CN106442786A - High performance liquid chromatography-tandem mass spectrometry detection method for paralytic shellfish poisoning toxins - Google Patents
High performance liquid chromatography-tandem mass spectrometry detection method for paralytic shellfish poisoning toxins Download PDFInfo
- Publication number
- CN106442786A CN106442786A CN201610858591.5A CN201610858591A CN106442786A CN 106442786 A CN106442786 A CN 106442786A CN 201610858591 A CN201610858591 A CN 201610858591A CN 106442786 A CN106442786 A CN 106442786A
- Authority
- CN
- China
- Prior art keywords
- mass spectrometry
- liquid chromatography
- detection method
- tandem mass
- toxins
- 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.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a high performance liquid chromatography-tandem mass spectrometry detection method for paralytic shellfish poisoning toxins. The method is characterized by comprising the following steps of 1 extracting, 2 purifying, 3 chromatographic condition determining and 4 mass spectrometry condition determining. By detecting a referential positive sample with a certificate through the established method, a satisfactory result is obtained, and the recycling rate is shown in the table 6 (please see the table in the description). The liquid chromatography-tandem mass spectrometry detection method for the PSP toxins is easy and convenient to operate, high in sensitivity, good in reproducibility and high in accuracy and can meet the daily PSP toxin detection requirements.
Description
Technical field
The present invention relates to the high performance liquid chromatography-tandem mass detection method of Paralytic Shellfish Toxins.
Background technology
Ocean shellfish poison toxin is to be produced by various natural marine algae, and shellfish is after filter food toxic algae or its spore, malicious
Element accumulation in shellfish body forms shellfish poison toxin, and shellfish has no intoxicating phenomenon in itself.Taking place frequently with red tide, by shellfish poison toxin
The marine product poisoning causing continuously emerges, and common ocean shellfish poison toxin has Paralytic Shellfish Toxins, diarrhetic shellfish poison poison
Element, neurotoxic shellfish poison's toxin, amnesic shellfish poisoning toxin, wherein Paralytic Shellfish Toxins are to be distributed the widest, danger so far
The maximum class ocean shellfish poison toxin of evil.
PSP toxin is the derivative of a class tetrahydrochysene purine.It has now been discovered that toxin have kind more than 20, according to R4 group
Difference can be divided into four classes, is carbamate toxoid (Carbamate toxins) respectively, including saxitoxin
(STX), N-STX (neoSTX), GTX1-4 (GTX1-4)；N- sulphonyl carbamyl toxoid (N-
Sulfocarbamoyl toxins), including B1-2, C1-4；Deamination formoxyl toxoid (decarbamoyl toxins), bag
Include dcSTX, dcneoSTX, dcGTX1-4；Deoxidation deamination formoxyl toxoid (deoxydecarbamoyl toxins), including
DoSTX, doGTX2-3.
The detection method of PSP toxin mainly has Mouse bioassay, liquid chromatography and liquid chromatography-tandem mass spectrometry mass spectrum
Method.Mouse bioassay is limited with selective due to sensitivity, is mainly used in screening analysis.Liquid phase process needs to add in mobile phase
Enter buffer salt, have certain damage to instrument, and PSP lps molecule chromophore itself very weak it is impossible to directly use ultraviolet or
Fluoroscopic examination, generally carries out deriving the ring structure of PSP lps molecule is oxidized to fluorescence chromophore.Liquid chromatography-tandem mass spectrometry
Method is provided that compound structure information, and sensitivity is higher, easy and simple to handle, has high qualitative, quantitation capabilities, effectively makes up
State all kinds of defects of method presence, can be used as the confirmation detection method of PSP toxin.
Content of the invention
The technical problem to be solved is：A kind of high performance liquid chromatography-the series connection of Paralytic Shellfish Toxins is provided
Mass Spectrometry detection method.
For solving above-mentioned technical problem, the technical solution used in the present invention is：
The high performance liquid chromatography-tandem mass detection method of the Paralytic Shellfish Toxins that the present invention provides, including following step
Suddenly：
(1) sample preparation
(1) extract
Accurately weigh the sample after 2.0g homogeneous, have in plug centrifuge tube in 15mL, add 10mL 0.1mol/L hydrochloric acid molten
Liquid, is simultaneously introduced 2-8g sodium chloride and 2-8g calcium chloride, is vortexed and mixes, after ultrasonic extraction 5-10min, with 5000r/min centrifugation
10min；Take supernatant standby；
Continuously add 10mL 30% ethanol water and 2-8g calcium chloride in centrifuge tube, be vortexed and mix, then in 70-
80 DEG C of ultrasonic extractions 5-10min, are centrifuged 5min with 5000r/min, take supernatant standby；
To supernatant merge twice, to be clean as extract.
(2) purify
Successively with 3mL methyl alcohol, 3mL water balance solid-phase extraction column, took that 5mL extract is whole to cross post, flow velocity 1-3mL/min,
Respectively with 3mL water and 3mL methyl alcohol drip washing, fully dry up leacheate, contain 80% methanol-eluted fractions of 2% formic acid, eluent with 3mL
After nitrogen is blown to closely do, treat Mass Spectrometer Method with after 1mL mobile phase constant volume.
(3) chromatographic condition
Mobile phase A is water, and B is 95% acetonitrile solution, and A, B contain 2mM ammonium formate, 7.2mM formic acid (pH 2.5)；Stream
Fast 300 μ L/min；30 DEG C of column temperature；Sample size 10 μ L；Gradient elution：
Condition of gradient elution
| Time, min | Flow velocity, μ L/min | A | B |
| 0 | 300 | 30 | 70 |
| 10 | 300 | 80 | 20 |
| 10.1 | 300 | 30 | 70 |
| 18 | 300 | 30 | 70 |
(4) Mass Spectrometry Conditions
Ion gun：Electric spray ion source；Scan mode：ESI+；Monitoring mode：Polyion reaction monitoring；Source parameters：
Collision gas (CAD) 12, gas curtain gas (CUR) 10, GS1 60, GS2 70, electron spray voltage (IS) 5000, ion source temperature (TEM)
600；
MRM parameter is as follows：
MRM parameter
* it is quota ion.
The method detection PSP toxin method that the present invention sets up is easy and simple to handle, sensitivity is strong, favorable reproducibility, and accuracy is high, can
Required with meeting the daily detection for PSP toxin.
Meanwhile, the present invention passes through to improve sample pre-treatments technique, adds extractant-sodium chloride and chlorination in extract
Calcium, meanwhile, the residue after first time is extracted carries out second extraction with ethanol, and the rate of recovery greatly improves.
Specific embodiment
Embodiment 1
The high performance liquid chromatography-tandem mass detection method of the Paralytic Shellfish Toxins that the present embodiment provides, including following
Step：
(1) sample preparation
(1) extract
Accurately weigh the sample after 2.0g homogeneous, have in plug centrifuge tube in 15mL, add 10mL 0.1mol/L hydrochloric acid molten
Liquid, is simultaneously introduced 6g sodium chloride and 6g calcium chloride, is vortexed and mixes, and after ultrasonic extraction 10min, is centrifuged 10min with 5000r/min；
Take supernatant standby；
Continuously add 10mL 30% ethanol water and 6g calcium chloride in centrifuge tube, be vortexed and mix, then in 70-80
DEG C ultrasonic extraction 10min, is centrifuged 5min with 5000r/min, takes supernatant standby；
To supernatant merge twice, to be clean as extract.
(2) purify
Successively with 3mL methyl alcohol, 3mL water balance solid-phase extraction column, took that 5mL extract is whole to cross post, flow velocity 1-3mL/min,
Respectively with 3mL water and 3mL methyl alcohol drip washing, fully dry up leacheate, contain 80% methanol-eluted fractions of 2% formic acid, eluent with 3mL
After nitrogen is blown to closely do, treat Mass Spectrometer Method with after 1mL mobile phase constant volume.
(3) chromatographic condition
Mobile phase A is water, and B is 95% acetonitrile solution, and A, B contain 2mM ammonium formate, 7.2mM formic acid (pH 2.5)；Stream
Fast 300 μ L/min；30 DEG C of column temperature；Sample size 10 μ L；Gradient elution：
Condition of gradient elution
| Time, min | Flow velocity, μ L/min | A | B |
| 0 | 300 | 30 | 70 |
| 10 | 300 | 80 | 20 |
| 10.1 | 300 | 30 | 70 |
| 18 | 300 | 30 | 70 |
(4) Mass Spectrometry Conditions
Ion gun：Electric spray ion source；Scan mode：ESI+；Monitoring mode：Polyion reaction monitoring；Source parameters：
Collision gas (CAD) 12, gas curtain gas (CUR) 10, GS1 60, GS2 70, electron spray voltage (IS) 5000, ion source temperature (TEM)
600；
MRM parameter is as follows：
MRM parameter
* it is quota ion.
Embodiment 2 detects lower bound
6 points of calibration curves of PSP toxin standard items carry out quantitation.In linear scope, linearly well, R2More than 0.99.Inspection
Survey lower bound to calculate with 10 times of signal to noise ratios, as shown in the table, qualitative ion signal to noise ratio requires to reach more than 3.
The detection lower bound of PSP toxin
Embodiment 3 rate of recovery and precision
Add the PSP toxin mixed standard solution of 2 concentration levels in blank clam sample, each pitch-based sphere is carried out
6 parallel sample, the sample extraction set up according to the present invention, purification method, using HILIC chromatographic column, with liquid chromatography-tandem
Mass spectrum is detected, calculates the rate of recovery and precision, and result is as shown in the table.
Pitch-based sphere 1 is 5 μ g/kg.
Pitch-based sphere 2 is 50 μ g/kg.
The rate of recovery of PSP toxin and precision
Embodiment 4 is with reference to the detection of positive
Using the method set up, the reference positive with certificate is detected, shown in the rate of recovery.Liquid chromatogram-string
Mass Spectrometer Method PSP toxin method is easy and simple to handle for connection, sensitivity is strong, favorable reproducibility, and accuracy is high, can meet daily for PSP
The detection of toxin requires.
PSP is with reference to the rate of recovery of positive
Claims (1)
1. the high performance liquid chromatography-tandem mass detection method of Paralytic Shellfish Toxins is it is characterised in that comprise the following steps：
(1) sample preparation
(1) extract
Accurately weigh the sample after 2.0g homogeneous, have in plug centrifuge tube in 15mL, add 10mL 0.1mol/L hydrochloric acid solution, with
When add 2-8g sodium chloride and 2-8g calcium chloride, be vortexed and mix, after ultrasonic extraction 5-10min, 10min is centrifuged with 5000r/min；
Take supernatant standby；
Continuously add 10mL 30% ethanol water and 2-8g calcium chloride in centrifuge tube, be vortexed and mix, then at 70-80 DEG C
Ultrasonic extraction 5-10min, is centrifuged 5min with 5000r/min, takes supernatant standby；
To supernatant merge twice, to be clean as extract；
(2) purify
Successively with 3mL methyl alcohol, 3mL water balance solid-phase extraction column, took the whole post excessively of 5mL extract, flow velocity 1-3mL/min, difference
With 3mL water and 3mL methyl alcohol drip washing, fully dry up leacheate, contain 80% methanol-eluted fractions of 2% formic acid, eluent nitrogen with 3mL
After being blown to closely do, treat Mass Spectrometer Method with after 1mL mobile phase constant volume；
(3) chromatographic condition
Mobile phase A is water, and B is 95% acetonitrile solution, and A, B contain 2mM ammonium formate, 7.2mM formic acid (pH 2.5)；Flow velocity
300μL/min；30 DEG C of column temperature；Sample size 10 μ L；Gradient elution；
Condition of gradient elution
(4) Mass Spectrometry Conditions
Ion gun：Electric spray ion source；Scan mode：ESI+；Monitoring mode：Polyion reaction monitoring；Source parameters：Collision
Gas (CAD) 12, gas curtain gas (CUR) 10, GS1 60, GS2 70, electron spray voltage (IS) 5000, ion source temperature (TEM) 600；
MRM parameter is as follows：
MRM parameter
* it is quota ion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610858591.5A CN106442786A (en) | 2016-09-28 | 2016-09-28 | High performance liquid chromatography-tandem mass spectrometry detection method for paralytic shellfish poisoning toxins |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610858591.5A CN106442786A (en) | 2016-09-28 | 2016-09-28 | High performance liquid chromatography-tandem mass spectrometry detection method for paralytic shellfish poisoning toxins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106442786A true CN106442786A (en) | 2017-02-22 |
Family
ID=58169862
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610858591.5A Pending CN106442786A (en) | 2016-09-28 | 2016-09-28 | High performance liquid chromatography-tandem mass spectrometry detection method for paralytic shellfish poisoning toxins |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106442786A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107102081A (en) * | 2017-04-29 | 2017-08-29 | 连云港市海洋环境监测预报中心 | A kind of method that rapid screening differentiates a variety of paralytic shellfish poison's elements in red tide algae |
| CN116990415A (en) * | 2023-08-14 | 2023-11-03 | 中国人民解放军军事科学院军事医学研究院 | Non-targeted screening method for typical paralytic shellfish toxins in shellfish meat |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563024A (en) * | 2004-03-20 | 2005-01-12 | 中国科学院水生生物研究所 | Method for extracting toxin of synnema alga |
| CN101196517A (en) * | 2007-12-13 | 2008-06-11 | 上海交通大学 | Method for early detecting toxicity of paralytic shellfish poison |
-
2016
- 2016-09-28 CN CN201610858591.5A patent/CN106442786A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563024A (en) * | 2004-03-20 | 2005-01-12 | 中国科学院水生生物研究所 | Method for extracting toxin of synnema alga |
| CN101196517A (en) * | 2007-12-13 | 2008-06-11 | 上海交通大学 | Method for early detecting toxicity of paralytic shellfish poison |
Non-Patent Citations (6)
| Title |
|---|
| LIYANG ZHUO ET AL: "Determination of paralytic shellfish poisoning toxins by HILIC-MS/MS coupled with dispersive solid phase extraction", 《FOOD CHEMISTRY》 * |
| 刘红河 等: "麻痹性贝类毒素的高效液相色谱-串联质谱测定方法研究", 《实用预防医学》 * |
| 吴振兴 等: "麻痹性贝毒毒素的高效液相色谱－串联质谱检测方法研究", 《海洋湖沼通报》 * |
| 沈月新 主编: "《水产食品学》", 30 April 2001, 中国农业出版社 * |
| 王连生 等译: "《环境有机化学》", 31 January 2004, 化学工业出版社、环境科学与工程出版中心 * |
| 陈剑刚 等: "固相萃取-高效液相色谱-串联质谱法测定贝类产品中麻痹性贝类毒素", 《中国卫生检验杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107102081A (en) * | 2017-04-29 | 2017-08-29 | 连云港市海洋环境监测预报中心 | A kind of method that rapid screening differentiates a variety of paralytic shellfish poison's elements in red tide algae |
| CN116990415A (en) * | 2023-08-14 | 2023-11-03 | 中国人民解放军军事科学院军事医学研究院 | Non-targeted screening method for typical paralytic shellfish toxins in shellfish meat |
| CN116990415B (en) * | 2023-08-14 | 2024-04-12 | 中国人民解放军军事科学院军事医学研究院 | Non-targeted screening method for typical paralytic shellfish toxins in shellfish meat |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110146632B (en) | Liquid chromatography-mass spectrometry detection method for various marine biotoxins in aquatic products | |
| CN102928528B (en) | High performance liquid chromatography-mass spectrometry detecting method of 16 fat soluble saxitoxins in shellfish meet | |
| CN103543221A (en) | Method for simultaneously detecting multiple mycotoxins in milk | |
| CN106324144B (en) | The method of chlorate, perchlorate and bromate in hydrophilic Interaction Chromatography-tandem mass spectrometry detection milk powder and milk power for infant and young children | |
| Chen et al. | Simultaneous screening for lipophilic and hydrophilic toxins in marine harmful algae using a serially coupled reversed-phase and hydrophilic interaction liquid chromatography separation system with high-resolution mass spectrometry | |
| CN105021737B (en) | It is a kind of at the same detect milk product in dicyandiamide and content of melamine method | |
| CN110174470B (en) | High-flux detection method for marine biotoxin in aquatic product | |
| CN106872617B (en) | Rapid extraction and LC-MS-MS detection method of benzimidazole and thiazole residual drugs in aquatic products | |
| CN109342624A (en) | The method that Solid Phase Extraction pre-treatment combination LC-MS technology detects 15 kinds of antibiotic in aquaculture system simultaneously | |
| CN107144645A (en) | It is a kind of while detecting the method for a variety of mycotoxins | |
| CN106153783A (en) | The Liquid Chromatography-Tandem Mass Spectrometry detection method of diarrhetic shellfish poison toxin | |
| CN106442786A (en) | High performance liquid chromatography-tandem mass spectrometry detection method for paralytic shellfish poisoning toxins | |
| CN111103387A (en) | Method for measuring chloroacetic acid, dichloroacetic acid and trichloroacetic acid in soil | |
| CN103808814B (en) | A kind of method for detecting Phthalic Acid Esters in Food | |
| CN110261517A (en) | Utilize the method for N- nitrosamine compound in GPC-GC/MS/MS measurement meat products | |
| CN106645451A (en) | Detection method of ochratoxin A in cereals | |
| CN101865887B (en) | Method for detecting nitromidazole residue in royal jelly by using high performance liquid chromatography tandem mass spectrum | |
| CN110702816B (en) | Liquid chromatography-mass spectrometry detection method for detecting acidic azo colorant in dry animal aquatic product | |
| CN105784905B (en) | Ochratoxin B Liquid Chromatography-Tandem Mass Spectrometry detection method in pet food | |
| CN109507306B (en) | Method for determining acetyl tributyl citrate in water-based adhesive | |
| CN112858536A (en) | LC-MS (liquid chromatography-Mass Spectrometry) detection method for saxitoxin in bivalve aquatic products | |
| CN109633071B (en) | Method for detecting Saisentong copper in water by using UPLC-MS/MS method | |
| CN105806979B (en) | The liquid chromatography-tandem mass of ochratoxin A in dog chew | |
| CN107991402B (en) | Method for simultaneously detecting 6 groups of azo sulfonic pigment isomers in bean products | |
| CN111474279A (en) | Method and kit for detecting macrolide antibiotic compounds |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170222 |
|
| RJ01 | Rejection of invention patent application after publication |