Detection reagent for amatoxin in blood and detection method thereof

Abstract

The invention relates to a reagent for detecting amatoxin in blood and a detection method thereof. The detection reagent for the amatoxin in the blood comprises an extraction tube, a dilution tube and a standard gradient solution, the extraction tube contains 1.5 mL of acetonitrile, the dilution tube contains 400 [mu] L of a 1% acetic acid aqueous solution, and the gradient concentrations of the standard gradient solution are 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL. The method provided by the invention effectively solves the problem of poor signal stability of amatoxin in a blood sample, and meanwhile, complex steps such as solid-phase extraction do not need to be adopted in the detection process, so that the detection time is saved, and a technical support is provided for emergency monitoring of public health emergencies.

Description

Technical Field

[0001] The invention belongs to the field of detection, and specifically relates to a detection reagent and a detection method of amatoxin in blood. Background Art

[0002] Mushroom poisoning incidents occur frequently in my country, among which acute liver failure poisoning caused by amatoxin has the highest mortality rate. After the toxin enters the human body, it enters the liver from the gastrointestinal tract through the portal vein, inhibiting mRNA transcription and protein synthesis. Amatoxin includes amatoxin and phalloidin. Amatoxin is a bicyclic octapeptide, and phalloidin is a bicyclic heptapeptide. Both are cyclic peptide toxins. The difference between the same type of toxin-like peptides lies in the difference in the substituents. High temperature and acid and alkali cannot destroy them.

[0003] At present, the detection of amatoxin in blood is mainly carried out by liquid chromatography-mass spectrometry / mass spectrometry. However, since amatoxin contains oxygen atoms and sulfur atoms with multiple unpaired lone pairs of electrons, and the blood contains a large number of sodium ions without empty electron orbitals, these sodium ions are easily captured by oxygen atoms and sulfur atoms, increasing the probability of amatoxin forming a [M+Na]+ peak, thereby affecting the signal stability and causing quantitative deviations in the results. On the other hand, the detection of amatoxin in blood is currently mainly carried out by solid phase extraction, which has a long detection cycle and is not conducive to the short-term needs of emergency poisoning inspections.

[0004] In order to solve the above technical problems, it is necessary to develop a detection reagent and a detection method for quickly and accurately detecting amatoxin in the blood. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the present invention discloses a detection reagent kit and a detection method for amatoxin in blood, which has high detection accuracy and is quick and easy to operate.

[0006] The technical objectives of the present invention are achieved through the following technical solutions:

[0007] The present invention provides a detection reagent for amatoxin in blood, comprising an extraction tube, a dilution tube and a standard gradient solution, wherein the extraction tube contains 1.5 mL of acetonitrile, the dilution tube contains 400 μL of a 1% acetic acid aqueous solution, and the gradient concentrations of the standard gradient solution are 1, 5, 10, 20, 50, and 100 ng / mL.

[0008] Furthermore, the amatoxin described in the present invention comprises at least one of α-amanitin, β-amanitin, γ-amanitin, carboxytrihydroxyphalloidin, dihydroxyphalloidin and carboxydihydroxyphalloidin.

[0009] Another technical purpose of the present invention is to provide a method for detecting amatoxin in blood, comprising the following steps:

[0010] S1. Take 500 μL of serum sample and add it to the extraction tube of claim 1, mix well and centrifuge;

[0011] S2, take 100 μL of the supernatant into the dilution tube of claim 1, mix well, and transfer to the injection bottle through the membrane;

[0012] S3. Using a liquid chromatography time-of-flight mass spectrometer to measure the standard gradient solution and the sample to be tested, to obtain the concentration of amatoxin in the sample to be tested.

[0013] Furthermore, the centrifugation temperature of the present invention is 4±0.5°C, the rotation speed is 10000r / min, and the centrifugation time is 5±1min.

[0014] Furthermore, the conditions of the liquid chromatography of the present invention are: mobile phase A is acetonitrile, mobile phase B is 0.01% formic acid aqueous solution; the mobile phase flow rate is 0.4 mL / min, the sample injection volume is 5 μL, the column temperature is 40°C, and the chromatographic column is an XSelect HSST3 column (150 mm×3.0 mm, 2.5 μm); the liquid chromatography elution gradient is: 0-1.5 min, 10%-25% B; 4-5 min, 25% B; 5-6 min, 25%-90% B; 6-9 min, 90% B; 9-12 min, 90%-10% B.

[0015] Furthermore, the mass spectrometry conditions of the present invention are: using an electrospray ionization source in positive ion mode ESI + , capillary voltage 3.0 kV, ion source temperature 120 ° C, desolvation gas temperature 550 ° C, cone gas flow rate 20 L / h, desolvation gas flow rate 800 L / h, acquisition mode is MS E mode, resolution greater than 20,000; mass range: 50-1200Da; Lock Spray dual spray ion source: leucine enkephalin solution, ESI + It is 556.2771Da.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a simple, rapid and efficient method and kit for quantitatively detecting amatoxin in blood. Compared with the prior art, this method effectively solves the problem of poor stability of amatoxin signals in blood samples. At the same time, the detection process does not require complex steps such as solid phase extraction, which saves detection time and provides technical support for emergency monitoring of public health emergencies. DETAILED DESCRIPTION

[0018] In order to better illustrate the technical purpose, technical solution and advantages of the present invention, the present invention is further described in conjunction with specific embodiments.

[0019] Instruments and reagents used in the embodiments of the present invention:

[0020] Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (Waters G2-XS, USA), AllegraX-30R centrifuge (Beckman Coulter, USA), M37610-33CN vortex mixer (IKA, Germany), MS204TS / 02 electronic balance (METTLER TOLEDO, Switzerland); α-amanitin, β-amanitin, γ-amanitin, dihydroxyphalloidin, carboxy-dihydroxyphalloidin standards were purchased from Cayman Chemical, USA, with a purity of ≥90%; carboxy-trihydroxyphalloidin was purchased from Fuzhou Qinpeng Biotechnology Co., Ltd. with a concentration of 10 μg / mL; acetonitrile, ammonium acetate, and formic acid (chromatographic grade, Thermo Fisher Scientific, USA).

[0021] Example 1

[0022] The method for detecting amatoxin in blood of the present invention comprises the following steps:

[0023] S1. Take 500 μL of serum sample and add it to extraction tube A. Vortex mix for 1 min, and then centrifuge at 4°C and 10,000 rpm for 5 min.

[0024] S2. Transfer 100 μL of supernatant to extraction tube B, vortex for 1 min to mix, and then transfer to the injection bottle through the membrane;

[0025] S3. Use a liquid chromatography time-of-flight mass spectrometer to measure the standard gradient solution and the sample to be tested to obtain the concentration of amatoxin in the sample to be tested.

[0026] The conditions of liquid chromatography in the step are: mobile phase A is acetonitrile, mobile phase B is 0.01% formic acid aqueous solution. The mobile phase flow rate is 0.4mL / min, the sample injection volume is 5μL, the column temperature is 40°C, and the chromatographic column is XSelect HSS T3 column (150mm×3.0mm, 2.5μm). The liquid chromatography elution gradient is: 0-1.5min, 10%-25% B; 4-5min, 25% B; 5-6min, 25%-90% B; 6-9min, 90% B; 9-12min, 90%-10% B.

[0027] The mass spectrometry conditions in the step are as follows: using an electrospray ionization source in positive ion mode (ESI +), capillary voltage 3.0 kV, ion source temperature 120 °C, desolvation gas temperature 550 °C, cone gas flow rate 20 L / h, desolvation gas flow rate 800 L / h, acquisition mode MS E mode, resolution greater than 20,000; mass range: 50-1200Da; Lock Spray dual spray ion source: leucine enkephalin solution, ESI + It is 556.2771Da.

[0028] The mass spectrometry parameters of the analyte are shown in Table 1:

[0029] Table 1. Mass spectrometry parameters of six amatoxins

[0030]

[0031] The reagent kit for detecting six types of amatoxins in blood of Example 1 includes the following substances: the extraction tube A contains 1.5 mL of acetonitrile, the dilution tube B contains 400 μL of 1% acetic acid aqueous solution, and the concentrations of the standard gradient solution are 1, 5, 10, 20, 50, and 100 ng / mL.

[0032] The concentration of the six amatoxin standards was used as the horizontal coordinate, and the peak area of ​​the six amatoxins was used as the vertical coordinate to perform linear regression to obtain the standard curve. The standard curve and correlation coefficient are shown in Table 2:

[0034] Table 2. Retention time, linear range, linear equation and correlation coefficient of six amatoxins

[0035]

[0036] The mixed standard solutions of 6 kinds of amatoxins at 3 levels with different contents were added to the matrix samples, and each added concentration was measured 6 times in parallel to determine the precision and accuracy. The recovery rates of 6 kinds of amatoxins in blood were 74.1%-110.6%, and the coefficient of variation CV was 0.1%-6.7%. The results are shown in Table 3. The accuracy and precision of the method can meet the requirements of food poisoning residue analysis.

[0037] Table 3. Test results of precision and coefficient of variation of 6 kinds of amatoxins (n=6)

[0038]

[0039]

[0040] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the protection scope of the present invention. In addition, the present invention may also be implemented in other ways. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solution of the present invention may be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. A reagent for detecting amatoxin in blood, characterized in that: It comprises an extraction tube, a dilution tube and a standard gradient solution, wherein the extraction tube contains 1.5 mL of acetonitrile, the dilution tube contains 400 μL of a 1% acetic acid aqueous solution, and the gradient concentrations of the standard gradient solution are 1, 5, 10, 20, 50, and 100 ng / mL.

2. The detection reagent according to claim 1, characterized in that The amatoxin comprises at least one of α-amanitin, β-amanitin, γ-amanitin, carboxytrihydroxyphalloidin, dihydroxyphalloidin and carboxydihydroxyphalloidin.

3. A method for detecting amatoxin in blood, characterized in that: The following steps are involved: S1. Take 500 μL of serum sample and add it to the extraction tube of claim 1, mix well and centrifuge; S2, take 100 μL of the supernatant into the dilution tube of claim 1, mix well, and transfer to the injection bottle through the membrane; S3. Using a liquid chromatography time-of-flight mass spectrometer to measure the standard gradient solution and the sample to be tested, to obtain the concentration of amatoxin in the sample to be tested.

4. The detection method according to claim 3, characterized in that: The centrifugal temperature was 4±0.5℃, the rotation speed was 10000r / min, and the centrifugal time was 5±1min.

5. The detection method according to claim 3, characterized in that: The conditions of the liquid chromatography are as follows: mobile phase A is acetonitrile, mobile phase B is 0.01% formic acid aqueous solution; the mobile phase flow rate is 0.4 mL / min, the sample injection volume is 5 μL, the column temperature is 40° C., and the chromatographic column is an XSelect HSS T3 column (150 mm×3.0 mm, 2.5 μm); the liquid chromatography elution gradient is as follows: 0 to 1.5 min, 10% to 25% B; 4 to 5 min, 25% B; 5 to 6 min, 25% to 90% B; 6 to 9 min, 90% B; 9 to 12 min, 90% to 10% B.

6. The detection method according to claim 3, characterized in that: The mass spectrometry conditions are: using electrospray ionization source positive ion mode ESI + , capillary voltage 3.0 kV, ion source temperature 120 ° C, desolvation gas temperature 550 ° C, cone gas flow rate 20 L / h, desolvation gas flow rate 800 L / h, acquisition mode is MS E mode, resolution greater than 20,000; mass range: 50-1200Da; Lock Spray dual spray ion source: leucine enkephalin solution, ESI + It is 556.2771Da.

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