阅读说明：本技术 对真菌毒素进行定量检测的方法 (Method for quantitative detection of mycotoxins ) 是由 张峰 冯峰 徐美霞 于 2020-12-15 设计创作，主要内容包括：本发明公开了一种对真菌毒素进行定量检测的方法。该方法包括：将样品进行干燥粉碎处理,以便得到样品粉末；将所述样品粉末与乙腈涡旋混匀,超声提取,加0.2g PSA净化,涡旋混匀2min,离心,以便得到净化提取物；将所述净化提取物进行复溶处理,以便得到待测液；以及将所述待测液进行色谱-质谱联用检测,以便获得所述真菌毒素含量。该方法具有高选择性、高灵敏度、定性定量分析准确等特点。(The invention discloses a method for quantitatively detecting mycotoxin. The method comprises the following steps: drying and crushing the sample to obtain sample powder; mixing the sample powder and acetonitrile by vortex, performing ultrasonic extraction, adding 0.2g of PSA for purification, mixing by vortex for 2min, and centrifuging to obtain purified extract; redissolving the purified extract to obtain a solution to be detected; and carrying out chromatography-mass spectrometry detection on the liquid to be detected so as to obtain the content of the mycotoxin. The method has the characteristics of high selectivity, high sensitivity, accurate qualitative and quantitative analysis and the like.)

1. A method for quantitatively detecting mycotoxins, comprising:

drying and crushing the sample to obtain sample powder;

mixing the sample powder and acetonitrile by vortex, performing ultrasonic extraction, adding 0.2g of PSA for purification, mixing by vortex for 2min, and centrifuging to obtain purified extract;

redissolving the purified extract to obtain a solution to be detected; and

and (3) carrying out chromatography-mass spectrometry detection on the solution to be detected so as to obtain the content of the mycotoxin.

2. The method of claim 1, wherein the sample powder is mixed with an isotopically labeled internal standard prior to said vortexing.

3. The method of claim 1, wherein the mass to volume ratio of the sample powder to acetonitrile is 1 g: 10 ml.

4. The method according to claim 1, wherein the chromatography-mass spectrometry detection chromatographic conditions are as follows:

a chromatographic column: agilent SB-C18 chromatographic column, 2.1 × 50mm, 1.8 μm;

the column temperature is 25 ℃;

the flow rate of the mobile phase is 0.2 mL/min;

the amount of sample was 5. mu.L.

5. The method according to claim 1, wherein the chromatography-mass spectrometry detection chromatography mobile phase is: phase A is 0.1% formic acid solution in water, and phase B is methanol.

6. The method of claim 1, wherein the chromatography-mass spectrometry detection is performed by a chromatography gradient elution procedure as follows: 0-5 min, 20-60% of B.

7. The method of claim 1, wherein the mass spectrometric conditions for the chromatography-mass spectrometric detection are:

electrospray ionization source (ESI): positive ion mode (ESI)⁺)；

Multiple reaction monitoring mode (MRM);

a triple quadrupole mass analyzer;

fragmentation voltage: 380V;

temperature of the drying gas: 250 ℃, dry air flow: 14L/min;

temperature of sheath gas: 250 ℃, sheath gas flow: 11L/min;

nozzle voltage: 2000V;

residence time: 65 ms.

8. The method of claim 1, wherein the mycotoxin is aflatoxin.

9. The method of claim 1, wherein the sample is a lonicera confusa material.

Technical Field

The invention relates to the field of analytical chemistry, in particular to a method for quantitatively detecting mycotoxins.

Background

The lonicera confusa is mostly grown and planted in a southern rainy region, the temperature is high, the lonicera confusa is harvested from june to july every year, the rainfall is high in the southern area, fresh flowers are not dried in time or storage and transportation conditions are not proper, and mycotoxin toxic compounds are generated due to the fact that the honeysuckle is easy to mildew.

Thus, methods for detection of mycotoxins in lonicera confusa are under study and improvement.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, one purpose of the invention is to provide a simple, rapid and accurate method for quantitatively detecting mycotoxin, wherein a sample is extracted by adding acetonitrile aqueous solution, PSA (pressure swing adsorption) is purified, and the filtered sample is subjected to machine detection. Can be applied to the detection of mycotoxin in the lonicera confusa medicinal material and related products, and provides reference for the quality control of the Chinese medicinal material.

According to one aspect of the invention, a method for the quantitative detection of mycotoxins is provided. According to an embodiment of the invention, the method comprises: drying and crushing the sample to obtain sample powder; mixing the sample powder and acetonitrile by vortex, performing ultrasonic extraction, adding 0.2g of PSA for purification, mixing by vortex for 2min, and centrifuging to obtain purified extract; redissolving the purified extract to obtain a solution to be detected; and carrying out chromatography-mass spectrometry detection on the liquid to be detected so as to obtain the content of the mycotoxin.

The method for quantitatively detecting the mycotoxin provided by the embodiment of the invention has the characteristics of high selectivity, high sensitivity, accurate qualitative and quantitative analysis and the like. The pretreatment of the sample is simple, the time and the cost are saved, and the working efficiency can be improved.

In addition, the method for quantitatively detecting mycotoxin according to the above embodiment of the present invention may further have the following additional technical features:

according to an embodiment of the invention, the sample powder is mixed with an isotopically labelled internal standard before the vortexing.

According to the embodiment of the invention, the mass volume ratio of the sample powder to the acetonitrile is 1 g: 10 ml.

According to the embodiment of the invention, the chromatographic conditions of the chromatography-mass spectrometry combined detection are as follows: a chromatographic column: agilent SB-C18 chromatographic column, 2.1 × 50mm, 1.8 μm; the column temperature is 25 ℃; the flow rate of the mobile phase is 0.2 mL/min; the amount of sample was 5. mu.L.

According to the embodiment of the invention, the chromatographic mobile phase of the chromatography-mass spectrometry detection is as follows: phase A is 0.1% formic acid solution in water, and phase B is methanol.

According to the embodiment of the invention, the chromatographic gradient elution procedure of the chromatography-mass spectrometry detection is as follows: 0-5 min, 20-60% of B.

According to the embodiment of the invention, the mass spectrum conditions of the chromatography-mass spectrometry combined detection are as follows: electrospray ionization source (ESI): positive ion mode (ESI)⁺) (ii) a Multiple reaction monitoring mode (MRM); a triple quadrupole mass analyzer; fragmentation voltage: 380V; temperature of the drying gas: 250 ℃, dry air flow: 14L/min; temperature of sheath gas: 250 ℃, sheath gas flow: 11L/min; nozzle voltage: 2000V; residence time: 65 ms.

According to an embodiment of the invention, the mycotoxin is aflatoxin.

According to the embodiment of the invention, the sample is a lonicera confusa medicinal material.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a schematic MRM chromatogram of 4 aflatoxins according to one embodiment of the invention;

FIG. 2 shows a schematic representation of a precursor ion scan of aflatoxin B1 in accordance with one embodiment of the present invention;

FIG. 3 shows a schematic precursor ion scan of aflatoxin B1 in accordance with one embodiment of the present invention;

FIG. 4 shows a schematic of a possible cleavage pathway for the major fragment of aflatoxin B2 in accordance with one embodiment of the present invention;

FIG. 5 shows a schematic diagram of a possible fragmentation pathway for the major fragment ion of aflatoxin G1 in accordance with one embodiment of the present invention;

FIG. 6 shows a schematic of a possible cleavage pathway for the major fragment of aflatoxin G2 in accordance with one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

According to one aspect of the invention, a method for the quantitative detection of mycotoxins is provided. According to an embodiment of the invention, the method comprises: drying and crushing the sample to obtain sample powder; mixing the sample powder and acetonitrile by vortex, performing ultrasonic extraction, adding 0.2g of PSA for purification, mixing by vortex for 2min, and centrifuging to obtain purified extract; redissolving the purified extract to obtain a solution to be detected; and carrying out chromatography-mass spectrometry detection on the liquid to be detected so as to obtain the content of the mycotoxin.

The method for quantitatively detecting the mycotoxin provided by the embodiment of the invention has the characteristics of high selectivity, high sensitivity, accurate qualitative and quantitative analysis and the like. The pretreatment of the sample is simple, the time and the cost are saved, and the working efficiency can be improved.

According to an embodiment of the invention, the sample powder is mixed with an isotopically labelled internal standard before the vortexing. Therefore, the detection accuracy is high.

According to the embodiment of the invention, the mass volume ratio of the sample powder to the acetonitrile is 1 g: 10 ml. Thereby, sufficient extraction of the compound is facilitated.

According to the embodiment of the invention, the chromatographic conditions of the chromatography-mass spectrometry combined detection are as follows: a chromatographic column: agilent SB-C18 chromatographic column, 2.1 × 50mm, 1.8 μm; the column temperature is 25 ℃; the flow rate of the mobile phase is 0.2 mL/min; the amount of sample was 5. mu.L. Therefore, the detection accuracy and sensitivity are high.

According to the embodiment of the invention, the chromatographic mobile phase of the chromatography-mass spectrometry detection is as follows: phase A is 0.1% formic acid solution in water, and phase B is methanol. This results in a high response in the test compound system.

According to the embodiment of the invention, the chromatographic gradient elution procedure of the chromatography-mass spectrometry detection is as follows: 0-5 min, 20-60% of B. Therefore, the separation effect of the compound to be detected is good, the peak-off time is appropriate, and the peak pattern is good.

According to the embodiment of the invention, the mass spectrum conditions of the chromatography-mass spectrometry combined detection are as follows: electrospray ionization source (ESI): positive ion mode (ESI)⁺) (ii) a Multiple reaction monitoring mode (MRM); a triple quadrupole mass analyzer; fragmentation voltage: 380V; temperature of the drying gas: 250 ℃, dry air flow: 14L/min; temperature of sheath gas: 250 ℃, sheath gas flow: 11L/min; nozzle voltage: 2000V; residence time: 65 ms. Therefore, the detection accuracy and sensitivity are high.

According to an embodiment of the invention, the mycotoxin is aflatoxin.

According to the embodiment of the invention, the sample is a lonicera confusa medicinal material.

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.

Example 1

The method of the embodiment of the invention is used for detecting aflatoxin in lonicera confusa medicinal material, and comprises the following steps:

1. experimental methods

1.1 sample pretreatment

Drying and crushing the lonicera confusa medicinal material,Sieving, precisely weighing 0.5G of medicinal powder, and adding 10 μ G/kg aflatoxin G2¹³C₁₇Adding 5mL of 60% acetonitrile into the internal standard substance, performing vortex mixing, performing ultrasonic extraction for 10min, adding 0.2g of PSA for purification, performing vortex mixing for 2min, centrifuging (8000rps/min, 10min), taking the supernatant, blowing nitrogen to be nearly dry, redissolving 20% methanol, fixing the volume to 2mL, filtering, and performing detection on a computer.

1.2 measurement method

1.2.1 chromatographic conditions

A chromatographic column: agilent SB-C18 column (2.1X 50mm, 1.8 μm); the column temperature is 25 ℃; mobile phase a was 0.1% formic acid and phase B was methanol at a flow rate of 0.2mL/min, and the gradient elution procedure was as follows: 0-5 min, 20% -60% B; the amount of sample was 5. mu.L.

1.2.2 Mass Spectrometry conditions

Electrospray ionization source (ESI): positive ion mode (ESI)⁺) (ii) a Multiple reaction monitoring mode (MRM); a triple quadrupole mass analyzer; fragmentation voltage: 380V; temperature of the drying gas: 250 ℃, dry air flow: 14L/min; temperature of sheath gas: 250 ℃, sheath gas flow: 11L/min; nozzle voltage: 2000V. Residence time: 65 ms. The mass spectrometric parameters of the individual compounds are shown in Table 1.

TABLE 1 Mass spectrometric parameters of the four aflatoxins

Quantitative ions

*Quantitative ion

1.2.3 matrix Effect

Taking a lonicera confusa medicinal material which is not polluted by aflatoxin, preparing a blank matrix solution according to the extraction method, preparing the aflatoxin solution with the same concentration by using a pure solvent and the blank matrix solution respectively, observing the matrix effect under the conditions of 1, 10 and 100 mu g/L as low, medium and high 3 addition concentrations, carrying out on-machine detection according to a 3.2.3 analysis method, and calculating the matrix effect according to a formula (1). Where Am is the response of the target compound in the matrix and As is the response of the target in pure solvent.

Mi(％)＝Am/As×100％ (1)

2 results and discussion

2.1 chromatographic Condition optimization

Experiments investigate the SB-C18 column and the phenyl column, the four compounds are weaker in retention on the phenyl column, and have better retention and separation effects on the SB-C18 column, and the four compounds can be completely separated from the matrix when the retention time exceeds two minutes, so that the matrix effect is reduced, and the accuracy of qualitative and quantitative determination is improved. Therefore, an Agilent SB-C18 ultra performance liquid chromatography column was selected. In addition, the influence of different mobile phase compositions on the separation and response of the four aflatoxins is also examined. The separation and response of methanol and acetonitrile as organic phase and water and 0.1% formic acid as aqueous phase were compared, respectively, with the highest response of the target in the methanol-0.1% formic acid system. The specific reason is analyzed as follows: the target substance is ionized in a positive ion mode, formic acid is beneficial to the ionization of the target substance to form positive ions, the ionization efficiency can be obviously improved, and methanol is a proton type solvent and can also promote the ionization of the target substance. FIG. 1 is MRM chromatogram (right) of 4 aflatoxins in matrix labeling and corresponding blank matrix solutions under optimized conditions.

2.2 optimization of Mass Spectrometry conditions and cleavage rules

In the process of optimizing the mass spectrum acquisition parameters, the responses of all the target objects in the positive ion mode and the negative ion mode are respectively compared, and the response of all the substances in the positive mode is higher than that in the negative mode. With respect to the selection of the parent ion, each target can form [ M + H ]]⁺、[M+Na]⁺And [ M + K]⁺Three kinds of parent ions, among them, [ M + Na ] as shown in FIG. 2]⁺The abundance is the largest, but the stability of ions is obviously improved after the sodium adduct is formed, stable fragment ions are not easily formed in a product ion mode, and the abundance of each fragment ion is obviously lower than that of [ M + H ]]⁺Abundance of fragment ions, so [ M + H ] is selected]⁺In the form of a precursor ion.

The four compounds of aflatoxins B1, B2, G1 and G2 are all derivatives of dihydrofuran oxanaphthalenone. The compound has the structure of anisole, has low bond energy and is easy to lose one methyl ([ M + H-CH ]₃]⁺313 > 298, m/z, stepsStep 1). The aflatoxin B1 has a lactone structure in the structure, so that neutral loss and loss of CO are easy to occur₂([M+H-CO₂]⁺313 > 269, m/z, step 3). In addition, the alicyclic ketone undergoes alpha cleavage and i cleavage to cause loss of neutrality (-CO) after ring cleavage, which occurs in steps 2, 4 and 5 in the figure, and ring closure reaction may occur due to generation of radicals in the processes 2 and 5. In the reaction of the step 4, the ring-closing reaction is difficult to occur due to the overlarge steric hindrance. FIG. 3 is a possible cleavage pathway for the major fragment ion of aflatoxin B1.

The aflatoxin B2 has a structure similar to aflatoxin B1, and can also form a product with lost neutrality and loss of one CO₂([M+H-CO₂]⁺315 > 271, m/z, step 4). However, the double bond on one side of the furan ring is saturated and the cleavage process shows some difference. The tetrahydrofuran ring is easily cleaved, so the ring-opened product has a relatively high abundance and can form a dehydrated product (315 > 297, [ M + H-H ]₂O]⁺M/z, step 3) and the deethylenized product (315 > 287, [ M + H-C)₂H₄]⁺M/z, step 2) and forming carbenium ions, which are primary and secondary carbenium ions, respectively, the latter being relatively more stable and more abundant than the former. FIG. 4 is a possible cleavage pathway for the major fragment of aflatoxin B2.

A possible cleavage pathway for the major fragment ion of aflatoxin G1 is shown in FIG. 5. Aflatoxins G1, G2 are structurally similar to the two above-mentioned substances, but two lactone structures are present in the two substance structures. The bond energy of the ester bond is low, and both ester bonds are easily broken. Three double bonds, namely a stable pi-pi conjugated structure, can be formed by dehydration in the step 1. The fragment ions formed are lost through neutrality ([ M + H-CO)]⁺311 > 283, m/z, step 2) forming a more abundant ion 283(m/z), or losing a neutral molecule CO₂(311 > 267, m/z, step 3). In addition, the lactone bond in the heteronaphthalene ring is also easy to break (step 4), the formed fragment ions have an active alpha-H to link two carbonyl groups (electron-withdrawing groups), the fragment ions can be induced to generate i-break (step 5), and the bond induced by electron transfer and charge center is broken (step 6) to form mass chargeThe fragment ion with the ratio of 243 is the basic peak of the mass spectrum of the aflatoxin G1.

The possible cleavage pathway of the major fragment of aflatoxin G2 is shown in fig. 6. Among them, there are theoretically two possible generation routes of fragment ions 313 and 285(m/z), namely, cleavage dehydration and deithylene of tetrahydrofuran ring and cleavage dehydration and decarbonylation of lactone ring. Both pathways are similar to the above-mentioned substances and can theoretically occur. In addition, fragment ion 245(m/z) is the most abundant daughter ion of aflatoxin G2, and the response is relatively stable. Form [ M + H]⁺The quasi-molecular ion peak of (1) is cracked by i to open a tetrahydrofuran ring, then rearranged into a tertiary carbocation ion by a secondary carbocation ion to form a more stable structure, and then cracked by i to lose a hydroxyl tetrahydrofuran to form fragment ions with the mass-to-charge ratio of 245 (m/z).

The aflatoxins B1, B2, G1 and G2 are similar in structure and are derivatives of dihydrofurocoumarin. The four compounds are easy to lose neutrality and CO in the cracking process₂、H₂O and other small molecular compounds. In addition, in the mass spectra of the four compounds, two identical fragment ions appeared, and the mass-to-nuclear ratio was 115 to 128 (m/z). The two common fragment ions have certain guiding significance for the non-target screening of unknown potential harmful substances.

2.3 matrix Effect

Aflatoxin G2 was chosen for the experiment¹³C₁₇Is composed of¹³And the C stable isotope internal standard is used for correcting the loss of the target in the pretreatment and detection processes. The internal standard substance has no interference in qualitative and quantitative ion channels, and the compound is artificially synthesized and does not exist in nature, so that the detection of the internal standard substance cannot be influenced. The four compounds in the experiment are similar in structure, so that it is sufficient to select an isotope to eliminate interference. Because the chlorogenic acid compound in the lonicera confusa medicinal material has higher content, and PSA has obvious adsorption performance on polyphenol acid, and can be used for reducing the matrix effect in detection, 0.2g of PSA is added in the pretreatment process to purify the extracting solution.

2.4 methodological validation

2.4.1 Linear relationship, detection Limit (LOD) and quantitation Limit (LOQ)

The substrate has a significant impact on target detection, so the substrate-labeling approach was chosen to best fit the standard curve for quantitative analysis. Preparing a mixed solution with the mass concentration of 100-0.1 mu g/L, and taking the peak area of the target object as a vertical coordinate (Y) and the corresponding mass concentration as a horizontal coordinate (X) to obtain a standard curve. The detection limit and the quantification limit were the contents of the target at signal-to-noise ratios of 3 and 10, respectively, and the results are shown in table 2.

TABLE 24 Linear regression equation, Linear Range, correlation coefficient, and detection and quantitation limits for aflatoxins

2.4.2 recovery and precision of spiking

The quantitative limits of 1 time, 2 times and 4 times of each compound are used as the blank sample standard adding levels, five groups of parallel experiments are carried out on each adding level, the detection is carried out according to the extraction analysis methods of 3.2.2 and 3.2.3, and the recovery rate results and the relative standard deviation are shown in table 3.

Table 34 recovery normalized to the relative standard deviation (n ═ 6) for the compounds

2.5 actual sample analysis

The established UHPLC-MS/MS method is applied to detect 11 lonicera confusa medicinal material samples in the market, and the result shows that 25.1 microgram/kg of aflatoxin B is detected in one sample of the 11 lonicera confusa medicinal materials, and the rest samples are negative and do not exceed the pharmacopoeia requirements.

In conclusion, the method provided by the embodiment of the invention establishes a UHPLC-MS/MS method for detecting aflatoxin in lonicera confusa medicinal material, and discusses and researches mass spectrum cracking ways and main mass spectrum fragments of four aflatoxins. The method has the characteristics of high selectivity, high sensitivity, accurate qualitative and quantitative analysis and the like. The pretreatment of the sample is simple, the time and the cost are saved, and the working efficiency can be improved. Meanwhile, the aflatoxin pollution analysis of the medicinal materials can be realized by monitoring characteristic fragments of the target object under the condition without a standard substance. A positive sample is detected in the experiment, and the supervision of the medicinal materials in the processes of drying, transportation and storage is recommended to be enhanced.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.