CN118440091A - Separation method of aflatoxins B2 and G2 and chiral isomers thereof - Google Patents
Separation method of aflatoxins B2 and G2 and chiral isomers thereof Download PDFInfo
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention relates to a separation method of aflatoxin B2 and aflatoxin G2 and enantiomers thereof. Aflatoxin B2, G2 and chiral isomers thereof are prepared by a chiral resolution method, and are respectively aflatoxin B2, aflatoxin B2', aflatoxin G2 and aflatoxin G2' (AFB 2, AFB2', AFG2 and AFG 2'). Then, the above-mentioned compounds are identified and structurally confirmed by means of ultraviolet, mass spectrum, nuclear magnetism, circular dichroism and other methods, and it is verified that AFB2 'and AFG2' are two newly discovered compounds which are respectively enantiomers with AFB2 and AFG 2. The newly discovered AFB2 'and AFG2' are subjected to cytotoxicity test, and the test results show that both compounds have cytogenetic toxicity, are very likely to have carcinogenicity, hepatotoxicity and photosensitivity to mammals, can cause chromosome damage in vitro to the mammals, have nonspecific genetic toxicity to the mammals, and can also have mutagenicity in the mammals.
Description
Technical Field
The invention belongs to the field of analysis and detection, and particularly relates to a separation method of aflatoxin B2 and aflatoxin G2 and chiral isomers thereof, in particular to a separation preparation method, a structure confirmation and a cytotoxicity experiment of aflatoxin B2 and aflatoxin G2 and chiral enantiomers thereof.
Background
The aflatoxins are derivatives of dihydrofurocoumarin and are mainly produced by secondary metabolism of aspergillus flavus (A. Flavus) and aspergillus parasiticus (A. Paconiticus), are the most studied mycotoxins, have definite carcinogenicity, can cause serious liver injury and even cause liver cancer, and are the mycotoxins with the greatest toxicity and extremely outstanding harm to human health. International cancer research institute (International Agency for Research on Cancer) classified aflatoxins (aflatoxins B1, B2, G1, G2) (AFB 1, AFB2, AFG1, AFG 2) as class 1 carcinogens in 2012, wherein the molecular formulas of AFB1, AFB2, AFG1, AFG2 were C17H12O6、C17H14O6、C17H12O7、C17H14O7, molecular weights, respectively: 312.27, 314.29, 328.27, 330.29.
Aflatoxins B1, B2, G1, G2 all have 2 chiral centers, and it is currently believed that these four aflatoxins all have only one chiral configuration, namely AFB1, AFG1, AFB2, AFG2 in fig. 1. According to the invention, in an actual sample, the existence of isomers of aflatoxin B2 and aflatoxin G2 is found, the isomer monomer compounds of the aflatoxin B2 and the aflatoxin G2 are respectively obtained by establishing a proper chiral separation method, and then the two new aflatoxin compounds are found by qualitative methods such as ultraviolet, mass spectrum, nuclear magnetism, circular dichroism and the like. Through further structural confirmation, two new aflatoxin compounds are respectively enantiomer with aflatoxin B2 and aflatoxin G2, and are respectively named as aflatoxin B2 enantiomer (AFB 2 ') and aflatoxin G2 enantiomer (AFG 2') in the invention, and the structural formulas are shown in figure 1.
The discovery of aflatoxin B2 and enantiomers of aflatoxin G2 (AFB 2 'and AFG 2') breaks through the original knowledge of only one chiral configuration of aflatoxin. Because the limit of aflatoxin is very low, the detection methods of aflatoxin (national food safety standard and Chinese pharmacopoeia) in legal methods all adopt immunoaffinity columns for purification. The purification process of the immunoaffinity column is that an antibody which is designed aiming at the three-dimensional structure of aflatoxin and can be specifically combined with the immunoaffinity column is firstly used for preparing the immunoaffinity column, the aflatoxin in a sample can be combined with the antibody which specifically recognizes the aflatoxin in the immunoaffinity column, the buffer solution is used for eluting and removing impurities, and finally the purification method of the high-purity aflatoxin solution is obtained by destroying the three-dimensional structure of the antibody. Since the immunoaffinity column is designed for the three-dimensional structure of aflatoxin, when the legal test method is adopted, neither the enantiomers of aflatoxin B2 and G2 (AFB 2 'nor AFG 2') can be combined with the antibody of aflatoxin, and therefore, the aflatoxin is eluted as impurities, cannot enter the final test sample solution, and cannot be detected. This may also be why the aflatoxin B2 and G2 enantiomers (AFB 2 'and AFG 2') have not been found to date. Meanwhile, when the aflatoxin control sold in the market is detected by adopting the method, the enantiomer (AFB 2') containing the aflatoxin B2 is found, which indicates the universality of the existence of the aflatoxin enantiomer.
Disclosure of Invention
The invention aims to establish a separation method of aflatoxin B2, aflatoxin G2 and chiral isomers (AFB 2 'and AFG 2'), in particular to a separation method for separating and preparing aflatoxin B2 and aflatoxin G2 chiral isomers (AFB 2 'and AFG 2'), and the structural confirmation of the two compounds is carried out by technical means such as ultraviolet, mass spectrum, nuclear magnetism, circular dichroism and the like, and the structural identification result shows that the two novel compounds are respectively enantiomers of aflatoxin B2 and aflatoxin G2, and the enantiomers are respectively named as aflatoxin B2 enantiomers (AFB 2 ') and aflatoxin G2 enantiomers (AFG 2'). Cytotoxicity experiments are carried out on four aflatoxins of AFB2, AFB2', AFG2 and AFG2' obtained by preparation, and the results show that the two newly discovered compounds AFB2 'and AFG2' have cytogenotoxicity, and further analysis shows that the two compounds AFB2 'and AFG2' are highly likely to have carcinogenicity to mammals, hepatotoxicity to mammals, photosensitivity to mammals, in-vitro chromosome damage to mammals and nonspecific genotoxicity to mammals, and mutagenicity in mammals can also exist.
The aim of the invention can be achieved by the following scheme:
The invention provides enantiomers of aflatoxin B2 (AFB 2 '), G2 (AFG 2');
the enantiomer of aflatoxin B2 (AFB 2') has the formula:
the enantiomer G2 of aflatoxin G2 (AFG 2') has the formula:
the invention provides a separation method of aflatoxins B2 and G2 and enantiomers thereof, which comprises the following steps:
s1, sample extraction and purification:
Soaking and extracting a sample by using an extraction solvent, carrying out rotary evaporation and concentration on an extracting solution, carrying out liquid-liquid extraction on an obtained concentrated solution by using the extraction solvent, wherein an organic phase layer in the obtained extracting solution is a sample enriched with aflatoxin, and concentrating to obtain a purified aflatoxin sample;
s2, chiral separation of samples
Chiral separation is carried out by liquid chromatography or supercritical fluid chromatography, wherein the chromatographic column is polysaccharide bonded silica gel or polysaccharide coated silica gel chiral chromatographic column to obtain aflatoxins B2, G2 and enantiomers thereof.
As one embodiment of the present invention, in the step S1, the sample comprises one of a naturally contaminated sample obtained, a fermentation culture using aflatoxin-producing Aspergillus, and a chemical synthesis.
In step S1, as an embodiment of the present invention, the volume ratio of the sample to the extraction solvent is 1:3-10. The extraction solvent is an aqueous solution containing acetonitrile and/or methanol; in the aqueous solution, the volume ratio of methanol or/and acetonitrile is 70-90%, and the volume ratio of water is 10-30%. The extraction solvent also comprises acid, and the dosage is 0-2% of the volume of the aqueous solution; the acid can be selected from one or more of small molecular organic acids such as formic acid, acetic acid, oxalic acid, etc.
In step S1, the extraction time is 30-120min, and ultrasonic or vibration extraction is used as an embodiment of the present invention. The extraction times are 1-4 times; the extraction may be repeated 2-3 times according to the effect of the extraction, and then the extracts are combined.
In step S1, the temperature of the spin concentration is controlled to be 60 ℃ or lower, preferably 50-60 ℃, and the organic solvent is removed to obtain a concentrated solution with a solvent mainly comprising water.
In step S1, the extraction solvent includes one or more of dichloromethane, chloroform and ethyl acetate, and the extraction times are 1-4 times. And (3) liquid-liquid extraction is repeated for 2-3 times, an organic phase layer in the obtained extract is a sample enriched with aflatoxin, and the organic phase solutions extracted for multiple times are combined and concentrated to obtain the purified aflatoxin sample.
In step S2, the aflatoxin B2 and its enantiomer and/or aflatoxin G2 and its enantiomer in the sample are chiral separated by liquid chromatography or supercritical fluid chromatography. And (3) during chiral separation, obtaining a liquid chromatography or supercritical fluid chromatography map, sequentially obtaining compound samples according to the occurrence time of peaks in the map, and carrying out structural identification to obtain aflatoxins B2 and G2 and enantiomers thereof. The method for identifying the structure comprises one or more of ultraviolet detection, high-resolution mass spectrum detection, nuclear magnetism detection and circular dichroism detection.
During separation and purification, an ultraviolet or diode array detector is adopted for detection, the detection wavelength is 360nm, each component in the detection is collected, and then mass spectrum detection is carried out on each collected component, wherein the component with the molecular weight of 314.29 is AFB2 or AFB2', and the component with the molecular weight of 330.29 is AFG2 or AFG2'. Carrying out chiral analysis on the separated AFB2 or AFB2 'component by adopting a known AFB2 standard substance, wherein the retention time is AFB2 consistent with the standard substance, and the other component is AFB2'; and (3) carrying out chiral analysis on the separated AFG2 or AFG2 'component by adopting a known AFG2 standard substance, wherein the retention time is AFG2 consistent with the standard substance, and the other component is AFG2'.
In step S2, when the separation apparatus is a liquid chromatograph, a normal phase or reverse phase separation mode is used.
When a normal phase separation mode is adopted, the chromatographic column is a silica gel matrix chiral chromatographic column provided with polysaccharide coating or bonding; the mobile phase comprises a mobile phase A (comprising one or more of normal hexane and petroleum ether) and a mobile phase B (comprising one or more of ethanol, isopropanol, dichloromethane, ethyl acetate and chloroform), a binary or multi-component mobile phase system is formed for chromatographic separation, wherein the use range of each solvent can be regulated and controlled within the volume range of 5-95%, and the composition of the whole mobile phase is 100%.
When the reverse phase separation mode is employed, the chromatographic column is a silica gel matrix chiral chromatographic column equipped with polysaccharide linkages, and the mobile phase is a combination of water and an organic phase (including one or more of methanol, acetonitrile, isopropanol). Wherein the proportion of water can be adjusted within the range of 5-95%, and the proportion of the organic phase can be adjusted within the range of 5-95%.
The mobile phase of the normal phase/separation phase separation mode also comprises an additive, which is used for regulating and controlling the separation condition, wherein the additive comprises organic acid or alkali, and specifically comprises one or more of formic acid, acetic acid, ammonia water, diethylamine and triethylamine. The proportion of the additives in the mobile phase can be controlled in the range from 0 to 1.0% by mass.
As an embodiment of the present invention, in step S2, when the separation device is a supercritical fluid chromatograph, the chromatographic column is a silica gel matrix chiral chromatographic column equipped with polysaccharide coating or bonding, the mobile phase adopts a combination of (subcritical or supercritical) CO 2 and an organic phase (including one of methanol, ethanol, isopropanol, and acetonitrile), wherein the proportion of CO 2 can be regulated in the range of 50-99% by volume, the proportion of the organic phase can be regulated in the range of 1-50% by volume, and the composition of CO 2 and the organic phase is 100%,
The mobile phase also comprises an additive, wherein the additive comprises organic acid or alkali, and specifically comprises one or more of formic acid, acetic acid, ammonia water, diethylamine and triethylamine. The proportion of the additives in the mobile phase can be controlled in the range from 0 to 1.0% by mass.
As one embodiment of the invention, in the step S2, when the content of aflatoxin in the aflatoxin sample obtained in the step S1 is lower than 50% (refer to chromatographic conditions in the third method in GB 5009.22-2016 national food safety Standard-determination of aflatoxins B and G in food; the content determination is carried out by adopting a method of detecting by ultraviolet or diode array detector at 360nm and normalizing the area, the measured content is lower than 50%), and the aflatoxin sample also comprises a refining and separating step. The refined separation can remove impurities except for aflatoxin samples, and after obtaining corresponding aflatoxin-rich samples, concentration treatment is carried out, so that corresponding refined separation samples can be obtained, wherein the contents of AFB2 and AFB2 'and the contents of AFG2 and AFG2' can be respectively confirmed by LC-MS, the respective contents are determined by peak areas under 360nm, and whether the contents are AFB2 (AFB 2 ') and AFG2 (AFG 2') are respectively confirmed by molecular weights 314.29 and 330.29. If the purification and separation are not performed, the chiral separation effect is impaired because the amount of impurities is too large.
As one embodiment of the present invention, the method of refining and separating includes one of chromatography column, TLC thin layer plate, liquid chromatography, supercritical fluid chromatography. During refining and separation, according to AFB2 and AFG2 samples, components which are reserved for different time periods are collected. At the same time, molecular weights 314.29 and 330.29 were used to confirm whether AFB2 (AFB 2 ') and AFG2 (AFG 2'), respectively.
As one embodiment of the invention, when the refining separation method adopts a chromatographic column or a TLC thin-layer plate, the packing (separation medium) of the column chromatography comprises one or more of silica gel, diol, amino and cyano, and the eluent comprises one of petroleum ether-ethyl acetate, dichloromethane-methanol, dichloromethane-ethanol and dichloromethane-isopropanol eluent combination or other eluent combinations with equal eluting strength. Wherein, the volume ratio of petroleum ether in the petroleum ether-ethyl acetate stripping agent combination is 5-95%, the volume ratio of ethyl acetate is 5-95%, the volume ratio of dichloromethane in the dichloromethane-methanol, dichloromethane-ethanol and dichloromethane-isopropanol eluent combination is 5-95%, and the volume ratio of methanol, ethanol and isopropanol is 5-95%.
In one embodiment of the present invention, when liquid chromatography is used for the purification and separation, a normal phase or reverse phase separation mode may be used.
Wherein when the normal phase separation mode is adopted, the type (packing) of the chromatographic column comprises one or more of silica gel, diol, amino packing and cyano packing, the mobile phase (or eluent) comprises a mobile phase A (one or more of normal hexane and petroleum ether) and a mobile phase B (one or more of ethanol, isopropanol, dichloromethane, ethyl acetate and chloroform), a binary or multi-component mobile phase system is formed for chromatographic separation, wherein the use range of each solvent can be controlled within the volume range of 5-95%, and the composition of the whole mobile phase is 100%.
Wherein when the reverse phase separation mode is adopted, the type (filler) of the chromatographic column comprises one or more of silica gel bonded C8-C30, phenyl filler and cyano filler, and the mobile phase is a combination of water and organic phase (one or more of methanol, acetonitrile and isopropanol). Wherein the proportion of water can be adjusted within the range of 5-95% by volume and the proportion of the organic phase can be adjusted within the range of 5-95%.
The mobile phase of the normal phase/separation phase separation mode also comprises an additive, which is used for regulating and controlling the separation condition, wherein the additive comprises organic acid or alkali, and specifically comprises one or more of formic acid, acetic acid, ammonia water, diethylamine and triethylamine. The proportion of the additives in the mobile phase can be controlled in the range from 0 to 1.0% by mass.
As an embodiment of the present invention, when supercritical fluid chromatography is used for refining separation, the type of column (packing) includes one or more of silica gel, diol, amino packing, cyano packing, phenyl packing, C18-bonded silica gel. The mobile phase adopts a combination of CO 2 (subcritical state or supercritical state) and an organic phase (one of methanol, ethanol, isopropanol and acetonitrile), wherein the proportion of CO 2 can be regulated and controlled within the range of 50-99%, the proportion of the organic phase can be regulated and controlled within the range of 1-50%, and the composition of CO 2 and the organic phase is 100%.
The mobile phase also comprises an additive, wherein the additive comprises organic acid or alkali, and specifically comprises one or more of formic acid, acetic acid, ammonia water, diethylamine and triethylamine. The proportion of the additives in the mobile phase can be controlled in the range from 0 to 1.0% by mass.
The invention also provides a structural identification method of aflatoxins B2 and G2 (respectively abbreviated as AFB2 and AFG 2) and enantiomers thereof (respectively abbreviated as AFB2 'and AFG 2'), which comprises the following steps:
a1, AFB2 and AFB2' chiral isomer identification and structure confirmation
Carrying out analysis of ultraviolet, mass spectrum, nuclear magnetism and circular dichroism on single compounds of AFB2 and enantiomer (AFB 2 ') thereof obtained through chiral resolution, comparing analysis information of the two compounds, confirming absolute configurations of AFB2 and AFB2', and identifying that AFB2' is chiral isomer of AFB2 and the two are chiral enantiomer;
a2, AFG2 and AFG2' chiral isomer identification and structure confirmation
Carrying out analysis of ultraviolet, mass spectrum, nuclear magnetism and circular dichroism on single compounds of AFG2 and enantiomer (AFG 2 ') thereof obtained through chiral resolution, comparing analysis information of the two compounds, confirming absolute configurations of AFG2 and AFG2', and identifying that AFG2' is chiral isomer of AFG2 and the two are chiral enantiomer;
The samples AFB2 and AFB2' in the step A1 are pure products with single configuration or samples with single configuration accounting for main content, wherein the content of the samples is not less than 90% by mass; the ultraviolet detection method can be tested by adopting an ultraviolet spectrophotometer or by adopting other equipment to connect ultraviolet detectors in series (such as liquid chromatography to connect ultraviolet detectors in series); the analysis method of the mass spectrum can adopt the technologies of time-of-flight high-resolution mass spectrum, ion trap high-resolution mass spectrum, tandem quadrupole mass spectrum and the like for detection; the nuclear magnetic resonance detection method comprises the following steps: one or more detection methods such as nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum, two-dimensional spectrum and the like are combined; the round two chromatography is detected by a single round two chromatograph, and also can be detected by equipment combined with other equipment (such as a liquid chromatography-round two chromatograph combined instrument);
the samples AFG2 and AFG2' in the step A2 are pure products with single configuration or samples with single configuration accounting for main content, wherein the content of the samples is not less than 90% by mass; the ultraviolet detection method can be tested by adopting an ultraviolet spectrophotometer or by adopting other equipment to connect ultraviolet detectors in series (such as liquid chromatography to connect ultraviolet detectors in series); the analysis method of the mass spectrum can adopt the technologies of time-of-flight high-resolution mass spectrum, ion trap high-resolution mass spectrum, tandem quadrupole mass spectrum and the like for detection; the nuclear magnetic resonance detection method comprises the following steps: one or more detection methods such as nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum, two-dimensional spectrum and the like are combined; the round two chromatography is detected by a single round two chromatograph, and also can be detected by equipment combined with other equipment (such as a liquid chromatography-round two chromatograph combined instrument);
Ultraviolet detection and identification: the aflatoxin compound and the corresponding isomer sample are respectively analyzed and detected by adopting the following chromatographic conditions: c18 bonded silica gel chromatographic column, methanol-water mobile phase system, 200-400nm full scan detection, 360nm single wavelength detection, if the same chromatographic retention time and 2D characteristic absorption spectrum appear, the enantiomer is proved to exist in the sample.
The invention also provides application of enantiomers of aflatoxin B2 and aflatoxin G2 in pollution detection of foods, feeds and Chinese medicinal materials.
The invention relates to the work of purification preparation, structure confirmation, cytogenetic toxicity test and the like of AFB2, AFB2', AFG2 and AFG2' (wherein AFB2', AFG2' are two newly discovered aflatoxin compounds), provides corresponding reliable basis for controlling the pollution of aflatoxins in foods, feeds, chinese medicinal materials and the like, and provides reliable technical guarantee for guaranteeing the edible safety of the foods, feeds, chinese medicinal materials and the like.
Compared with the prior art, the invention has the following beneficial effects:
(1) A pretreatment method for extracting aflatoxin and enantiomer thereof in one step and a purification treatment method are established;
(2) A chiral separation method of aflatoxin B2, aflatoxin G2 and enantiomers thereof is established, and the enantiomers of aflatoxin B2 and the enantiomers of aflatoxin G2 are prepared by separation through the method;
(3) The absolute configurations of two compounds separated by the method are identified by qualitative technologies such as ultraviolet, high-resolution mass spectrum, nuclear magnetism, circular dichroism and the like, and the absolute configurations are taken as two new compounds discovered for the first time, so that the types of aflatoxin compounds are supplemented;
(4) Four aflatoxin compounds (including two newly discovered AFB2', AFG 2') were tested for predicted bacterial back mutations by using the Derek and Sarah combination of Lhasa, all of which showed genotoxicity;
(5) The separation method provides a basis for more accurately evaluating the pollution condition of samples such as food, feed, chinese herbal medicine decoction pieces and the like to aflatoxin, and provides a more accurate and reliable technical guarantee for carrying out separation and purification of aflatoxin B2, aflatoxin G2 and enantiomers thereof.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 is a schematic diagram of the structure of 6 aflatoxins;
FIG. 2 shows chiral separation patterns of aflatoxin B2 and its enantiomer (upper panel) and aflatoxin G2 enantiomer (lower panel);
FIG. 3 is an ultraviolet analysis chart of aflatoxins B2, G2 and chiral isomers thereof (upper AFB2, lower AFG 2);
FIG. 4 is an AFB2MS/MS secondary spectrum;
FIG. 5 is a MS/MS secondary spectrum of the AFB2 enantiomer;
FIG. 6 is an AFG2MS/MS secondary spectrum;
FIG. 7 is a MS/MS secondary spectrum of the AFG2 enantiomer;
FIG. 8 shows the assignment of 1H NMR data of AFB2 to NOESY;
FIG. 9 shows the assignment of 1H NMR data for the AFB2 enantiomer to NOESY;
FIG. 10 shows the assignment of 1H NMR data to AFG2 and NOESY correlation;
FIG. 11 shows the assignment of 1H NMR data for the AFG2 enantiomer to NOESY;
FIG. 12 is an AFB2 1H NMR spectrum;
FIG. 13 is an AFB2 enantiomer 1H NMR spectrum;
FIG. 14AFG2 1H NMR spectrum;
FIG. 15 is an AFG2 enantiomer 1H NMR spectrum;
FIG. 16 is a NOESY spectrum of AFB 2;
FIG. 17 is a NOESY spectrum of the AFB2 enantiomer;
FIG. 18 is an AFG2NOESY spectrum;
FIG. 19 is a NOESY spectrum of the AFG2 enantiomer;
FIG. 20 is a circular dichroism spectrum of AFB2 and AFB2 enantiomer;
FIG. 21 is a circular dichroism spectrum of AFG2 and AFG2 enantiomer;
FIG. 22 shows the predicted genotoxicity of AFB2 and AFB2 enantiomer.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples. The following examples, which are presented to provide those of ordinary skill in the art with a detailed description of the invention and to provide a further understanding of the invention, are presented in terms of implementation and operation. It should be noted that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made on the premise of the inventive concept are all within the protection scope of the present invention.
Example 1
The embodiment provides a separation method of aflatoxins B2 and G2 and enantiomers thereof, which comprises the following steps:
1. Sample extraction and purification:
Performing sterilization treatment on a sample of AFB2/AFB2 'and a sample of AFG2/AFG2' produced by fermenting and culturing aspergillus (strain is Aspergillus Flavus and the number is CGMCC 3.4408), soaking for 1 hour by using a mixed solvent of acetonitrile, water and formic acid (wherein the volume ratio of acetonitrile, water and formic acid is 80:20:1), performing ultrasonic extraction for 30 minutes, repeatedly extracting for 2 times, and combining 3 times of extracting solutions; concentrating the combined and extracted solution by rotary evaporation, wherein the temperature of rotary evaporation is controlled at 55 ℃ to obtain a concentrated solution mainly containing water; and then carrying out liquid-liquid extraction by using chloroform with the same volume as that of the aflatoxin concentrated solution, repeating for 2 times, and combining and concentrating the organic phase obtained by extraction to obtain the purified aflatoxin sample.
2. Sample refining separation
The content of aflatoxin in the extracted and purified sample is lower than 50 percent (refer to chromatographic conditions in a third method in GB 5009.22-2016 national food safety Standard-determination of aflatoxins B and G in food; the content determination is carried out by adopting a diode array detector, a 360nm detection and an area normalization method), and further refining and separation are carried out to remove impurities of non-aflatoxin samples.
The refining separation adopts a liquid chromatography reverse separation mode: the preparation conditions are as follows: chromatographic column: 20 x 250mm,5 μm, C18, flow rate: 18mL/min, detection wavelength: 360nm, mobile phase A: water, mobile phase B: acetonitrile;
gradient conditions (volume fraction) were as follows:
| Time (min) | 0 | 25 | 25.2 | 28 | 28.2 | 30 |
| A(％) | 70 | 40 | 5 | 5 | 70 | 70 |
| B(％) | 30 | 60 | 95 | 95 | 70 | 70 |
Under the preparation conditions, collecting samples with the components of AFG2/AFG2 'and the retention time of 17-20min, collecting samples with the components of AFB2/AFB2' and the retention time of 22-25min, and concentrating to obtain corresponding refined and separated samples.
Wherein the content of AFB2 and AFB2 'and AFG2' after refining can be determined by LC-MS, respectively, determining the content of each of the above components by peak area at 360nm, and determining whether AFB2 (AFB 2 ') and AFG2 (AFG 2') by molecular weights 314.29 and 330.29, respectively.
3. Chiral separation of samples
Carrying out isocratic elution on the sample rich in AFB2/AFB2 'obtained in the steps 1 and 2 by adopting a supercritical fluid chromatograph and a chiral chromatographic column equipped with polysaccharide bonded silica gel, taking 80% carbon dioxide (subcritical state or supercritical state) -20% ethanol in volume ratio as a mobile phase, adding 0.2% ammonia water in mass ratio into the mobile phase to obtain a map shown in figure 2, sequentially obtaining samples according to the appearance time of peaks in the map, namely, sample 1 and sample 2 (later verification, sample 1 is AFB2, sample 2 is AFB 2'), namely, separating a mixture of the AFB2/AFB2', and respectively obtaining single compounds of the AFB2 and the AFB 2';
carrying out isocratic elution on the sample rich in AFG2/AFG2' obtained in the steps 1 and 2 by adopting a supercritical fluid chromatograph and a chiral chromatographic column equipped with polysaccharide bonded silica gel, wherein the mobile phase is 80% carbon dioxide (subcritical state or supercritical state) -20% ethanol in volume ratio, and ammonia water with the mass ratio of 0.2% is added into the mobile phase to separate the mixture of AFG2/AFG2', so as to obtain single compounds of AFG2 and AFG2', respectively;
Carrying out chiral analysis on the separated AFB2 or AFB2 'component by adopting a known AFB2 standard substance, wherein the retention time is AFB2 consistent with the standard substance, and the other component is AFB2'; and (3) carrying out chiral analysis on the separated AFG2 or AFG2 'component by adopting a known AFG2 standard substance, wherein the retention time is AFG2 consistent with the standard substance, and the other component is AFG2'.
As shown in FIG. 2, the polysaccharide-bonded silica gel chromatographic column, the supercritical fluid chromatographic system and the CO 2 -ethanol-diethylamine system are adopted to separate the aflatoxin B2 and the enantiomer thereof, and the aflatoxin G2 and the enantiomer thereof.
Aflatoxin B2 enantiomer (AFB 2 ') Aflatoxin G2 enantiomer (AFG 2')
Example 2
The invention also provides a structural identification method of aflatoxin B2, aflatoxin G2 and enantiomers thereof, which comprises the following steps:
1. ultraviolet detection and identification
The aflatoxin compound with single configuration after chiral separation preparation is respectively analyzed and detected by adopting the following chromatographic conditions (C18 bonded silica gel chromatographic column, methanol-water mobile phase system, 200-400nm full-scan detection and 360nm single-wavelength detection). As shown in fig. 3, the results show that aflatoxin B2 and its chiral isomer (upper graph) have the same chromatographic retention time and 2D characteristic absorption spectrum, indicating that the pair of compounds have the same absorption characteristics and very similar or identical structural characteristics; aflatoxin G2 and its chiral isomers (lower panel) have the same chromatographic retention time and 2D characteristic absorption profile, indicating that the pair of compounds have the same characteristic absorption and very similar or identical structural features.
2. High resolution mass spectrometry detection
The high performance liquid chromatography tandem mass spectrometry is adopted to respectively analyze the aflatoxin B2, the aflatoxin B2 chiral isomer, the aflatoxin G2 and the aflatoxin G2 chiral isomer which are prepared by the chiral separation method, and the results are shown in figures 4-7. AFB2 and AFB2 enantiomer have the same MS/MS secondary spectrogram, which shows that the two have the same chemical structure; AFG2 and AFG2 enantiomer have the same MS/MS secondary spectrum, indicating that both have the same chemical structure.
3. Nuclear magnetic detection
The AFB2 and AFB2 enantiomer, AFG2 and AFG2 enantiomer were separated by the above method and subjected to 1H NMR and NOESY tests, and the nuclear magnetic data assignment of the four compounds was shown in fig. 8-11, respectively, wherein the assignment of two pairs of enantiomer 1H NMR data (fig. 12-15) and NOESY (fig. 16-19) correlated completely, indicating that they are highly likely to be enantiomers.
4. Round two chromatographic detection
AFB2 and AFB2 enantiomer, AFG2 and AFG2 enantiomer were subjected to round dichroism chromatography respectively, and the results are shown in fig. 20-21. The results show that the Cotton effect in AFB2 and AFB2 enantiomers are completely opposite (the symmetry of the two curves is good, which indicates that the two compounds are enantiomers with each other), indicating that the two compounds are enantiomers with each other; the Cotton effect in AFG2 and AFG2 enantiomers are diametrically opposed, indicating that they are enantiomers with respect to each other. The configuration of both enantiomers was determined by circular dichroism.
The analysis and identification of the aflatoxin B2 and the chiral isomer thereof, the aflatoxin G2 and the chiral isomer thereof are carried out by detection means such as ultraviolet, mass spectrum, nuclear magnetism and circular dichroism, and the like, and the compounds separated by the separation method are two novel compounds and are enantiomers with the aflatoxin B2 and the aflatoxin G2 respectively.
Example 3
The invention also provides a computer toxicity prediction method of aflatoxin B2, aflatoxin G2 and enantiomers thereof, which comprises the following steps:
The bacterial back mutation test was predicted for four compounds using the Lhasa company Derek and Sarah combination, and the results of the AFB2 enantiomer showed genotoxicity in both Derek and Sarah predictions; the AFG2 enantiomer showed genotoxicity in the predicted result of Derek (fig. 22).
More than 60 toxicology endpoints of the compound were predicted using Lhasa company Derek software, and as a result, both the AFB2 enantiomer and the AFG2 enantiomer showed a high probability of carcinogenicity to mammals, hepatotoxicity to mammals, photosensitivity to mammals, damage to chromosomes in vitro, nonspecific genotoxicity to mammals, and the possibility of mutagenicity in vivo in mammals.
The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.
Claims (10)
1. An enantiomer of aflatoxin B2, G2, characterized in that,
The enantiomer of aflatoxin B2 has the structural formula:
the enantiomer of aflatoxin G2 has the structural formula:
2. A process for the separation of enantiomers of aflatoxins B2, G2 as claimed in claim 1, comprising the steps of:
s1, sample extraction and purification:
after the sample is soaked and extracted, the extracting solution is subjected to rotary evaporation concentration and liquid-liquid extraction, an organic phase layer in the obtained extracting solution is a sample enriched with aflatoxin, and the purified aflatoxin sample is obtained after the organic phase layer is concentrated;
s2, chiral separation of samples
Chiral separation is carried out by liquid chromatography or supercritical fluid chromatography, wherein the chromatographic column is a chiral chromatographic column of polysaccharide bonded silica gel or polysaccharide coated silica gel.
3. The method according to claim 2, wherein in step S1, the sample comprises one of a naturally contaminated sample obtained, a fermentation culture of aspergillus using aflatoxin-producing aspergillus, and a chemical synthesis.
4. The separation method according to claim 2, wherein in step S1, the extraction solvent is an aqueous solution containing acetonitrile and/or methanol; in the aqueous solution, the volume ratio of methanol or/and acetonitrile is 70-90%, and the volume ratio of water is 10-30%.
5. The separation method according to claim 2, wherein in step S1, the extraction solvent comprises one or more of dichloromethane, chloroform and ethyl acetate, and the number of extraction is 1 to 4.
6. The separation method according to claim 2, wherein in step S2, when liquid chromatography is used, a normal phase or reverse phase separation mode is used;
When a normal phase separation mode is adopted, the chromatographic column is a silica gel matrix chiral chromatographic column provided with polysaccharide coating or bonding; the adopted mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A comprises one or more of normal hexane and petroleum ether, and the mobile phase B comprises one or more of ethanol, isopropanol, dichloromethane, ethyl acetate and chloroform;
When the reverse phase separation mode is adopted, the chromatographic column is a chiral chromatographic column with a polysaccharide-bonded silica gel matrix, the adopted mobile phase comprises water and an organic phase, and the organic phase comprises one or more of methanol, acetonitrile and isopropanol.
7. The separation method according to claim 2, wherein in step S2, when supercritical fluid chromatography is used, the chromatographic column is a silica gel matrix chiral chromatographic column equipped with polysaccharide coating or bonding, the mobile phase comprises CO 2 in subcritical or supercritical state and an organic phase, and the organic phase comprises one or more of methanol, ethanol, isopropanol, and acetonitrile.
8. The separation method according to claim 2, wherein in step S2, when the content of aflatoxin in the aflatoxin sample obtained in step S1 is lower than 50%, the aflatoxin sample further comprises a refining separation step; the refining separation method comprises one of chromatographic column, TLC thin layer plate, liquid chromatography, and supercritical fluid chromatography.
9. A method for identifying the structure of aflatoxins B2 and G2 and enantiomers thereof, which is characterized by comprising the following steps: the single compounds of aflatoxin B2, G2 and enantiomers thereof obtained by chiral resolution according to the separation method of claim 2 are respectively subjected to ultraviolet, mass spectrum, nuclear magnetism and circular dichroism analysis, and the absolute configuration of the aflatoxins B2, G2 and enantiomers thereof is confirmed by respectively comparing analysis information of the two compounds.
10. Use of enantiomers of aflatoxin B2, G2 according to claim 1 for pollution detection of food, feed, chinese herbal medicine.
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