CN105259292A - Method for measuring paralysis shellfish poison in aquatic products - Google Patents

Abstract

The invention provides a method for measuring paralysis shellfish poison in aquatic products. The method comprises the steps of extraction, freezing, redissolving, purification and liquid chromatography-tandem mass spectrometer detection. The method has the advantages that sample pretreatment is simple, quick and economical, and sensitivity and recovery rate are high. The method is suitable for simultaneous, qualitative and quantitative detection of multiple kinds of paralysis shellfish poison in aquatic products, and provides effective technical support for guaranteeing quality safety of shellfish aquatic products in China.

Description

A kind of method measuring paralytic shellfish poisoning (PSP) in aquatic products

Technical field

The present invention relates to aquatic products detection field, relate to a kind of method measuring paralytic shellfish poisoning (PSP) in aquatic products specifically.

Background technology

Paralytic shellfish poisoning (PSP) (PSTs) is the water-soluble extremely strong Small molecular secondary metabolite of a class that in water body, planktonic algae or microorganism produce.Its basic structure is the how folded hexatomic ring of four aquation purine of two guanidine radicals.According to the difference of substituted radical, mainly be divided into four classes: 1. carbamate toxoid (Carbamatetoxins), comprise saxitoxin (Saxitoxin, STX), N-STX (neoSTX, and GTX1-4 (Gonyautoxins, GTXl-4) NEO); 2. N-sulphonyl carbamyl toxoid (N-sulfocarbamoyltoxins), comprises GTX5 (B1), GTX6 (B2) and C1-C4; 3. deamination formoxyl toxoid (Decarbamoyltoxins), comprises dcSTX, dcneoSTX, dcGTX1-4; 4. deoxidation deamination formoxyl toxoid (Deoxydecarb-amoyltoxins), comprises doSTX, doneoSTX and doGTX1 etc., and wherein, carbamate toxoid is a toxoid of outbalance.

PSTs is the efficient sodium-ion channel neurotoxin of a class.After human body has taken in the marine product polluted by PSTs, clinical initial stage main manifestations has been that mouth, lip, tongue are numb, and paralysis symptom appears in four limbs, and sensation occurs abnormal, and people becomes weak and chaotic; Later stage main manifestations is nauseating, and vomiting, is short of breath, headache fever, dysphagia, dysarteriotony, and severe patient occurs clouding of consciousness even dead.Wherein, the distribution of carbamate toxoid is the widest, toxicity is the strongest, and its Typical Representative STX accounts for more than 85% of PSTs distribution.Although itself toxicity of N-sulphonyl carbamyl toxoid is less, very easily slough sulfonyl and carbamic acid ester chain generates the very strong STX of toxicity and deamination formoxyl toxoid by biological conversion in vivo.In addition, up-to-date Biotransformation experiments finds and confirms in shellfish body, there is a class C1/C2 toxin.

For ensureing the public not by the threat of PSTs, world many countries, area and relevant international organization all carry out strict control and control to shellfish aquatic products, and have formulated the PSTs limit standard of corresponding shellfish aquatic products and goods thereof.The safety evaluatio being different from other toxin is the toxicity assessment being based upon toxin standard items itself, and the limit standard of PSTs is the toxicological evaluation based on zoopery method.

In biosome current at present, PSTs limit standard is that the United Nations's health organization (WHO) obtains according to mouse test method (MBA).The LOD value of this method and occur that the minimum virulence of pathological characteristics is 80 μ gSTXeq/100g, namely 400MU, MBA are also confirmed as the standard method of PSTs safety evaluatio.This standard is included China and adopts in interior most countries, is also considered to the minimum standard of PSTs safety problem.Research shows, although do not comprise all PSTs (5-10 kind PSTs being detected at most in current known a kind of biosome) known at present in physical environment and single creature body, but for this toxoid that some has found, its character we and unclear, be also just difficult to carry out safety and toxicity assessment to it.In addition; different PSTs has different toxicity; although some toxin is as lower in itself toxicity of N-sulphonyl carbamyl toxoid, the molecular conformation of high poison can be formed by environmental induction or biological conversion, and this conversion is very common in environmental and biological materials.Therefore, because PSTs has toxicity diversity and changeableness, make this anatoxic accurate quantification is detected to have occurred no small difficulty.Although MBA method is standard detecting method general at present, its sensitivity is low makes this analytical approach endure dispute to the fullest extent with poor accuracy.2009, PSTs Limited Doses controlled within 7.5 μ gSTXeq/100g, well below the LOD of MBA by EFSA's suggestion.Therefore, the suitable detection method possessing more high sensitivity and accuracy must be found to substitute MBA, for PSTs Research on Safety Assessment provides means and prerequisite.

At present, MBA and HPLC-FLD method has become the standard detecting method that the standards setting organizations such as FSA (FSA), AOAC, U.S.'s shellfish administration of health plan (NSSP) are defined as PSTs, has set up the monitoring system of relative maturity.The appearance of LC-MS method is considered to the suitable replacer of MBA and HPLC-FLD method, because of its to HPLC-FLD method the consuming time and large shortcoming of operation easier large two done obvious improvement.Still there is certain deficiency for the LC-MS detection method of PSTs in the world at present, the recovery of component as each in pretreatment mode contratoxin is widely different, does not have suitable purification method well to limit the impact etc. of matrix effect on compound mensuration.

Therefore the quantitative detecting method establishing a set of polycomponent PSTs of system is necessary, to guarantee fish quality.

Summary of the invention

The object of the present invention is to provide a kind of method measuring paralytic shellfish poisoning (PSP) in aquatic products, the method specifically comprises the steps:

(1) take the sample after smashing to pieces, add extraction solution, centrifugal after vortex and sonic oscillation extract, after supernatant merging is freezing, take out lower floor's freeze drying near dry;

(2) redissolve, purify with adsorbent, vortex oscillation is centrifugal, and upper liquid filtering membrane is to be analyzed;

(3) analyte sample fluid carries out the qualitative and quantitative analysis of paralytic shellfish poisoning (PSP) by Liquid Chromatography-Tandem Mass Spectrometry instrument.

Wherein said sample can be the shellfish aquatic products such as scallop, clam, mussel;

Extraction solution described in step (1) is the acetonitrile solution (acetonitrile: water=80:20, volume ratio) containing 0.1% (mass percent concentration) formic acid;

In described step (2), purifying adsorbent is C18 silica gel and acidic alumina, and the weight ratio of the two is 1:1;

In step (3) survey paralytic shellfish poisoning (PSP) and be: saxitoxin (STX), N-STX (neoSTX), GTX1-4 (GTX1-4), N-sulphonyl carbamyl gonyatoxin-2 (C1) and N-sulphonyl carbamyl gonyatoxin-3 (C2);

Liquid phase analysis condition in described step (3) is specific as follows:

The hydrophilic post of chromatographic column: TSK-gelAmide-80 (150 × 2mm, 3 μm); Column temperature: 30 DEG C; Flow velocity: 0.3mL/min; Sample size: 5 μ L;

Mobile phase: A phase: acetonitrile; B phase: 3.6mM formic acid, 2mM ammonium formate aqueous solution (pH3.5)

Gradient elution program: 0min, 15%B; 0-10min, 45%B; 10-11min, 45%B; 11-14min, 15%B, 14-19, min15%B;

The mass spectrophotometry condition used in step (3) is specific as follows:

Ion gun pattern: positive ion mode (ESI+); Spray voltage: 4.0kv (ESI+); Heat block temperature: 300 DEG C; Ion transfer tube temperature: 350 DEG C; Sheath gas: nitrogen, flow 35arb; Assisted gas: nitrogen, flow 20arb; Collision gas: argon gas, impact pressure 1.5mTorr.

Specifically, a kind of method measuring paralytic shellfish poisoning (PSP) in aquatic products of the present invention, comprises the steps:

(1) the shellfish biological sample collected washes, take out soft tissue, put into tissue mashing machine to smash to pieces, sample thief adds the acetonitrile solution (80:20 containing 0.1% formic acid, v/v) extract, ultrasonic extraction less than 10min, 4500rpm15 DEG C centrifugal 10min under frozen water state after vortex oscillation 1min; By the supernatant obtained after centrifugal, freezing 4h under-20 DEG C of conditions, discards rapidly upper organic phase (<1min) after taking-up, and lower floor's freeze drying is near dry;

Be wherein 1-3:1 (ml:g) containing the acetonitrile solution of 0.1% formic acid and the ratio of sample;

(2) redissolve and constant volume with the formic acid water that mass percent concentration is 0.1%, add C18 that mass ratio is 1:1 and acidic alumina purifies it as adsorbent, vortex oscillation 1min, less than the 15 DEG C centrifugal 10min of 4500rpm, it is to be analyzed that upper liquid crosses 0.22 μm of nylon leaching film;

(3) detected sample that step (2) obtains is analyzed by Liquid Chromatography-Tandem Mass Spectrometry instrument, obtain the testing result of 8 kinds of paralytic shellfish poisoning (PSP)s respectively.

The invention provides that a kind of pre-treating method simple economy is quick, plain detection method that sensitivity and the high liquid phase tandem mass spectrometry of accuracy measure 8 kinds of paralytic shellfish poison in aquatic products.

A kind of method measuring paralytic shellfish poisoning (PSP) in aquatic products of the present invention compared with prior art, has following technological merit and effect: pre-treatment adopts that the method improved is more simple and efficient, time of saving sample preparation and cost, decrease the consumption of organic solvent.Sample simultaneously after process is very clean, and after continuous sample introduction, sensitivity does not have significant change.In the inventive method, the quantitative limit of different toxin in different substrates is respectively: STX, 0.33-1.65 μ g/kg; NEO, 0.69-2.59 μ g/kg; GTX1,4.14-5.52 μ g/kg; GTX4,2.70-6.08 μ g/kg; GTX2,2.01-2.82 μ g/kg; GTX3,1.60-4.79 μ g/kg; C1,2.27-3.33 μ g/kg; C1,1.35-2.69 μ g/kg; The recovery is between 81.52% ~ 116.50%, and relative standard deviation is all less than 19.10%.As can be seen here, sensitivity and precision all can meet quantitative testing requirement.

Accompanying drawing explanation

Fig. 1 is that (wherein the concentration of each Component Standard solution is respectively: STX:39.69 μ g/mL for Selective reaction monitoring (SRM) collection of illustrative plates of 8 kinds of paralytic shellfish poisoning (PSP) standard solution; NEO:41.37 μ g/mL; GTX2:90.29 μ g/mL; GTX3:38.31 μ g/mL; GTX1:49.69 μ g/mL; GTX4:16.21 μ g/mL; C1:107.82 μ g/mL; C2; 32.23 μ g/mL.)；

Embodiment

The present invention is further illustrated for embodiment given below.The present invention is described in conjunction with most preferred embodiment, but after reading example of the present invention, those skilled in the art can understand and in disclosed enforcement, make many changes and also can obtain same or similar result, all belong to the spirit and scope of the present invention.In particular, the alternative reagent disclosed herein of some reagent and obtain identical or similar results.All similar replacements or modification are all considered to the spirit and scope of the present invention, and all above-mentioned equivalent form of values all belong to the scope that claims of the present invention limits.

Embodiment 1

1, materials and methods

1.1 key instruments and reagent

Liquid Chromatography-Tandem Mass Spectrometry instrument: ThermoTSQQuantumUltra triple level Four bar mass spectrometer, electric spray ion source (ESI) (ThermoFisherScientific, USA);

The hydrophilic post of liquid-phase chromatographic column: TSK-gelAmide-80 (150 × 2mm, 3 μm):

Tissue mincer: IKAT-18 (IKA, Germany);

Vortex instrument: its Lindberg Optic Design A/S of Haimen;

Ultrasonator: Ningbo Xin Zhi company;

Refrigerated centrifuge: HeraeusMultifuge1L-R (ThermoFisherScientific, Germany);

Freeze drier: HetoPowerDryLL3000, Shanghai Hui Fen Electronic Science and Technology Co., Ltd.;

Nitrogen evaporator: HGC-24 Xi'an Jing great checkout equipment company limited;

Ultrasonic washing instrument: Shanghai High Kudos Science Instrument Co., Ltd.;

Ultrapure water machine: Milli-Q, German Millipore company;

1.2 standard items and reagent

8 kinds of standard items toxin (STX, NEO, GTX1/4, GTX2/3, C1/C2) are purchased from marine life research institute of Canadian NRC; Formic acid, acetonitrile, C18, acidic alumina and dSPE adsorption stuffing are purchased from traditional Chinese medicines chemical reagent company limited.

2, experimental technique

2.1 sample-pretreating method

(1) market collect scallop, clam, mussel samples with water clean, take out soft tissue, put into tissue mashing machine to smash to pieces, get 1g ± 0.01g shellfish meat and add the acetonitrile water (80:20 of 2mL containing 0.1% formic acid, v/v) extract, ultrasonic extraction less than 10min, 4500rpm15 DEG C centrifugal 10min under frozen water state after vortex oscillation 1min.Repeatedly extract 2 times, merged by the supernatant obtained after centrifugal, freezing 4h under-20 DEG C of conditions, discards rapidly upper organic phase (<1min) after taking-up, and lower floor's freeze drying is near dry.

(2) redissolve with 0.1% formic acid water and be settled to 1mL, adding 50mgC18 and 50mg acidic alumina adsorbent and purify it, vortex oscillation 1min, less than the 15 DEG C centrifugal 10min of 4500rpm, it is to be analyzed that upper liquid crosses 0.22 μm of nylon leaching film.

(3) detected sample obtained is analyzed by Liquid Chromatography-Tandem Mass Spectrometry instrument, obtains the result of carbamate toxoid and N-sulphonyl carbamyl toxoid residual quantity respectively.

2.2 liquid chromatography mass conditions

(1) liquid-phase condition:

The hydrophilic post of chromatographic column: TSK-gelAmide-80 (150 × 2mm, 3 μm); Column temperature: 30 DEG C;

Flow velocity: 0.3mL/min; Sample size: 5 μ L;

Mobile phase: A phase: acetonitrile; B phase: 3.6mM formic acid, 2mM ammonium formate aqueous solution (pH3.5)

Gradient elution program: 0min, 15%B; 0-10min, 45%B; 10-11min, 45%B; 11-14min, 15%B, 14-19, min15%B.

(2) Mass Spectrometry Conditions:

Ion gun pattern: positive ion mode (ESI+); Spray voltage: 4.0kv (ESI+); Heat block temperature: 300 DEG C; Ion transfer tube temperature: 350 DEG C; Sheath gas: nitrogen, flow 35arb; Assisted gas: nitrogen, flow 20arb; Collision gas: argon gas, impact pressure 1.5mTorr.

Fig. 1 is shown in by the SRM collection of illustrative plates of the standard solution obtained under above-mentioned condition.

The mass spectrometry parameters of 8 kinds of paralytic shellfish poisoning (PSP)s is in table 1.

The mass spectrometry parameters of each toxin of table 1

* quantitative fragmention, what do not mark * is then that the auxiliary alternative ion of qualitative ion excludes in table

3, result and discussion

Use acetonitrile/water system not only to have very high extraction efficiency to PSTs, and better can precipitate the protein in shellfish tissue, sample is effectively purified.

3.1 linear relationships and detection limit, quantitative limit

The standard working solution of 8 kinds of paralytic shellfish poisoning (PSP)s is configured with bare substrate (scallop, clam, mussel).Preparation standard solution concentration is as follows: STX:39.69 μ g/mL; NEO:41.37 μ g/mL; GTX2:90.29 μ g/mL; GTX3:38.31 μ g/mL; GTX1:49.69 μ g/mL; GTX4:16.21 μ g/mL; C1:107.82 μ g/mL; C2; 32.23 μ g/mL.Investigate the linear degree (R of the range of linearity ²) and quantitative limit (LOD), detection limit (LOQ), dependent linearity scope and LOD, LOQ value are in table 2.Result shows that the detection method of the present embodiment is feasible.

The linear relationship of 8 kinds of paralytic shellfish poisoning (PSP)s and detection limit, quantitative limit in table 2 different substrates

3.2, the recovery and Precision Experiment

Adopt matrix after extracting front matrix mark-on and purification to add target mode, both ratio is the recovery obtained through method aftertreatment.Select the interpolation concentration that three groups suitable, often organize 6 parts of Duplicate Samples, carry out in a few days repeatability (Intra-day) and in the daytime reappearance (Inter-day) mensuration, with the recovery and RSD value thereof for standard is evaluated, the results are shown in Table 3.Result illustrates that the reappearance of this detection method is better.

4, conclusion

The present invention uses the method for 8 kinds of paralytic shellfish poisoning (PSP) content in the sample-pretreating method of improvement and liquid phase tandem mass spectrometry Simultaneously test aquatic products, sensitivity and stability high, solve technical barrier of the prior art, for ensureing that China's fish quality provides good technical support.

Claims (2)

1. measure a method for paralytic shellfish poisoning (PSP) in aquatic products, it is characterized in that the method specifically comprises the steps:

(1) take the sample after smashing to pieces, add extraction solution, centrifugal after vortex and sonic oscillation extract, after supernatant merging is freezing, take out lower floor's freeze drying near dry;

(2) redissolve, purify with adsorbent, vortex oscillation is centrifugal, and upper liquid filtering membrane is to be analyzed;

(3) analyte sample fluid carries out the qualitative and quantitative analysis of paralytic shellfish poisoning (PSP) by Liquid Chromatography-Tandem Mass Spectrometry instrument.

Wherein the extraction solution described in step (1) is the acetonitrile solution containing 0.1% formic acid;

In described step (2), purifying adsorbent is C18 silica gel and acidic alumina, and the weight ratio of the two is 1:1;

In step (3) survey paralytic shellfish poisoning (PSP) and be: saxitoxin, N-STX, GTX1-4, N-sulphonyl carbamyl gonyatoxin-2 and N-sulphonyl carbamyl gonyatoxin-3.

2. the method for paralytic shellfish poisoning (PSP) in mensuration aquatic products according to claim 1, is characterized in that the liquid phase analysis condition in described step (3) is specific as follows:

The hydrophilic post of chromatographic column: TSK-gelAmide-80; Column temperature: 30 DEG C; Flow velocity: 0.3mL/min; Sample size: 5 μ L;

Mobile phase: A phase: acetonitrile; B phase: the 2mM ammonium formate aqueous solution of 3.6mM formic acid, pH3.5

Gradient elution program: 0min, 15%B; 0-10min, 45%B; 10-11min, 45%B; 11-14min, 15%B, 14-19, min15%B;

The mass spectrophotometry condition used in step (3) is specific as follows:

Ion gun pattern: positive ion mode ESI+; Spray voltage: 4.0kvESI+; Heat block temperature: 300 DEG C; Ion transfer tube temperature: 350 DEG C; Sheath gas: nitrogen, flow 35arb; Assisted gas: nitrogen, flow 20arb; Collision gas: argon gas, impact pressure 1.5mTorr.