CN102928529B - High performance liquid chromatography mass spectrometry detecting method of 16 fat soluble saxitoxins in seawater - Google Patents
High performance liquid chromatography mass spectrometry detecting method of 16 fat soluble saxitoxins in seawater Download PDFInfo
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- CN102928529B CN102928529B CN201210388457.5A CN201210388457A CN102928529B CN 102928529 B CN102928529 B CN 102928529B CN 201210388457 A CN201210388457 A CN 201210388457A CN 102928529 B CN102928529 B CN 102928529B
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- absorbent packet
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Abstract
The invention discloses a high performance liquid chromatography mass spectrometry method of 16 fat soluble saxitoxins in seawater. The detecting method comprises the following steps of: making an absorbing bag, using the absorbing bag, extracting the saxitoxins, testing the condition and the mode, qualitatively testing and quantitatively testing. According to the detecting method, resin is adopted to actively absorb the saxitoxins in the seawater, the specific adsorption of different hierarchies of the seawater is carried out, and the detection to the 16 fat soluble saxitoxins in the seawater is analyzed in the extraction step. Compared with the traditional method, the detecting method has small sampling quantity, cost reduction, operation simpliness and convenience and strong stability.
Description
Technical field
The present invention relates to a kind of detection method of fat-soluble saxitoxin, particularly the LC-MS detection method of 16 kinds of fat-soluble saxitoxins in a kind of seawater.
Background technology
Marine biotoxins (saxitoxin) refer in particular to mainly by the poisonous micro-algae in ocean or microorganisms, can be in sea life especially bivalve shellfish enrichment, other biological is comprised to the mankind produce a large micromolecular toxic chemical substance of harm.For these biotoxins, researcher's initial stage is mainly divided into six large classes according to caused toxicity symptom: paralytic shellfish poisoning (PSP) (Paralytic Shellfish Poisoning, PSP), research of diarrhetic shellfish poisons (Diarrhetic Shellfish Poisoning, DSP), nerve saxitoxin (Neurotoxic Shellfish Poisoning, NSP), Toxin-Domoic Acid (Amnesic Shellfish Poisoning, ASP), west adds ichtyhotoxisin (Ciguatera Fish Poisoning, CFP), blue-green algae toxin.But, along with the going deep into of research, new biotoxin kind is constantly found, and multiple toxin often forms with toxin A ZA and PTX association as OA, but has different mechanism of toxication, and original sorting technique can not meet the demand of management and scientific research.Therefore, 2004, by FAO (Food and Agriculture Organization of the United Nation), the common bivalve software biotoxin working group of setting up of the World Health Organization (WHO) and Intergovernmental Oceanographic Commission is divided into eight large classes by saxitoxin, be respectively saxitoxin group (Saxitoxin, STX), domoic acid group (Domoic acid, DA), okadaic acid toxin group (Okadaic acid, OA), former many formic acid toxin group (Azaspiracid, AZA), short and naked dinoflagellate toxin group (Brevetoxin, BTX), clam toxin group (Pecenotoxins, PTX), Patinopecten yessoensis toxin group (Yessotoxin, YTX) and epimino toxoid (Cyclic imines, CIs).In addition, whether palytoxin (Palytoxins, P1TX) and west add ichtyhotoxisin (Ciguatoxins, CTX) and also are being considered to draw to do in saxitoxin.
In existing 8 large class saxitoxins, STX and DA toxin group are more soluble in water, comparatively speaking OA, AZA, BTX, PTX, YTX, CIs are polyether substance, tool thermal stability, soluble in the nonpolar organic reagents such as methyl alcohol, ether, therefore be unified and be referred to as fat-soluble saxitoxin (lipophilic phycotoxins, LPs).The serious harm of saxitoxin has caused the close attention of a plurality of countries, and the developed country headed by adding etc. with America and Europe has formulated saxitoxin monitoring state plan in succession.Traditional method for supervising is mainly regularly the bivalve shellfish in relevant marine site to be carried out to the content of saxitoxin and toxiferous algae kind and quantity to monitor; risk with assessment saxitoxin; to Protection of consumer safety, guarantee that shellfish industry develops in a healthy way to have made positive contribution.Yet traditional means need to gather a large amount of shellfishes and algae sample could be made effective early warning to saxitoxin, not only expends a large amount of manpower and materials, and ageing not strong.2004, people's reported first such as MacKenzie a kind of solid phase adsorption toxin tracer technique (Solid phase adsorption toxin tracking, SPATT), mainly to utilize specific adsorption material to adsorb target toxin, wash-out in laboratory, concentrated and purify and detect by LC-MS then.SPATT technology is compared with classic method, not only reduce in a large number sampling analysis workload, but also can monitoring objective marine site the variation of different water layer saxitoxins, simultaneously in conjunction with the situation of change of mussel poisoning element and poisonous algae, saxitoxin is had to early warning effect, there is very large development prospect.The main still Mouse bioassay of existing detection technique, there is animal welfare and detect limit for height and can not be clear and definite the discrimination shellfish poison problem that becomes to grade, although mass spectrum detection has also started the detection for saxitoxin, detect kind more single, or pre-treating method is still needed further perfect.Therefore set up a kind of quick, sensitive, multiple fat-soluble saxitoxin Simultaneous Detection becomes particularly important accurately.
Summary of the invention
Technical matters to be solved by this invention is for the deficiencies in the prior art, provide a kind of with strong points, simple to operate, detection speed fast, detect wide, reduced testing cost, the LC-MS detection method of 16 kinds of fat-soluble saxitoxins in the seawater that is easy to apply.
The present invention is achieved by the following technical solutions.
In the LC-MS detection methods of 16 kinds of fat-soluble saxitoxins, comprise the making of absorbent packet, the use of absorbent packet, toxin extraction, condition determination and mode, qualitative determination and quantitative measurement, concrete steps are as follows:
(1) making of absorbent packet: use polyester screen cloth, be sewn into cloth bag, after packing resin into, seal, one jiao of square cloth bag fixedly nylon buckle, absorbent packet is soaked in methyl alcohol, then with distilled water immersion, remove methyl alcohol, put into immediately the polybag of sealing, under 4 ℃ of conditions, the short time preserves, stand-by;
(2) use of absorbent packet: absorbent packet is tied up on long rope, divide upper, middle and lower-ranking, interlayer is every at least surpassing more than 5 meters, otherwise the corresponding minimizing number of plies is once fixed 3 and above absorbent packet for every layer, rope is lain on the floating bar of fixed position, absorbent packet is taken out at interval of 7-10 days, then put into new absorbent packet, the absorbent packet distilled water flushing of extraction, put into the polybag of numbering in advance, under 4 ℃ of conditions, the short time preserves;
(3) toxin extraction: resin extender is proceeded in Sha Xin filter post, with 50-70mL deionized water, wash away salinity, moisture content positive pressure blowing in post, add 20mL methyl alcohol, filter in evaporative flask, add again 20mL methyl alcohol to repeat to extract once, extract makes methyl alcohol volatilization at 40 ℃, remaining 1-2mL water, with centrifugal after 5mL dichloromethane extraction, take out dichloromethane layer, use again 5mL methylene chloride re-extract once, after merging twice dichloromethane extraction layer, with nitrogen, dry up, methanol constant volume with 1.0mL 80%, membrane filtration with 0.22 μ m, sample introduction high performance liquid chromatography-tandem mass is analyzed,
(4) condition determination and mode: liquid chromatography for the testing sample-quadrupole rod mass spectrum of connecting is measured, instrument parameter condition, in Table 1, selects reaction monitoring parent ion, daughter ion and collision energy in Table 2;
The mass spectrographic instrument parameter condition of table 1 liquid chromatography-series connection quadrupole rod
Table 2 is selected reaction monitoring parent ion, daughter ion, collision energy and scan mode
(5) qualitative determination
Under same test condition, in sample solution, the retention time of fat-soluble saxitoxin and standard substance relative deviation of retention time are in ± 5%, and the relative abundance of the qualitative ion detecting, in should the standard solution suitable with concentration, the relative abundance of qualitative ion be consistent, and base peak should meet table 3 requirement with time strong fragmention abundance ratio;
Table 3 base peak and time strong fragmention abundance ratio requirement
| Inferior strong fragmention relative abundance % | ＞50 | ＞20～50 | ＞10～20 | ≤10 |
| Allow relative deviation % | ±20 | ±25 | ±30 | ±50 |
(6) quantitative measurement
Hybrid standard working fluid and sample solution equal-volume sample introduction are measured, by external standard method, used standard working curve to carry out quantitatively sample, in the range of linearity that in sample solution and standard solution, the response of fat-soluble saxitoxin all should detect at instrument.
The present invention's beneficial effect compared with prior art:
1) detection method of the present invention adopts the initiatively saxitoxin in adsorbing seawater of resin, and can carry out specific adsorption to the different layerings of seawater, after through extraction step, analyze the detection of 16 kinds of fat-soluble saxitoxins in seawater.And compare with classic method, sampling quantity is few, reduces costs, simple to operation, and stability is strong.
2) using high performance liquid chromatography tandem mass spectrum detection method of the present invention has significantly improved detection efficiency, and once experiment can be analyzed 16 kinds of fat-soluble saxitoxins, and the setting that detects index more meets the objective situation that current saxitoxin pollutes.
3) detection method of the present invention adopts C
18chromatographic column, using high performance liquid chromatography tandem mass spectrum detection method, has higher sensitivity, and detection limit is all no more than 10ng/ml, is enough to guarantee the monitoring requirement of daily saxitoxin.
4) using high performance liquid chromatography tandem mass spectrum detection method of the present invention has excellent qualitative function, only need on instrument, complete the structure of standard mass spectrum picture library, subsequent analysis can not need reference substance, only need to detect sample, sample test result and mass-spectrogram storehouse relatively can be confirmed to the target additive in sample.
Embodiment
Below by embodiment, technical scheme of the present invention is further explained, but protection scope of the present invention is not subject to any pro forma restriction of embodiment.
In the LC-MS detection methods of 16 kinds of fat-soluble saxitoxins, its step is as follows:
(1) making of absorbent packet: be the polyester screen cloth of 48 μ m with aperture, being sewn into the length of side is the square cloth bag of 6cm * 6cm, packs sealing after 20g DIAION HP20 macroporous absorbent resin into, one jiao of square cloth bag fixedly Buddhist nun buckle.Absorbent packet is soaked in methyl alcohol to 24h, then use distilled water immersion 2h, take out, renew distilled water and repeat 3 times, put into the polybag of sealing, under 4 ℃ of conditions, the short time preserves, stand-by.
(2) use of absorbent packet: absorbent packet is tied up on long rope, divide upper, middle and lower-ranking, interlayer is every 6 meters, once fix 3 absorbent packets for every layer, rope is lain on the floating bar of fixed position, at interval of 8 days, absorbent packet is taken out, put into again new absorbent packet, the absorbent packet distilled water flushing extracting, puts into the polybag of numbering in advance, and under 4 ℃ of conditions, the short time preserves;
(3) toxin extraction: resin extender is proceeded in Sha Xin filter post, wash away salinity with 50mL deionized water, moisture content positive pressure blowing in post, add 20mL methyl alcohol, filter in evaporative flask, then add 20mL methyl alcohol to repeat to extract once.Extract boils off methyl alcohol at 40 ℃, remaining 1-2mL water, with centrifugal after 5mL dichloromethane extraction, take out dichloromethane layer, 5mL methylene chloride re-extract once, dries up with nitrogen after merging twice dichloromethane extraction liquid again, by the methanol constant volume of 1.0mL 80%, with the membrane filtration of 0.22 μ m, sample introduction high performance liquid chromatography-tandem mass is analyzed.
(4) measure: liquid chromatography for testing sample-series connection quadrupole rod mass spectrum is measured, instrument parameter condition, in Table 1, selects reaction monitoring parent ion, daughter ion and collision energy in Table 2;
The mass spectrographic instrument parameter condition of table 1 liquid chromatography-series connection quadrupole rod
Table 2 is selected reaction monitoring parent ion, daughter ion, collision energy and scan mode
(5) qualitative determination
Under same test condition, in sample solution, the retention time of fat-soluble saxitoxin and standard substance relative deviation of retention time are in ± 5%, and the relative abundance of the qualitative ion detecting, in should the standard solution suitable with concentration, the relative abundance of qualitative ion be consistent, and base peak should meet table 3 requirement with time strong fragmention abundance ratio;
Table 3 base peak and time strong fragmention abundance ratio requirement
| Inferior strong fragmention relative abundance % | ＞50 | ＞20～50 | ＞10～20 | ≤10 |
| Allow relative deviation % | ±20 | ±25 | ±30 | ±50 |
(6) quantitative measurement
Hybrid standard working fluid and sample solution equal-volume sample introduction are measured, by external standard method, used standard working curve to carry out quantitatively sample, in the range of linearity that in sample solution and standard solution, the response of fat-soluble saxitoxin all should detect at instrument;
(7) test experience that adopts the present embodiment method to carry out.
State Aquatic Product Quality Monitoring Test Centre application this method in 2011 years to east, Jiangnan, Shandong monitoring point (35 ° of 53 ' 57.11 " N, 120 ° 7 ' 42.42 " E), western monitoring point (35 ° of 53 ' 57.62 " N, 120 ° 7 ' 53.47 " E) carried out the monitoring and early warning for saxitoxin in the seawater in one year, result shows that the saxitoxin content of the third season is the highest, wherein OA average is higher than 80ng/ml, DTX-1 content average is 46ng/ml, PTX-2 content average is 92.7ng/ml, YTX content average is 10ng/ml, AZA class total content average reaches 40ng/ml, GYM and SPX are all in 20ng/ml left and right, BTX class total content average reaches 50ng/ml.
Claims (1)
1. a LC-MS detection method for 16 kinds of fat-soluble saxitoxins in seawater, comprises the making of absorbent packet, the use of absorbent packet, toxin extraction, condition determination and mode, qualitative determination and quantitative measurement is characterized in that concrete steps are as follows:
(1) making of absorbent packet: use polyester screen cloth, be sewn into cloth bag, after packing resin into, seal, one jiao of square cloth bag fixedly nylon buckle, absorbent packet is soaked in methyl alcohol, then with distilled water immersion, remove methyl alcohol, put into immediately the polybag of sealing, under 4 ℃ of conditions, the short time preserves, stand-by;
(2) use of absorbent packet: absorbent packet is tied up on long rope, divide upper, middle and lower-ranking, interlayer is every at least surpassing more than 5 meters, otherwise the corresponding minimizing number of plies is once fixed 3 above absorbent packets for every layer, rope is lain on the floating bar of fixed position, absorbent packet is taken out at interval of 7-10 days, then put into new absorbent packet, the absorbent packet distilled water flushing of extraction, put into the polybag of numbering in advance, under 4 ℃ of conditions, the short time preserves;
(3) toxin extraction: resin extender is proceeded in Sha Xin filter post, with 50-70mL deionized water, wash away salinity, moisture content positive pressure blowing in post, add 20mL methyl alcohol, filter in evaporative flask, add again 20mL methyl alcohol to repeat to extract once, extract makes methyl alcohol volatilization at 40 ℃, remaining 1-2mL water, with centrifugal after 5mL dichloromethane extraction, take out dichloromethane layer, 5mL methylene chloride re-extract once again, after merging twice dichloromethane extraction layer, with nitrogen, dry up, methanol constant volume with 1.0mL80%, membrane filtration with 0.22 μ m, sample introduction high performance liquid chromatography-tandem mass is analyzed,
(4) condition determination and mode: liquid chromatography for the sample to be tested-quadrupole rod mass spectrum of connecting is measured, instrument parameter condition, in Table 1, selects reaction monitoring parent ion, daughter ion and collision energy in Table 2;
The mass spectrographic instrument parameter condition of table 1 liquid chromatography-series connection quadrupole rod
Table 2 is selected reaction monitoring parent ion, daughter ion, collision energy and scan mode
(5) qualitative determination
Under same test condition, in sample solution, the retention time of fat-soluble saxitoxin and standard substance relative deviation of retention time are in ± 5%, and the relative abundance of the qualitative ion detecting, in should the standard solution suitable with concentration, the relative abundance of qualitative ion be consistent, and base peak should meet table 3 requirement with time strong fragmention abundance ratio;
Table 3 base peak and time strong fragmention abundance ratio requirement
(6) quantitative measurement
Hybrid standard working fluid and sample solution equal-volume sample introduction are measured, by external standard method, used standard working curve to carry out quantitatively sample, in the range of linearity that in sample solution and standard solution, the response of fat-soluble saxitoxin all should detect at instrument.
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| CN105651575B (en) * | 2016-01-18 | 2018-09-28 | 浙江大学 | The desorption apparatus and its method of saxitoxin in macroporous absorbent resin |
| CN107024547A (en) * | 2016-02-01 | 2017-08-08 | 徐静 | The long-term detection method for preserving fat-soluble saxitoxin in oyster sample |
| CN111272901A (en) * | 2020-03-14 | 2020-06-12 | 广西壮族自治区水产科学研究院 | High-resolution mass spectrometry detection method for lipophilic toxins in shellfish |
| CN113092610B (en) * | 2021-03-31 | 2022-05-10 | 中国海洋大学 | Method for extracting gymnodinotoxin from marine microalgae |
| CN115980234B (en) * | 2022-10-27 | 2024-08-30 | 福州大学 | Sample pretreatment method for benzodiazepine novel drug detection |
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