CN112816578B - Detection method of amino-containing small molecule mushroom toxin and kit - Google Patents
Detection method of amino-containing small molecule mushroom toxin and kit Download PDFInfo
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Abstract
The invention relates to the technical field of analysis and detection, and provides a detection method of amino-containing small molecule mushroom toxin and a kit. The invention adopts fluorenylmethoxycarbonyl chloride to perform derivatization on amino-containing micromolecule mushroom toxin, uses dichloromethane to perform enrichment purification, and analyzes the liquid to be detected through liquid chromatography-tandem mass spectrometry. The invention adopts the fluorenylmethoxycarbonyl acyl chloride to perform derivatization on the amino-containing micromolecule mushroom toxin, the derivatization reaction speed is high, the required time is short, the efficiency is far higher than that of the existing detection method, the chromatographic peak shape and the separation degree of a derivatization product can be effectively improved, and the accuracy and the sensitivity of the method are obviously improved; according to the invention, the amino-containing micromolecular mushroom toxin derivative is enriched and purified, so that the matrix effect of a sample can be reduced, and the accuracy of a detection result is improved. The kit provided by the invention can conveniently and quickly realize the detection of the amino-containing micromolecular mushroom toxin.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a detection method of amino-containing small molecule mushroom toxin and a kit.
Background
In recent years, poisoning accidents caused by eating wild mushrooms by mistake tend to spread in China, so that the wild mushrooms attract social attention and become a very prominent problem in the field of food safety. The national food safety risk assessment center in 2020 discovers that 31.8% of food-borne poisoning events caused by poisonous mushroom poisoning live at the head of various food-borne poisoning inducing factors based on data statistics of a food-borne disease outbreak monitoring system in China between 2003-2017. More than 100 kinds of mushroom toxins are identified at present, and cyclic peptides, small molecule compounds and the like are involved. Wherein the amanitic acid (Ibotenic acid) is a small molecule amino acid mushroom toxin, and the muscovil is decarboxylated compound. The main toxigenic bacteria of these two toxins are Amanita muscarius (Amanita muscaria) and Amanita pantherina (Amanita pantoea). After the poisonous mushroom is ingested, the nervous and mental symptoms are manifested as dizziness, tension, excitement, sensation change, muscle twitch and the like. Amanitic acid is rapidly excreted in the urine in its maternal form, partly converted into cyromotoxin. In addition, Yamaura et al, a Japanese scholarer, isolated two lethal small amino acid mushroom toxins from Amanita globosa: 2-amino-5-hexynoic acid (2-amino-5-hexynoic acid) and 2-amino-4-pentynoic acid (2-amino-4-pentynoic acid) (Toxicology,1986,38, 161. sup. -. 173). The two amino acid mushroom toxins have strong water solubility and polarity, but the metabolism and lethal mechanism in animals are not clear. In view of the current situation that toxic events of the poisonous mushrooms in China occur frequently, a robust mushroom toxin detection method is developed, and the method has important significance for tracing the mushroom toxins and identifying the poisonous mushrooms.
At present, only a few studies are carried out on detection methods of amanitic acid and drosophila melanogaster at home and abroad, and no reports on detection methods of 2-amino-5-hexynic acid and 2-amino-4-pentynic acid exist. Quantitative detection of amanithine and muscardine through liquid chromatography tandem mass spectrometry has been reported (Forensic diagnostics, 2013, 322-7664; food safety quality detection, 2019,22, 7656-7664). However, both toxins produce only one effective secondary mass spectrum product ion in the electrospray ion source (amanitic acid: 159 → 113; cyroma venosa: 115 → 98), are poorly characterized and are highly susceptible to false positive results. Kenji Tsujikawa et al derivatize amanithine and Symphytum officinale in two steps using dansyl chloride for an overall derivatization time of about 150min, and the derivatized products were detected by liquid chromatography UV (Journal of chromatography B,2007,852, 430-435). Xu et al used a similar derivatization scheme at 5% NaHCO3The reaction was carried out at 60 ℃ for 30min under the conditions to give 2-dansyl chloride-amanitin and 2-dansyl chloride-venomous flybush, followed by quantitation by liquid Chromatography tandem mass spectrometry (Journal of Chromatography B,2020,1146,122128). However, such derivatization methods require heat treatment, have long reaction time and complicated operation, and only amanitic acid and cyromazine are detected, but lethal small molecule mushroom toxins such as 2-amino-5-hexynic acid and 2-amino-4-pentynic acid cannot be detected.
In view of this, it is an urgent need to solve the technical problem to develop a detection method for mushroom toxins containing amino small molecules, which is simple in operation and accurate in quantification.
Disclosure of Invention
In view of the above, the invention provides a detection method for mushroom toxins containing small amino molecules and a kit. The detection method provided by the invention has the advantages of simple pretreatment, time saving, rapidness, high sensitivity and high accuracy.
In order to achieve the above object, the present invention provides the following technical solutions:
a detection method of amino-containing small molecule mushroom toxin comprises the following steps:
(1) extracting a sample to be detected by using an extraction solvent, and centrifuging the obtained extracting solution to obtain a supernatant;
(2) mixing the supernatant, a sodium tetraborate buffer solution and a fluorenylmethoxycarbonyl chloride solution for derivatization reaction, and performing first extraction on the obtained derivatization reaction liquid by using an organic solvent to obtain a lower layer solution; the organic solvent comprises one or more of n-pentane, n-hexane, cyclohexane and petroleum ether;
(3) adjusting the pH value of the lower layer solution to be acidic, and then carrying out second extraction by using dichloromethane to obtain dichloromethane extraction liquid;
(4) drying the dichloromethane extraction liquid by using nitrogen, and redissolving to obtain a solution to be detected;
(5) performing liquid chromatography tandem mass spectrometry on the liquid to be detected to obtain a spectrogram, and calculating according to the area of a spectrogram peak and a standard curve to obtain the content of the amino-containing micromolecular mushroom toxin in the sample to be detected; the standard curve is a relation curve of the concentration of the amino-containing small molecule mushroom toxin and the peak area of a derivative product of the amino-containing small molecule mushroom toxin.
Preferably, the amino-containing small molecule mushroom toxin is one or more of amanitic acid, cyromazine, 2-amino-5-hexynic acid, 2-amino-4-pentynic acid and 2-amino-4, 5-hexadienoic acid.
Preferably, the sample to be detected is mushroom, urine or plasma, and when the sample to be detected is mushroom, the extraction solvent is acetonitrile water solution or methanol water solution; when the sample to be detected is urine or blood plasma, the extraction solvent is acetonitrile; the extraction is ultrasonic extraction or oscillation extraction, and the extraction time is 5-10 min.
Preferably, the volume ratio of the supernatant to the sodium tetraborate buffer to the fluorenylmethoxycarbonyl chloride solution is 1-5: 1: 1-2; the concentration of the sodium tetraborate buffer solution is 0.1-0.5 mol/L, and the pH value of the sodium tetraborate buffer solution is 8-9; the concentration of the fluorenylmethoxycarbonyl chloride solution is 0.5-5 mg/mL; the temperature of the derivatization reaction is 25-45 ℃, and the time is 10-20 min.
Preferably, the number of times of the first extraction is 1-3, and the volume ratio of the organic solvent for single extraction to the derivatization reaction solution is 1-3: 1.
Preferably, the regulator for regulating the pH value of the lower layer solution is a hydrochloric acid solution, and the pH value of the lower layer solution is regulated to 2-3; the volume ratio of the lower layer solution to the dichloromethane is 1: 1-2.
Preferably, the solvent for redissolution is an aqueous methanol solution.
Preferably, the mass spectrum used for the liquid chromatography tandem mass spectrometry is a triple quadrupole mass spectrum, and the adopted mode is a multi-reaction monitoring mass spectrum mode;
the liquid chromatography conditions of the liquid chromatography tandem mass spectrometry comprise: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is methanol, the mobile phase B is an ammonium acetate aqueous solution containing ammonia water, the mass fraction of the ammonia water in the ammonium acetate aqueous solution containing ammonia water is 0.05%, and the concentration of the ammonium acetate is 5 mmol/L; the flow rate of the mobile phase is 0.3-0.4 mL/min; the chromatographic column is a ZORBAX eclipse plus C18 column, and the column temperature is 40 ℃;
the mass spectrum conditions of the liquid chromatography tandem mass spectrometry comprise: the detection mode is an electrospray ion source negative ion mode, the spray voltage is-4500V, the ion source temperature is 500 ℃, the air curtain pressure is 40psi, the sheath gas pressure is 50psi, and the auxiliary gas pressure is 50 psi.
Preferably, the mass spectrum parameters of the derivative product of the amino-containing small molecule mushroom toxin are shown in the table 1:
TABLE 1 Mass Spectrometry parameters for derivatized products of amino-containing Small molecule Mushroom toxins
The invention also provides a kit for detecting the amino-containing small molecule mushroom toxin, which comprises an extraction reagent, a derivatization reagent, a first extraction reagent, a second extraction reagent and a redissolution reagent which are independently packaged; the extraction reagent is acetonitrile, acetonitrile water solution or methanol water solution, and the derivatization reagent comprises sodium tetraborate buffer solution and fluorenylmethoxycarbonyl chloride solution; the first extraction reagent comprises one or more of n-pentane, n-hexane, cyclohexane and petroleum ether; the second extraction reagent is dichloromethane, and the redissolution reagent is methanol aqueous solution.
The invention provides a method for detecting amino-containing micromolecule mushroom toxins, which comprises the steps of firstly extracting a sample to be detected, centrifuging an extracting solution to obtain a supernatant, derivatizing the supernatant in a sodium tetraborate buffer solution and a fluorenylmethoxycarbonyl acyl chloride solution, carrying out first extraction, second extraction, blow-drying and redissolution on the obtained derivatization reaction solution to obtain a liquid to be detected, and carrying out liquid chromatography tandem mass spectrometry on the liquid to be detected. The detection method provided by the invention adopts fluorenylmethoxycarbonyl chloride (Fmoc-Cl) to perform derivatization on the amino-containing micromolecule mushroom toxin, the derivatization reaction speed is high, the required time is short, the reaction does not need to be heated, the complete derivatization can be performed only within 10min at room temperature, the efficiency is far higher than that of the existing detection method, the detection time is greatly shortened, the detection flux is improved, the chromatographic peak shape and the separation degree of the derivatization product can be effectively improved, and the accuracy and the sensitivity of the method are obviously improved.
The detection method provided by the invention adjusts the pH value of the lower layer solution obtained by the first extraction, and then adopts dichloromethane to enrich and separate the derivatization product containing the amino micromolecule mushroom toxin, and the invention can reduce the solubility of the derivatization product in the reaction liquid by adjusting the pH value of the system, thereby improving the extraction efficiency, increasing the enrichment times and improving the detection sensitivity; according to the invention, the amino-containing micromolecular mushroom toxin derivatives can be separated and enriched by dichloromethane extraction, the matrix effect of a sample is reduced, and the accuracy of a detection result is improved.
The detection method provided by the invention adopts liquid chromatography tandem mass spectrometry to detect the liquid to be detected, has the advantages of simple operation, high sample flux, high sensitivity, high detection accuracy and the like, can accurately quantify the trace amount of amino-containing micromolecular mushroom toxin in the sample to be detected, and is suitable for tracing the micromolecular mushroom toxin and identifying the mushroom containing toxin.
In addition, the method provided by the invention can be used for simultaneously detecting various amino-containing small molecule mushroom toxins, greatly reduces the detection difficulty, and is time-saving and rapid.
The invention also provides a kit for detecting the amino-containing small molecule mushroom toxin, and the detection of the amino-containing small molecule mushroom toxin can be conveniently and rapidly realized by utilizing the kit.
Drawings
FIG. 1 is a representative MRM profile of the derivatized products of four amino-containing small molecule mushroom toxins (all at 20ng/mL) in a urine sample;
FIG. 2 is a standard graph of four amino-containing small molecule mushroom toxins in urine.
Detailed Description
The invention provides a detection method of amino-containing small molecule mushroom toxin, which comprises the following steps:
(1) extracting a sample to be detected by using an extraction solvent, and centrifuging the obtained extracting solution to obtain a supernatant;
(2) mixing the supernatant, a sodium tetraborate buffer solution and a fluorenylmethoxycarbonyl chloride solution for derivatization reaction, and performing first extraction on the obtained derivatization reaction liquid by using an organic solvent to obtain a lower layer solution; the organic solvent comprises one or more of n-pentane, n-hexane, cyclohexane and petroleum ether;
(3) adjusting the pH value of the lower layer solution to be acidic, and then carrying out second extraction by using dichloromethane to obtain dichloromethane extraction liquid;
(4) drying the dichloromethane extraction liquid by using nitrogen, and redissolving to obtain a solution to be detected;
(5) performing liquid chromatography tandem mass spectrometry on the liquid to be detected to obtain a spectrogram, and calculating according to the spectrogram peak area and standard curve data to obtain the content of the amino-containing micromolecular mushroom toxin in the sample to be detected; the standard curve is a relation curve of the concentration of the amino-containing small molecule mushroom toxin derivative and the peak area of the amino-containing small molecule mushroom toxin derivative.
In the invention, the amino-containing small molecule mushroom toxin is preferably one or more of amanitic acid, cyromazine, 2-amino-5-hexynic acid, 2-amino-4-pentynic acid and 2-amino-4, 5-hexadienic acid; the detection method provided by the invention can be used for simultaneously detecting various small molecule mushroom toxins in a sample.
The method comprises the steps of extracting a sample to be detected by using an extraction solvent, and centrifuging the obtained extracting solution to obtain a supernatant. In the invention, the sample to be tested is preferably mushroom, plasma or urine; when the sample to be detected is mushroom, the extraction solvent is preferably acetonitrile water solution or methanol water solution, the volume fraction of acetonitrile in the acetonitrile water solution is preferably 50%, and the volume fraction of methanol in the methanol water solution is preferably 50%; when the sample to be detected is urine or blood plasma, the extraction solvent is preferably acetonitrile; when the sample to be detected is a mushroom sample, the mushroom sample is preferably crushed in advance, and the crushed material is extracted; when the sample to be detected is blood plasma or urine, the detection method provided by the invention is only used for detecting the content of the amino-containing small molecule mushroom toxin in the sample to be detected, and does not relate to subsequent diagnosis and treatment.
In the present invention, when the sample to be tested is mushroom, the ratio of the amount of the sample to be tested to the amount of the extraction solvent is preferably 1 g: 4-5 mL, wherein when the sample to be detected is blood plasma or urine, the volume ratio of the sample to be detected to the extraction solvent is preferably 1: 1; the extraction method is preferably ultrasonic extraction or oscillation extraction, and the extraction time is preferably 5-10 min; the present invention has no particular requirements on the power of the ultrasonic extraction and the frequency of the oscillatory extraction, and conditions well known to those skilled in the art can be employed.
In the invention, the rotation speed of the centrifugation is preferably more than 4000r/min, more preferably 5000-12000 r/min, and the time of the centrifugation is preferably 5 min.
After obtaining the supernatant, mixing the supernatant, a sodium tetraborate buffer solution and a fluorenylmethoxycarbonyl chloride solution for derivatization reaction to obtain a derivatization reaction solution. In the invention, the volume ratio of the supernatant, the sodium tetraborate buffer solution and the fluorenylmethoxycarbonyl chloride solution is preferably 1-5: 1: 1-2; the concentration of the sodium tetraborate buffer solution is preferably 0.1-0.5 mol/L, more preferably 0.2-0.3 mol/L, and the pH value of the sodium tetraborate buffer solution is preferably 8-9; the concentration of the fluorenylmethoxycarbonyl chloride solution is preferably 0.5-5 mg/mL, more preferably 1-4 mg/mL; the solvent of the fluorenylmethoxycarbonyl chloride solution is preferably acetone; the invention uses fluorenylmethoxycarbonyl chloride as a derivatization reagent and uses sodium tetraborate buffer solution to provide an alkaline environment for derivatization reaction.
In the invention, the temperature of the derivatization reaction is preferably 25-45 ℃, more preferably 25-30 ℃, and in the specific embodiment of the invention, the derivatization reaction is preferably directly carried out at room temperature without additional heating or cooling; the time of the derivatization reaction is preferably 10-20 min, and more preferably 10 min; the invention preferably mixes the supernatant, the sodium tetraborate buffer solution and the fluorenylmethoxycarbonyl chloride solution, then violently whirls and oscillates for 1min, and then the mixture is placed at room temperature for derivatization reaction; in the derivatization reaction process, the amino-containing small molecule mushroom toxin reacts with the fluorenylmethoxycarbonyl acyl chloride, and the obtained derivatization product has good chromatographic peak shape and high separation degree, and can obviously improve the detection accuracy and sensitivity.
In the present invention, the equation of the derivatization reaction is as follows:
after the derivatization reaction solution is obtained, the invention uses an organic solvent to carry out first extraction on the derivatization reaction solution to obtain a lower layer solution. In the invention, the organic solvent comprises one or more of n-pentane, n-hexane, cyclohexane and petroleum ether; the number of times of the first extraction is preferably 1-3, more preferably 3, and the volume ratio of the organic solvent for single extraction to the derivatization reaction solution is preferably 1-3: 1, more preferably 1.5: 1; the invention purifies the derivatization reaction liquid through first extraction to remove unreacted fluorenylmethoxycarbonyl chloride; after the first extraction is completed, the organic solvent layer and impurities, unreacted derivatizing reagent and the like are in the upper layer, and the derivatized product is in the lower layer solution.
After the first extraction is finished, the pH value of the obtained lower layer solution is adjusted to be acidic, and then dichloromethane is used for second extraction to obtain dichloromethane extraction liquid. In the invention, the regulator for regulating the pH value of the lower layer solution is preferably hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 1 mol/L; the pH value of the lower layer solution is preferably adjusted to 2-3; the frequency of the second extraction is preferably 1, and the volume ratio of the lower-layer solution to dichloromethane is preferably 1: 1-2; after extraction and delamination, taking a dichloromethane phase, namely the dichloromethane extract liquid. According to the invention, the solubility of the derivatization product in the solution is reduced by adjusting the pH value, the extraction rate of dichloromethane is improved, and the derivatization product is extracted into a dichloromethane phase through second extraction, so that the separation and enrichment of the derivatization product are realized, the matrix effect of a sample is reduced, and the accuracy of a detection result is improved.
After dichloromethane extraction liquid is obtained, the dichloromethane extraction liquid is re-dissolved after being dried by nitrogen, and the liquid to be detected is obtained. The specific conditions for drying the nitrogen gas are not particularly required, and the method known to those skilled in the art can be adopted. In the present invention, the redissolution solvent is preferably an aqueous methanol solution, and the volume fraction of methanol in the aqueous methanol solution is preferably 50%.
And after the liquid to be detected is obtained, performing liquid chromatography tandem mass spectrometry on the liquid to be detected to obtain a spectrogram. In the invention, the mass spectrum used for the liquid chromatography tandem mass spectrometry is preferably triple quadrupole mass spectrum, and the adopted mode is preferably a multiple reaction monitoring mass spectrum mode.
In the present invention, the liquid chromatography conditions of the liquid chromatography tandem mass spectrometry preferably include: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is methanol, the mobile phase B is an ammonium acetate aqueous solution containing ammonia water, the mass fraction of the ammonia water in the ammonium acetate aqueous solution containing ammonia water is 0.05%, and the concentration of the ammonium acetate is 5 mmol/L; the flow rate of the mobile phase is 0.3-0.4 mL/min; the chromatographic column is a ZORBAX eclipse plus C18 column, the specification of the chromatographic column is preferably 3.5 mu m multiplied by 150mm, the column temperature is 40 ℃, and the injection volume is preferably 5 mu L;
the mass spectrometry conditions of the liquid chromatography tandem mass spectrometry preferably include: the detection mode is an electrospray ion source negative ion mode, the spray voltage is-4500V, the ion source temperature is 500 ℃, the air curtain pressure is 40psi, the sheath gas pressure is 50psi, and the auxiliary gas pressure is 50 psi.
In a specific embodiment of the invention, the instrument used is preferably a Waters Acquity UPLC liquid chromatograph and an AB SCIEX 5500 triple quadrupole mass spectrometer.
In the invention, the mass spectrum parameters of the derivative product of the amino-containing small molecule mushroom toxin are shown in the table 1:
TABLE 1 Mass Spectrometry parameters for derivatized products of amino-containing Small molecule Mushroom toxins
Under the condition of mass spectrum, various derivatization products of the amino-containing small molecule mushroom toxin can be accurately detected, fig. 1 is a representative MRM map of four derivatization products of the amino-containing small molecule mushroom toxin (the concentration is 20ng/mL) in a urine sample, and as can be seen from fig. 1, the four derivatization products of the amino-containing small molecule mushroom toxin have better peak patterns in an MRM map and have better separation degree.
After a spectrogram is obtained, the content of the amino-containing small molecule mushroom toxin in the sample to be detected is calculated according to the peak area of the spectrogram and a standard curve; the standard curve is a relation curve of the concentration of the amino-containing small molecule mushroom toxin and the peak area of the amino-containing small molecule mushroom toxin derivative. The method for obtaining the standard curve has no special requirement, and the method well known by the technicians in the field can be adopted, in the specific embodiment of the invention, the standard substance containing the amino micromolecule mushroom toxin and the blank substrate are preferably used for preparing the gradient standard solution, the derivatization treatment and the purification are carried out according to the method in the scheme, then the detection is carried out, and the standard curve is drawn according to the concentration of the standard solution and the measured peak area.
The invention also provides a kit for detecting the amino-containing small molecule mushroom toxin, which comprises an extraction reagent, a derivatization reagent, a first extraction reagent, a second extraction reagent and a redissolution reagent which are independently subpackaged; the extraction reagent is acetonitrile, acetonitrile water solution or methanol water solution, and the derivatization reagent comprises sodium tetraborate buffer solution and fluorenylmethoxycarbonyl chloride solution; the first extraction reagent comprises one or more of n-pentane, n-hexane, cyclohexane and petroleum ether; the second extraction reagent is dichloromethane, and the redissolution reagent is methanol aqueous solution; when the kit is used, the method is used according to the scheme, and the kit can be used for conveniently and quickly detecting the amino-containing small molecule mushroom toxins in the sample.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1 optimization of derivatization reaction conditions
1. Instrumentation and equipment
Waters Acquity UPLC liquid chromatograph (Waters corporation, usa), AB SCIEX 5500 triple quadrupole mass spectrometer (AB SCIEX corporation, usa), vortex mixer, high speed centrifuge, nitrogen blow dryer, electronic analytical balance, pipette gun, etc.
2. Materials and reagents
Standard substances such as amanithine, cyromazine, 2-amino-5-hexynoic acid and 2-amino-4-pentynoic acid were purchased from Tianjin Alta; Fmoc-Cl, ammonium acetate (chromatographic grade), ammonia from Sigma-Aldrich; sodium tetraborate buffer (0.5mol/L, pH 8.0) was purchased from michelin, shanghai; chromatographically pure acetonitrile, methanol and acetone were purchased from Shanghai Tantake Technology, Inc.
3. Solution preparation
(1) Accurately weighing 5mg of amanithine, muscardine, 2-amino-5-hexynoic acid and 2-amino-4-pentynoic acid standard substances into volumetric flasks, adding ultrapure water to constant volume of 10mL, storing as standard stock solution at-20 deg.C in dark. The mixed working solution was obtained by gradient serial dilution of a standard stock solution with 50% aqueous methanol.
(2) Accurately weighing 100mg of fluorenylmethoxycarbonyl chloride (Fmoc-Cl) into a 50mL volumetric flask, carrying out constant volume by using acetone, shading and storing at room temperature.
(3) 0.3985g of sodium acetate solution is accurately weighed into a 1L volumetric flask, and the volume is fixed by using ultrapure water and then ultrasonic treatment is carried out for 5min to prepare 5mmol/L ammonium acetate aqueous solution which is prepared on site.
4. Derivatization process
(1) Investigation of derivatization time
Taking 200 mu L of urine sample or mushroom extract into a 2mL test tube, respectively adding 200 mu L of sodium tetraborate buffer solution (0.5mol/L, pH 8.0) and 400 mu L of Fmoc-Cl solution, violently shaking and vortexing for 1min, then placing the mixture at room temperature for reaction for 5, 10, 15 and 20min, then repeatedly extracting the mixture for 3 times by using n-pentane, removing the n-pentane layer, and taking the lower layer solution, wherein only the derivatization reaction condition is verified in the embodiment, in order to simplify the experiment operation, the lower layer solution is directly diluted and filtered for analysis without subsequent purification treatment, and the method specifically comprises the following steps: and diluting the obtained lower layer solution by using methanol multiple ratio, filtering the solution by using a membrane, analyzing the solution by using a liquid chromatography tandem mass spectrometer, and judging the derivatization efficiency by comparing the peak area.
(2) Examination of derivatization temperature
Putting 200 mu L of urine sample or mushroom extract into a 2mL test tube, respectively adding 200 mu L of sodium tetraborate buffer solution (0.5mol/L, pH 8.0) and 400 mu L of Fmoc-Cl solution, violently shaking and vortexing for 1min, then placing the mixture at 15 ℃, 25 ℃, 35 ℃, 45 ℃ and 55 ℃ for reaction for 10min, then repeatedly extracting the mixture for 3 times by using n-pentane, removing the n-pentane layer, analyzing the lower layer solution by using a filter membrane for liquid chromatography tandem mass spectrometry after methanol multiple dilution, and judging the derivatization efficiency by comparing the peak area size.
5. Liquid chromatography mass spectrometry conditions
(1) Chromatographic conditions
Mobile phase: methanol (A) and a 5mmol/L aqueous ammonium acetate solution (B) containing 0.05% ammonia water; the flow rate is 0.3 mL/min; the chromatographic column is a ZORBAX eclipse plus C18 column (3.5 μm. times.150 mm); the column temperature was 40 ℃; the injection volume was 5. mu.L. The gradient elution procedure was as follows: 0min, 10% A; 1min, 10% A; 9min, 100% A; 12min, 100% A; 12.2min, 10% A; 15min, 10% A.
(2) Conditions of Mass Spectrometry
Electrospray ion source negative ion mode (ESI)-) (ii) a The ion source parameters are: the spraying voltage is-4500V; the ion source temperature is 500 ℃; the air pressure of the air curtain is 8 psi; sheath gas pressure is 50 psi; the auxiliary air pressure was 50 psi; the air curtain air was 40 psi. Mass spectrometry parameters of derivatized products of amino-containing small molecule mushroom toxins are shown in Table 1 above.
6. Analysis of results
(1) The results of the tests performed on urine samples at different derivatization times are shown in Table 2.
TABLE 2 Effect of different derivatization times on the Signal response of four Mushroom toxins
According to the table 2, when the derivatization time is 5min, the signal response values of the four mushroom toxins are lower than those of other products after the derivatization time is more than 10min, the signal response values of the mushroom toxins are not very different, which indicates that the derivatization time is more than 10min, and the derivatization time of 10min is selected as the optimal reaction time for saving the detection time.
(2) The results of the measurements obtained with urine as the sample and at different derivatization temperatures are shown in table 3.
TABLE 3 Effect of different derivatization temperatures on the Signal response of four Mushroom toxins
As can be seen from Table 3, the signal response values of the four mushroom toxins have no obvious difference under the condition that the derivatization temperature is 25-45 ℃; when the derivatization temperature reaches 55 ℃, the signal values of the four mushroom toxins are reduced on the contrary, and presumably because at higher temperature, the derivatization reagent can continuously react with secondary amine in the mushroom toxins, so that other derivatization products are generated, and the detection signal is reduced. Therefore, room temperature conditions were chosen for the derivatization reaction.
(3) When mushroom is used as a sample and the derivatization condition of the mushroom extracting solution is optimized, the result is similar to that in the steps (1) to (2), subsequent derivatization is carried out at room temperature, and the derivatization time is set to be 10 min.
Example 2 extraction purification of amino-containing Small molecule Mushroom toxins
1. Effect of pH on extraction efficiency
Putting 200 mu L of urine sample or mushroom extract into a 2mL test tube, respectively adding 200 mu L of sodium tetraborate buffer solution (0.5mol/L, pH 8.0) and 400 mu L of Fmoc-Cl solution, violently shaking, vortexing for 1min, then placing at room temperature for reaction for 10min, then repeatedly extracting for 3 times with n-pentane, removing the n-pentane layer, adjusting the pH value of the lower layer solution to 2, 5 and 8 with 1mol/L hydrochloric acid or 1mol/L sodium hydroxide solution, then extracting with dichloromethane, re-dissolving the extract in 50% methanol aqueous solution after drying with nitrogen, analyzing with a liquid chromatography tandem mass spectrometer, and judging the influence of pH on the extraction efficiency by comparing the peak area.
2. Optimization of extraction method
(1) Direct derivatization of urine samples
Putting 200 mu L urine sample into a 2mL test tube, respectively adding 200 mu L sodium tetraborate buffer solution (0.5mol/L, pH 8.0) and 400 mu L Fmoc-Cl solution, violently shaking, vortexing for 1min, reacting for 10min at room temperature, repeatedly extracting for 3 times with n-pentane, removing the n-pentane layer, adjusting the pH of the lower layer solution to 2-3 with 1mol/L hydrochloric acid solution, then adding isovolumetric dichloromethane for extraction, re-dissolving the extract in 50% methanol aqueous solution after drying the nitrogen gas of the extract, analyzing by a liquid chromatography tandem mass spectrometer, and judging the extraction and purification efficiency by comparing the peak area.
(2) Ethyl acetate extraction purification of urine samples
Putting 200 mu L urine sample into a 2mL test tube, adding 2 times volume of ethyl acetate, violently vortexing, removing an ethyl acetate layer, repeating the operation for 3 times, then adding 200 mu L sodium tetraborate buffer solution (0.5M, pH 8.0) and 400 mu L Fmoc-Cl solution into the lower layer solution, violently vortexing for 1min, placing the lower layer solution at room temperature for reaction for 10min, repeatedly extracting for 3 times by using n-pentane, removing the n-pentane layer, adjusting the pH of the lower layer solution to 2-3 by using 1mol/L hydrochloric acid solution, then adding isovolumetric dichloromethane for extraction, re-dissolving the extract in 50% methanol aqueous solution after drying the nitrogen, carrying out analysis by using a liquid-phase chromatography tandem mass spectrometer, and judging the extraction and purification efficiency by comparing the peak area.
(3) Acetonitrile extraction and purification of urine sample
Putting 200 mu L urine sample into a 2mL test tube, adding equivalent volume of acetonitrile, performing vigorous vortex, centrifuging at 12000r/min for 5min, transferring supernatant into a 5mL test tube, adding 400 mu L sodium tetraborate buffer solution (0.5M, pH 8.0) and 400 mu L Fmoc-Cl solution, performing vigorous vortex for 1min, placing the test tube at room temperature for reaction for 10min, repeatedly extracting for 3 times by using n-pentane, removing an n-pentane layer, adjusting the pH of a lower layer solution to 2-3 by using 1mol/L hydrochloric acid solution, adding equivalent volume of dichloromethane for extraction, drying extract nitrogen, re-dissolving the extract in 50% methanol aqueous solution, performing analysis by using a liquid chromatography-mass spectrometer in series, and judging the extraction and purification efficiency by comparing peak areas.
(4) Extraction of mushroom samples
Accurately weighing 0.5g of fully crushed mushroom sample into a 5mL test tube, adding 2mL of different extraction solutions (acetonitrile, methanol, 50% methanol aqueous solution and 50% acetonitrile aqueous solution), carrying out ultrasonic extraction for 10min, then centrifuging for 5min at 12000r/min, taking 200 μ L of supernatant into a 2mL test tube, adding 200 μ L of sodium tetraborate buffer solution (0.5M, pH 8.0) and 400 μ L of Fmoc-Cl solution, violently shaking and whirling for 1min, then placing the solution at room temperature for reaction for 10min, then repeatedly extracting for 3 times by using n-pentane, removing an n-pentane layer, adjusting the pH of a lower layer solution to 2-3 by using 1mol/L of hydrochloric acid solution, then adding isometric dichloromethane for extraction, re-dissolving the extract in 50% methanol aqueous solution after nitrogen blow-drying, feeding a liquid chromatography-tandem mass spectrometer for analysis, and judging the extraction efficiency by comparing the peak area.
3. Analysis of results
(1) The test results of urine samples under different pH conditions and extraction times are shown in Table 4
TABLE 4 Effect of different extraction conditions on the Peak area response of 4 Mushroom toxins
As can be seen from table 4, the extraction yield of mushroom toxin by dichloromethane was low under the condition of pH 8; when the pH value is 5, the extraction recovery rate of the amanitic acid is less than 50 percent; when the pH value is 2, the recovery rate of the four mushroom toxins can reach more than 95 percent after the four mushroom toxins are extracted for 1 time by dichloromethane. Therefore, the samples were concentrated by enrichment using 1 extraction with dichloromethane at pH 2.
The test results of the mushroom extract liquid under different pH conditions and extraction times are similar to those in Table 4, and the results show that the recovery rate of four mushroom toxins can reach more than 95% when the pH value is 2 and the dichloromethane is used for extracting 1 time.
(2) The results of the different purification modes are shown in table 5:
TABLE 5 Effect of different clarification modes on the peak area response of 4 Agaricus campestris toxins
As can be seen from Table 5, the acetonitrile extraction purge significantly improved the signal response values for Symphytum, 2-amino-4-pentynoic acid and 2-amino-5-hexynoic acid at the same addition concentrations compared to the unpurified and ethyl acetate purge modes. Therefore, acetonitrile was chosen for extractive decontamination of biological samples.
(3) The extraction efficiency of the different extraction solutions on 4 toxins in mushrooms is shown in table 6:
TABLE 6 Effect of different extraction solutions on the area of the peak of the Mushroom toxin in the Mushroom sample
As can be seen from table 6, when acetonitrile or methanol was used as the extraction solution, the extraction efficiency was low for 4 toxins in the mushroom sample, and the peak area of each toxin was low; when 50% acetonitrile water or 50% methanol water solution was used as the extraction solvent, the extraction efficiency of 4 kinds of mushroom toxins was remarkably improved. Finally, 50% methanol in water was selected as the best extraction solution for the mushroom specimens.
Example 3 methodological validation of detection of amino-containing small molecule mushroom toxins
1. Linearity
The blank urine sample is prepared into standard solutions with the concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 and 100ng/mL by using a mixed standard and a standard containing four mushroom toxins. The optimal extraction method and derivatization conditions determined in example 1 and example 2 were used for processing, followed by analytical determination by a liquid chromatography tandem mass spectrometer. The results are shown in FIG. 2, where the peak area is taken as the Y-axis and the concentration of the mushroom toxin is taken as the X-axis, and a standard curve is plotted.
As can be seen from FIG. 2, R of the standard curves for the four mushroom toxins2Values were all greater than 0.99, with good linearity in the urine matrix.
In addition, mushroom and plasma are used as blank samples, the same method is adopted for processing and drawing a standard curve, and the result shows that R of the standard curves of the four mushroom toxins2Values were also both greater than 0.99, indicating good linearity in the mushroom and plasma matrices.
2. Recovery rate
And adding four kinds of mushroom toxins into blank urine, plasma and mushroom samples, testing the recovery rate, wherein the adding concentration is 1ng/mL, 10 ng/mL and 100ng/mL respectively, processing by adopting the optimal extraction method and the derivatization condition determined in the example 1 and the example 2, and then analyzing and determining by a liquid chromatography tandem mass spectrometer. The quantification was performed using the standard curve drawn in step 1 based on the peak area of the analyte, and the results are shown in table 7.
TABLE 7 recovery of four mushroom toxins in urine, plasma and mushrooms normalized
As can be seen from Table 7, the recovery rates of four mushroom toxins among urine, plasma and mushrooms were good, all between 83% and 104%.
3. Sensitivity of the probe
Limit of quantitation (LOQ) refers to the concentration at which the target quantitation ion is 10 times the signal-to-noise ratio (S/N10) in the corresponding solution of the sample matrix. By adding four kinds of mushroom toxin mixed standard solutions to urine, plasma and mushroom matrixes and carrying out gradient dilution, the quantitative limit of amanitic acid is 0.5ng/mL, the quantitative limit of cyromotoxin is 1ng/mL, and the quantitative limit of 2-amino-4-pentynic acid and 2-amino-5-hexynic acid is 0.1 ng/mL.
Example 4 Blind sample testing
After the urine, plasma and mushroom samples are subjected to blind sample labeling, other testers perform tests according to the detection method of the invention, and the extraction conditions and the derivatization conditions are the optimal conditions determined in the embodiments 1-2. The test results are shown in table 8:
TABLE 8 Blind sample labeling test results
The results in table 8 show that all quantitative results achieved satisfactory results, and the detection method provided by the present invention has high accuracy.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A detection method of amino-containing small molecule mushroom toxin is characterized by comprising the following steps:
(1) extracting a sample to be detected by using an extraction solvent, and centrifuging the obtained extracting solution to obtain a supernatant;
(2) mixing the supernatant, a sodium tetraborate buffer solution and a fluorenylmethoxycarbonyl chloride solution for derivatization reaction, and performing first extraction on the obtained derivatization reaction liquid by using an organic solvent to obtain a lower layer solution; the organic solvent comprises one or more of n-pentane, n-hexane, cyclohexane and petroleum ether;
(3) adjusting the pH value of the lower layer solution to be acidic, and then carrying out second extraction by using dichloromethane to obtain dichloromethane extraction liquid;
(4) drying the dichloromethane extraction liquid by using nitrogen, and redissolving to obtain a solution to be detected;
(5) performing liquid chromatography tandem mass spectrometry on the liquid to be detected to obtain a spectrogram, and calculating according to the area of a spectrogram peak and a standard curve to obtain the content of the amino-containing micromolecular mushroom toxin in the sample to be detected; the standard curve is a relation curve of the concentration of the amino-containing small molecule mushroom toxin and the peak area of a derivative product of the amino-containing small molecule mushroom toxin;
the amino-containing small molecule mushroom toxin is amanitic acid, cyromazine, 2-amino-5-hexynic acid, 2-amino-4-pentynic acid and 2-amino-4, 5-hexadienoic acid; the temperature of the derivatization reaction is 25-45 ℃, and the time is 10-20 min;
the sample to be detected is mushroom, urine or plasma, and when the sample to be detected is mushroom, the extraction solvent is acetonitrile water solution or methanol water solution; when the sample to be detected is urine or blood plasma, the extraction solvent is acetonitrile;
the liquid chromatography conditions of the liquid chromatography tandem mass spectrometry comprise: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is methanol, the mobile phase B is an ammonium acetate aqueous solution containing ammonia water, the mass fraction of the ammonia water in the ammonium acetate aqueous solution containing ammonia water is 0.05%, and the concentration of the ammonium acetate is 5 mmol/L; the chromatographic column is a ZORBAX eclipse plus C18 column; the gradient elution procedure was: 0min, 10% A; 1min, 10% A; 9min, 100% A; 12min, 100% A; 12.2min, 10% A; 15min, 10% A.
2. The detection method according to claim 1, wherein the extraction is ultrasonic extraction or oscillation extraction, and the extraction time is 5-10 min.
3. The detection method according to claim 1, wherein the volume ratio of the supernatant to the sodium tetraborate buffer to the fluorenylmethoxycarbonyl chloride solution is 1-5: 1: 1-2; the concentration of the sodium tetraborate buffer solution is 0.1-0.5 mol/L, and the pH value of the sodium tetraborate buffer solution is 8-9; the concentration of the fluorenylmethoxycarbonyl chloride solution is 0.5-5 mg/mL.
4. The detection method according to claim 1, wherein the number of times of the first extraction is 1 to 3, and the volume ratio of the organic solvent for single extraction to the derivatization reaction solution is 1 to 3: 1.
5. The detection method according to claim 1, wherein the pH of the lower layer solution is adjusted to 2 to 3 by using a hydrochloric acid solution as an adjusting agent for adjusting the pH of the lower layer solution; the volume ratio of the lower layer solution to the dichloromethane is 1: 1-2.
6. The detection method according to claim 1, wherein the solvent for reconstitution is an aqueous methanol solution.
7. The detection method according to claim 1, wherein the mass spectrum used in the LC-MS is triple quadrupole mass spectrum in a multiple reaction monitoring mass spectrum mode;
the flow rate of the mobile phase is 0.3-0.4 mL/min; the column temperature of the chromatographic column is 40 ℃;
the mass spectrum conditions of the liquid chromatography tandem mass spectrometry comprise: the detection mode is an electrospray ion source negative ion mode, the spray voltage is-4500V, the ion source temperature is 500 ℃, the air curtain pressure is 40psi, the sheath gas pressure is 50psi, and the auxiliary gas pressure is 50 psi.
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