CN110174469B - Detection method of three diarrheic shellfish poisoning types OA, DTX1 and DTX2 in seaweed - Google Patents

Abstract

The invention discloses a method for detecting three types of diarrheal shellfish poisoning, OA, DTX1 and DTX2 in seaweed, which can carry out risk warning in advance for the pollution of seaweed in feed and water environment. The adopted technical scheme is: The detection method for three types of diarrheal shellfish poisoning, OA, DTX1 and DTX2, is characterized by comprising the following steps: (1) pretreatment of seaweed samples: grinding and breaking the seaweed samples; (2) concentration and enrichment: weighing 1 g of pre- The treated seaweed samples were added with 10 mL of methanol, vortexed for 30 s, and after ultrasonic extraction for 10 min, centrifuged at 1000 r/min for 10 min. After centrifugation at 6000 r/min for 5 min, the supernatant was passed through a 0.22 μm PVDF membrane; (3) liquid chromatography tandem mass spectrometer was used for detection. Compared with the prior art, the present invention selects the PSA adsorbent as the purifying agent, which simplifies the pretreatment process, optimizes the purification effect, and the method of the present invention has stable linearity and moderate quantitative limit.

Description

Methods for the detection of OA, DTX1 and DTX2 in seaweed for diarrheal shellfish poisoning

technical field

The invention relates to a method for detecting toxins, in particular to a method for detecting three types of diarrheal shellfish poisons, OA, DTX1 and DTX2, in seaweed.

Background technique

Diarrhetic Shellfish Poisoning (DSP) is a lipid-soluble polycyclic ether biologically active substance produced by some species of the poisonous red tide algae Pneumonia and Prototheca. Its chemical structure is characterized by polyether or macrolide compounds. These components can be divided into three groups according to their carbon skeleton structures: (1) Acidic components: halichondric acid (OA) and its natural derivatives, finnolyxins (DTXs). (2) Neutral component: polyether lactone-saxitoxin (PTXI-Vn). (3) Other components: sulfonated poisons, toxin (YTX) and its derivatives. Among the acidic components, OA, DTX1 and DTX2 are the three most important components in DSP.

Diarrheal shellfish poisoning can be enriched in filter-feeding animals such as shellfish, so it is relatively easy to detect shellfish DSP. Even so, the detection methods of shellfish DSP mainly focus on OA. The main detection methods are: mouse biological test method, Immunoassays (Immunoassays), High-performance liquid chromatography (HPLC), Liquid chromatography-massspectrometry (LC/MS). At present, there is no direct detection of algal DSP, and no simultaneous detection of the three components of OA, DTX1 and DTX2. It can simultaneously detect the three components of OA, DTX1 and DTX2 in seaweed, which has a symbolic significance for seaweed research and aquaculture based on seaweed as bait.

SUMMARY OF THE INVENTION

In order to establish a rapid and effective detection of DSP in seaweed, the present invention discloses a method for detecting three types of diarrheal shellfish poisoning, OA, DTX1 and DTX2 in seaweed, which can carry out risk warning in advance for the pollution of seaweed in feed and water environment. , the technical solution adopted is:

The detection method of three kinds of diarrhea shellfish poisoning of OA, DTX1 and DTX2 in seaweed is characterized in that comprising the following steps:

(1) Pretreatment of seaweed samples: grinding and crushing seaweed samples;

(2) Concentration and enrichment: Weigh 1 g of pretreated seaweed sample, add 10 mL of methanol, vortex for 30 s, ultrasonically extract for 10 min, centrifuge at 1000 r/min for 10 min, take 1 mL of supernatant, add PSA for adsorption After vortex mixing for 30s, centrifuge at 6000 r/min for 5 min, and the supernatant was filtered through 0.22 μm PVDF membrane;

(3) Detection by liquid chromatography tandem mass spectrometer.

Further, the liquid chromatography conditions in step (3) are:

Column: ACQUITYTM UPLC BEH C18 (1.7μm, 2.1 mm i.d.×100 mm); column temperature: 40°C; mobile phase: A: acetonitrile; B: solution containing 0.1% formic acid (B); flow rate: 0.25 mL/min ; Injection volume: 10 μL; the gradient elution program table is

；

;

The mass spectrometry conditions are:

Automatic tuning, the optimized mass spectrometry conditions are as follows: ionization mode: positive ion (ESI-); ionization voltage: 2.50kV; cone voltage: 48 V; ion source temperature: 150 °C; cone backflush gas flow rate: 50 L /h; Desolvation gas temperature: 450°C; Desolvation gas flow rate: 900 L/h; Argon gas flow rate: 0.12 mL/min; Other parameters are

compound parent ion daughter ion Cone voltage collision energy DTX1 817 150.8;255.2* 30 50;45 DTX2 803 113;255.3* 30 50;45 OA 803 113;255.3* 30 50;45

* represents quantitative ions.

Compared with the prior art, according to the matrix characteristics of algae, the present invention selects PSA adsorbent as the purifying agent, which simplifies the pretreatment process and optimizes the purification effect, and the method of the invention has stable linearity and moderate quantitative limit.

Description of drawings

Figure 1 is a comparison chart of the effect of adding PSA adsorbent (centrifuge tube on the right) and without adding adsorbent (centrifuge tube on the left).

Fig. 2 is the blank spectrum of the commercially available bait algae 1 sample.

Figure 3 is the 0.8 μg/L standard spectrum.

Figure 4. The recovery chromatogram of adding 10 μg/kg to seaweed.

Figure 5 is the OA, DTX1 and DTX2 spectra of the commercially available Lemonella vulgaris 1 sample.

FIG. 6 is the OA, DTX1 and DTX2 spectra of commercially available Forage algae 2 samples.

FIG. 7 is the OA, DTX1 and DTX2 spectra of commercially available Lemony algae 3 samples.

FIG. 8 is the OA, DTX1 and DTX2 spectra of commercially available Lemony algae 4 samples.

FIG. 9 is the OA, DTX1 and DTX2 spectra of commercially available Alimenta 5 samples.

Detailed ways

Three types of diarrheal shellfish poisons, OA, DTX1 and DTX2, in the five bait algae currently on the market, were detected according to the following steps:

(1) Pretreatment of seaweed samples: grinding and crushing seaweed samples;

(2) Concentration and enrichment: Weigh 1 g of pretreated seaweed sample, add 10 mL of methanol, vortex for 30 s, ultrasonically extract for 10 min, centrifuge at 1000 r/min for 10 min, take 1 mL of supernatant, add PSA for adsorption After vortex mixing for 30s, centrifuge at 6000 r/min for 5 min, and the supernatant was filtered through 0.22 μm PVDF membrane;

(3) Detection by liquid chromatography tandem mass spectrometer.

Because the seaweed matrix is characterized by more pigments and organic acids, and lower oil content. Therefore, the use of PSA adsorbent can obtain better purification effect. Previously, the inventor also considered to carry out SPE column for purification, but found that the recovery rate was extremely low, and finally decided to use PSA adsorbent through numerous experiments. At the same time, when determining the addition amount of PSA adsorbent, it is found that the addition amount of 250mg has the best effect. Any more will affect the amount of the fixed solution on the machine, and when the addition amount is 50 mg, it can have a certain purification effect. The effect of adding PSA adsorbent (centrifuge tube on the right) and without adding adsorbent (centrifuge tube on the left) is shown in Figure 1.

Wherein, the liquid chromatography conditions in step (3) are:

Column: ACQUITYTM UPLC BEH C18 (1.7μm, 2.1 mm i.d.×100 mm); column temperature: 40°C; mobile phase: A: acetonitrile; B: solution containing 0.1% formic acid (B); flow rate: 0.25 mL/min ; Injection volume: 10 μL; the gradient elution program table is

；

;

The mass spectrometry conditions are:

Automatic tuning, the optimized mass spectrometry conditions are as follows: ionization mode: positive ion (ESI-); ionization voltage: 2.50kV; cone voltage: 48 V; ion source temperature: 150 °C; cone backflush gas flow rate: 50 L /h; Desolvation gas temperature: 450°C; Desolvation gas flow rate: 900 L/h; Argon gas flow rate: 0.12 mL/min; Other parameters are

compound parent ion daughter ion Cone voltage collision energy DTX1 817 150.8;255.2* 30 50;45 DTX2 803 113;255.3* 30 50;45 OA 803 113;255.3* 30 50;45

* represents quantitative ions.

The OA, DTX1 and DTX2 spectra of the five commercially available algae samples are shown in Figures 5-9, respectively. It can be seen that OA, DTX1 and DTX2 of the five commercially available algae samples were not detected.

The present invention has the following advantages:

(1) This method is linearly stable:

OA, DTX1 and DTX2 were all diluted to the standard series of 0.80, 5.00, 10.0, 20.0, 50.0 μg/L, and the following equations were obtained:

DTX1: R2 = 0.9968 Y = 75.5409*X±14.2469

DTX2: R2 = 0.9947 Y = 42.2391*X±11.7144

OA: R2 = 0.9972 Y = 82.2475*X±2.0798

The R2 of DTX1, DTX2, and OA were all greater than 0.99, indicating that the three diarrheal shellfish poisons had good linear stability.

(2) The quantitative limit of this method: DTX1, DTX2 and OA are all 10 μg/kg.

The following data are obtained by adding the commercially available bait algae 1 to the experiment:

The sample spectrum, standard spectrum and sample addition are shown in Figure 1-3.

Claims (1)

1. A method for detecting three diarrheic shellfish poisoning (OA), DTX1 and DTX 2) in seaweed is characterized by comprising the following steps:

(1) pretreatment of the seaweed sample: grinding and smashing the seaweed sample;

(2) concentration and enrichment: weighing 1g of pretreated seaweed sample, adding 10mL of methanol, mixing in a vortex for 30s, performing ultrasonic extraction for 10min, centrifuging at 1000r/min for 10min, taking 1 mL of supernatant, adding 50mg of PSA adsorbent, mixing in a vortex for 30s, centrifuging at 6000r/min for 5min, and filtering the supernatant with 0.22 mu m PVDF filter membrane;

(3) detecting by adopting a liquid chromatogram tandem mass spectrometer;

the conditions of the liquid chromatography are as follows:

a chromatographic column: ACQUITYTM UPLC BEH C18; temperature of the column: 40 ℃; mobile phase: a is acetonitrile; b, a solution containing 0.1 percent of formic acid; flow rate: 0.25 mL/min; sample introduction amount: 10 mu L of the solution; the gradient elution schedule is

Time (min) Flow rate (mL/min) A% B% 0.00 0.25 30 70 0.50 0.25 30 70 2.00 0.25 90 10 4.00 0.25 90 10 5.00 0.25 30 70

；

The mass spectrum conditions are as follows:

auto-tuning, the optimized mass spectrometry conditions are as follows: an ionization mode: a positive ion; ionization voltage: 2.50 kV; taper hole voltage: 48V; ion source temperature: 150 ℃; taper hole blowback air flow rate: 50L/h; desolventizing gas temperature: at 450 ℃; desolventizing air flow rate: 900L/h; argon flow rate: 0.12 mL/min; other parameters are

Compound (I) Parent ion Daughter ions Voltage of taper hole Collision energy DTX1 817 150.8；255.2* 30 50；45 DTX2 803 113；255.3* 30 50；45 OA 803 113；255.3* 30 50；45

Represents the quantification ion.

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