Beneficial effect: in aquatic products provided by the invention, the detection method of cyanophycean toxin β-methylamino-ALANINE content is simple to operate, analysis speed is fast, the problem that the method is complicated for biological specimen matrix in water environment, pretreatment technology difficulty is large, the pretreating process adopted can extract free state in aquatic products and the BMAA in conjunction with state, and high performance liquid chromatography-quadrupole rods tandem mass spectrometry method connection detection method detectability of employing is low, favorable reproducibility, highly sensitive, the recovery is better and degree of accuracy is high.
Accompanying drawing explanation
Fig. 1 is BMAA standard items is (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.But those skilled in the art will readily understand, concrete material proportion, process conditions and result thereof described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1
1. the extraction of sample
By even for cryodesiccated aquatic products crucian flesh of fish sample comminution, take 15mg dry powder and add 2mL0.1molL
-1trichloroacetic acid solution is in homogenised sample on ice, and ultrasonic power is 600-800W, in the centrifugal 10min of 12000r/m after ultrasonic 10min, pipettes supernatant liquor, as BMAA free state to be measured.Sediment fraction adds 6molL
-1hCL, and be fully hydrolyzed 24h under being placed in 110 DEG C of conditions, be cooled to natural temperature, filter through 0.22 μm of teflon filter, the hydrolysate filtrate obtained and free state BMAA two parts of collecting all under 55 DEG C of conditions nitrogen blow concentrated, to be analyzed in-20 DEG C of preservations.
2. analyte derivative
Before LC-MS analysis, the appropriate 20mM HCl of freezing sample step 1 preserved dissolves, 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 one 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: EclipseXDB-C18 post, 4.6 × 100mm × 5.0 μm, column temperature is 45 DEG C; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass Spectrometry Conditions: electric spray ion source: positive ion electrospray is from pattern ESI+; Dry gas temperature 320 DEG C of ion source temperatures; Gas flow rate 10Lmin
-1; Air pressure 40psi; Capillary voltage 3500V; Sheath temperature degree 320 DEG C; Sheath gas velocity 11Lmin
-1; Spray nozzle voltage 1000V.Select multiple-reaction monitoring pattern (MRM), target compound is determined with retention time and characteristic ion, and drawing standard curve, quantitatively detects with external standard method.
4. the drafting of typical curve, carries out quantitative measurement with external standard method
Accurately take BMAA toxin standard items, be dissolved in 50% acetonitrile solution, be mixed with the standard solution of a series of variable concentrations, with the concentration of BMAA toxin for horizontal ordinate, peak area after liquid phase-mass spectrometry analysis measures is that ordinate carries out regretional analysis, obtain regression equation y=20533x-1609.3, related coefficient is 0.9957.For object in working sample.In blank sample, add the standard solution of variable concentrations, after identical process, the interpolation concentration obtaining 3 times of signal to noise ratio (S/N ratio)s is detectability; Detecting of the method is limited to 6ng/g, and in the scope of concentration 0.05 μ g/mL ~ 1mg/mL, peak area and sample concentration are good linear relationship.
The stratographic analysis retention time of BMAA and quota ion are in 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 to be measured flesh of fish sample, after effectively extracting by step 1 pair sample, derivatization treatment is carried out by step 2, HPLC-MS detection is carried out again by step 3, and compare with above-mentioned obtained typical curve, finally obtain BMAA free state and the content in conjunction with state in crucian flesh of fish sample by calculating.
After adopting identical crucian flesh of fish sample to carry out freeze drying, take 15mg blank sample to be measured, in adding 2mL0.2molL on ice
-1trichloroacetic acid, adds standard items BMAA solution according to the addition of 0.5 μ g/g simultaneously.Carry out the content that identical pre-service measures BMAA, and carry out recovery calculating according to the following formula:
R=(C-C
0)/0.5×100%
R-recovery, %
BMAA content of toxins in aquatic products sample after 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 all carries 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% |
Embodiment 2
1. the extraction of sample
Cryodesiccated aquatic products river snail sample edible portion is pulverized evenly, takes 15mg dry powder and add 2mL0.1molL
-1trichloroacetic acid is in homogenised sample on ice, and ultrasonic power is 600-800W, in the centrifugal 10min of 12000r/m after ultrasonic 10min, pipettes supernatant liquor, as BMAA free state to be measured.Sediment fraction adds 6molL
-1hCL, and be fully hydrolyzed 24h under being placed in 110 DEG C of conditions, be cooled to natural temperature, filter through 0.22 μm of teflon filter, the hydrolysate filtrate obtained and free state BMAA two parts of collecting all under 55 DEG C of conditions nitrogen blow concentrated, to be analyzed in-20 DEG C of preservations.
2. analyte derivative
Before LC-MS analysis, the appropriate 20mMHCL of freezing sample step 1 preserved dissolves, 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 one 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: EclipseXDB-C18 post, 4.6 × 100mm × 5.0 μm, column temperature is 45 DEG C; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass Spectrometry Conditions: electric spray ion source: positive ion electrospray is from pattern ESI+; Dry gas temperature 320 DEG C of ion source temperatures; Gas flow rate 10Lmin
-1; Air pressure 40psi; Capillary voltage 3500V; Sheath temperature degree 320 DEG C; Sheath gas velocity 11Lmin
-1; Spray nozzle voltage 1000V.Select multiple-reaction monitoring pattern (MRM), target compound is determined with retention time and characteristic ion, and drawing standard curve, quantitatively detects with external standard method.
4. the drafting of typical curve, carries out quantitative measurement with external standard method
Accurately take BMAA toxin standard items, be dissolved in 50% acetonitrile solution, be mixed with a series of standard to be dissolved in, with the concentration of BMAA toxin for horizontal ordinate, peak area after liquid phase-mass spectrometry analysis measures is that ordinate carries out regretional analysis, regression equation is y=25608x-4231.5, and related coefficient is 0.9976.Carry out replicate determination (n=6) to 100 μ g/mLBMAA standard solutions, relative standard deviation RSD is 0.5%.In blank sample, add the standard solution of variable concentrations, after identical process, the interpolation concentration obtaining 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 portion to be measured, after effectively extracting by step 1 pair sample, derivatization treatment is carried out by step 2, HPLC-MS detection is carried out again by step 3, and compare with above-mentioned obtained typical curve, BMAA free state and the content in conjunction with state in river snail sample edible portion is finally obtained by converting.
After adopting identical river snail sample edible portion to carry out freeze drying, take 15mg blank sample to be measured, in adding 2mL0.1molL on ice
-1trichloroacetic acid, adds BMAA standard solution by 0.5 μ g/g addition.Carry out the content that identical pre-service measures BMAA, and carry out recovery calculating according to the following formula:
R=(C-C
0)/0.5×100%
R-recovery, %
BMAA content of toxins in aquatic products sample after 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 all carries 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 portion (μ g/g dry weight) | The recovery/% | RSD/% |
| BMAA | 0.63μg/g | 78.6% | 5.7% |
Embodiment 3
1. the extraction of sample
Cryodesiccated aquatic products river snail sample edible portion is pulverized evenly, takes 10mg dry powder and add 2mL0.2molL
-1trichloroacetic acid is in homogenised sample on ice, and ultrasonic power is 600W, in the centrifugal 15min of 8000r/m after ultrasonic 15min, pipettes supernatant liquor, as BMAA free state to be measured.Sediment fraction adds 3molL
-1hCL, and be fully hydrolyzed 18h under being placed in 110 DEG C of conditions, be cooled to natural temperature, filter through 0.22 μm of teflon filter, the hydrolysate filtrate obtained and free state BMAA two parts of collecting all under 70 DEG C of conditions nitrogen blow concentrated, to be analyzed in-20 DEG C of preservations.
2. analyte derivative
Before LC-MS analysis, the appropriate 10mM HCl of freezing sample step 1 preserved dissolves, 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 one 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: EclipseXDB-C18 post, 4.6 × 100mm × 5.0 μm, column temperature is 45 DEG C; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass Spectrometry Conditions: electric spray ion source: positive ion electrospray is from pattern ESI+; Dry gas temperature 320 DEG C of ion source temperatures; Gas flow rate 10Lmin
-1; Air pressure 40psi; Capillary voltage 3500V; Sheath temperature degree 320 DEG C; Sheath gas velocity 11Lmin
-1; Spray nozzle voltage 1000V.Select multiple-reaction monitoring pattern (MRM), target compound is determined with retention time and characteristic ion, and drawing standard curve, quantitatively detects with external standard method.
4. the drafting of typical curve, carries out quantitative measurement with external standard method
Accurately take BMAA toxin standard items, be dissolved in 50% acetonitrile solution, be mixed with a series of standard to be dissolved in, with the concentration of BMAA toxin for horizontal ordinate, peak area after liquid phase-mass spectrometry analysis measures is that ordinate carries out regretional analysis, regression equation is y=22574x-4581.3, and related coefficient is 0.9981.Carry out replicate determination (n=6) to 100 μ g/mLBMAA standard solutions, relative standard deviation RSD is 0.5%.In blank sample, add the standard solution of variable concentrations, after identical process, the interpolation concentration obtaining 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 portion to be measured, after effectively extracting by step 1 pair sample, derivatization treatment is carried out by step 2, HPLC-MS detection is carried out again by step 3, and compare with above-mentioned obtained typical curve, BMAA free state and the content in conjunction with state in river snail sample edible portion is finally obtained by converting.
Embodiment 4
1. the extraction of sample
Cryodesiccated aquatic products river snail sample edible portion is pulverized evenly, takes 20mg dry powder and add 2mL0.05molL
-1trichloroacetic acid is in homogenised sample on ice, and ultrasonic power is 800W, in the centrifugal 6min of 10000r/m after ultrasonic 5min, pipettes supernatant liquor, as BMAA free state to be measured.Sediment fraction adds 10molL
-1hCL, and be fully hydrolyzed 20h under being placed in 110 DEG C of conditions, be cooled to natural temperature, filter through 0.22 μm of teflon filter, the hydrolysate filtrate obtained and free state BMAA two parts of collecting all under 40 DEG C of conditions nitrogen blow concentrated, to be analyzed in-20 DEG C of preservations.
2. analyte derivative
Before LC-MS analysis, the appropriate 15mMHCL of freezing sample step 1 preserved dissolves, 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 one 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: EclipseXDB-C18 post, 4.6 × 100mm × 5.0 μm, column temperature is 45 DEG C; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass Spectrometry Conditions: electric spray ion source: positive ion electrospray is from pattern ESI+; Dry gas temperature 320 DEG C of ion source temperatures; Gas flow rate 10Lmin
-1; Air pressure 40psi; Capillary voltage 3500V; Sheath temperature degree 320 DEG C; Sheath gas velocity 11Lmin
-1; Spray nozzle voltage 1000V.Select multiple-reaction monitoring pattern (MRM), target compound is determined with retention time and characteristic ion, and drawing standard curve, quantitatively detects with external standard method.
4. the drafting of typical curve, carries out quantitative measurement with external standard method
Accurately take BMAA toxin standard items, be dissolved in 50% acetonitrile solution, be mixed with a series of standard to be dissolved in, with the concentration of BMAA toxin for horizontal ordinate, peak area after liquid phase-mass spectrometry analysis measures is that ordinate carries out regretional analysis, regression equation is y=24821x-3815.5, and related coefficient is 0.9978.Carry out replicate determination (n=6) to 100 μ g/mLBMAA standard solutions, relative standard deviation RSD is 0.5%.In blank sample, add the standard solution of variable concentrations, after identical process, the interpolation concentration obtaining 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 portion to be measured, after effectively extracting by step 1 pair sample, derivatization treatment is carried out by step 2, HPLC-MS detection is carried out again by step 3, and compare with above-mentioned obtained typical curve, BMAA free state and the content in conjunction with state in river snail sample edible portion is finally obtained by converting.
Claims (2)
1. the detection method of cyanophycean toxin β-methylamino-ALANINE content in aquatic products, is characterized in that: comprise the following steps:
(1) pre-service: cryodesiccated aquatic products are pulverized evenly, adds trichloroacetic acid solution homogenate on ice bath, centrifugal, obtain supernatant; After precipitation adds hydrochloric acid solution hydrolysis, membrane filtration, obtains filtrate; Merge supernatant and filtrate, concentrated, obtain concentrate; Wherein, 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
-1; Homogenate adopts ultrasonic 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
-1; Hydrolysis temperature is 110 DEG C, and hydrolysis time is 18h-24h; Filter sizes 0.22 μm or 0.45 μm; At method for concentration is used in 45 DEG C-70 DEG C, nitrogen blows concentrated;
(2) derivatization: concentrate is added dissolving with hydrochloric acid, gets 20 μ L derivatizations, obtains liquid to be measured;
(3) liquid chromatography-level Four bar-Electrospray Ionization Mass Spectrometry: the liquid to be measured adopting liquid chromatography-level Four bar-electrospray ionization mass spectrum detecting step (2), β-methylamino-ALANINE is determined with retention time and characteristic ion, drawing standard curve, detect with quantified by external standard method, chromatography-mass spectroscopy condition is respectively:
Liquid phase chromatogram condition: INSTRUMENT MODEL: Agilent1290/6460HPLC/MS/MS; Liquid-phase chromatographic column: EclipseXDB-C18 post, 4.6 × 100mm × 5.0 μm, column temperature is 45 DEG C; Mobile phase is A:0.1% ammoniacal liquor, and B is 28% acetonitrile water; Flow velocity is 0.60mL/min;
Mass Spectrometry Conditions: electric spray ion source: positive ion electrospray is from pattern ESI+; Dry gas temperature 320 DEG C of ion source temperatures; Gas flow rate 10Lmin
-1; Air pressure 40psi; Capillary voltage 3500V; Sheath temperature degree 320 DEG C; Sheath gas velocity 11Lmin
-1; Spray 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 step (2), the concentration of hydrochloric acid solution is 10mmolL
-1-20mmolL
-1.
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| 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;Thomas Kruger et al;《Toxicon》;20091013(第55期);547-557 * |
| Production of the Neurotoxin BMAA by a Marine Cyanobacterium;Sandra Anne Banack et al;《Marine Drugs》;20071206;第193-194页第4f节 * |
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