CN111474283A - Method and kit for qualitative/quantitative detection of target compound - Google Patents
Method and kit for qualitative/quantitative detection of target compound Download PDFInfo
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- CN111474283A CN111474283A CN202010446001.4A CN202010446001A CN111474283A CN 111474283 A CN111474283 A CN 111474283A CN 202010446001 A CN202010446001 A CN 202010446001A CN 111474283 A CN111474283 A CN 111474283A
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Abstract
The invention discloses a method and a kit for qualitatively/quantitatively detecting a target compound, wherein the method for qualitatively/quantitatively detecting the target compound comprises the following steps: providing a solution to be detected; contacting an extraction plate with the solution to be detected, and enriching the target compound in the extraction plate so as to obtain an enriched extraction plate; and performing qualitative/quantitative analysis and detection on the enriched extraction plate, wherein the extraction plate comprises: a substrate; and an adsorption layer which is coated on at least part of the surface of the substrate, is formed by crosslinking polymer, is porous and has a compound embedding space to be extracted. The method has the advantages that the extraction plate is utilized to have specific adsorption and enrichment effects on the compound to be extracted and the structural analogue thereof, the specificity of extraction and enrichment is strong, the detection steps are simple, and the detected background noise is low and the specificity is high.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for qualitatively/quantitatively detecting a target compound and a kit for qualitatively/quantitatively detecting the target compound.
Background
The concept of Ambient Mass Spectrometry (AMS) was first mentioned by the professor Cooks in 2004 when proposing desorption electrospray ionization (DESI) techniques, and mass spectrometry could be performed directly in atmospheric pressure environments. Since then, a series of mass spectrometric techniques such as direct Real Time analysis (DART) mass spectrometry, extractive electrospray (electrospray) mass spectrometry, and desorption corona ionization (DCBI) mass spectrometry have been reported in succession. The AMS technology has the advantages of simple operation, short analysis time, no or few sample pretreatment processes, small dosage and the like, and is widely applied to many fields of medicine analysis, biological analysis, food, environment, forensic identification and the like at present.
As one of the open Mass spectrometry techniques, Paper Spray Mass spectrometry (PS-MS) was first proposed in 2010, which has good potential for analysis of small molecule drugs in dry biological fluids without pre-processing the sample. Since then, various electrospray mass spectrometry technologies using a solid substrate as a carrier have been developed on the basis of a paper spray ion source, which may be referred to as solid substrate-electrospray mass spectrometry, and these solid substrates include, but are not limited to, toothpicks, bamboo tips, porous membranes, glass rods, tungsten filaments, etc., a sample is loaded on the surface of these solid substrates, a desorption solvent is added, and under the action of high voltage electricity, a spray is generated at the tip of the solid substrate, which directly enters the mass spectrometry for analysis, the analysis time is short, and the detection is rapid. However, when the solid substrate-electrospray mass spectrometry technology is used to analyze a target analyte in a complex sample, the method has the disadvantages of higher detection limit, low selectivity and the like while ensuring a faster analysis speed.
Thus, solid substrate-electrospray mass spectrometry techniques are in need of improvement.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a method for qualitative/quantitative detection of a target compound, which has low detection background noise, high specificity and simple detection process.
Thus, according to one aspect of the invention, there is provided a method for the qualitative/quantitative detection of a compound of interest. According to an embodiment of the invention, the method comprises: providing a solution to be detected; contacting an extraction plate with the solution to be detected, and enriching the target compound in the extraction plate so as to obtain an enriched extraction plate; and performing qualitative/quantitative analysis and detection on the enriched extraction plate, wherein the extraction plate comprises: a substrate; and an adsorption layer which is coated on at least part of the surface of the substrate, is formed by crosslinking polymer, is porous and has a compound embedding space to be extracted.
According to the detection method provided by the embodiment of the invention, the extraction plate is utilized to have specific adsorption and enrichment effects on the compound to be extracted and the structural analogue thereof, the specificity of extraction and enrichment is strong, the extraction step is simple, the detection sensitivity can be effectively improved, and the background interference is reduced.
In addition, the method for performing qualitative/quantitative detection on a target compound according to the above embodiment of the present invention may further have the following additional technical features:
according to an embodiment of the present invention, the method of preparing the extraction plate comprises: acidizing the substrate to obtain an acidized substrate; subjecting the acidified substrate to silanization treatment to silanize at least part of the surface of the acidified substrate so as to obtain a silanized substrate; performing double bond modification treatment on at least part of the surface of the silanized substrate so as to obtain a modified substrate; dissolving a compound to be extracted, a monomer compound and a cross-linking agent in a pore-foaming agent so as to obtain a pre-polymerization liquid; contacting at least part of the surface of the modified substrate with a prepolymerization solution to carry out a crosslinking reaction so as to obtain a crosslinked polymer embedded with a compound to be extracted; and performing elution treatment on the cross-linked polymer in which the compound to be extracted is embedded, and removing the compound to be extracted embedded on the cross-linked polymer, so as to obtain the compound extraction plate.
According to an embodiment of the present invention, the monomer compound is methacrylic acid and the cross-linking agent is ethylene glycol dimethacrylate.
According to an embodiment of the invention, the crosslinking reaction further comprises: an initiator which is Azobisisobutyronitrile (AIBN); the pore-foaming agent is a chloroform-methanol mixed solution, and preferably, the volume ratio of chloroform to methanol of the pore-foaming agent is 4-6: 1.
According to an embodiment of the present invention, the contacting is immersing at least a part of the extraction plate in the solution to be tested, and the contacting is performed for 10-60min under the condition of shaking at 180rpm, preferably: it is 30 min.
According to an embodiment of the present invention, the volume of the solution to be tested is 6-10ml, preferably 8 ml.
According to an embodiment of the invention, the analytical detection is an open electrospray mass spectrometry detection.
According to an embodiment of the invention, the extraction plate has at least one angular end, preferably said extraction plate is in the shape of an isosceles triangle.
According to an embodiment of the invention, the tip of the extraction plate is 3-8mm, preferably 5mm, from the mass spectrometry inlet.
According to an embodiment of the invention, the spray ionization conditions of the open electrospray mass spectrometry are a spray voltage of 2.5-5Kv, preferably 3.5Kv, and a spray desorption solvent of (0-1)% acetic acid in methanol, the volume of the desorption solvent being 10-30 μ L, preferably 20 μ L.
According to the embodiment of the invention, the detection conditions of the open electrospray mass spectrometry are as follows: the detection mode is as follows: multiple Reaction Monitoring (MRM); atomizing gas pressure: 55 psi; auxiliary gas pressure: 50 psi; air curtain pressure: 20 psi; ion source temperature: 550 ℃; residence time: 100 ms.
According to an embodiment of the present invention, the target compound is a quinolone compound.
According to another aspect of the invention, the invention provides a kit for qualitative/quantitative detection of a target compound, which is characterized by comprising an extraction plate prepared by the method, wherein the extraction plate comprises a substrate and an adsorption layer coated on at least part of the surface of the substrate, the adsorption layer is formed by crosslinked polymers, is porous and has a compound to be extracted embedding space, and a desorption solvent is a methanol solution of 20 mu L (0-1)% acetic acid.
According to an embodiment of the present invention, the target compound is a quinolone compound.
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 flow diagram of a method for qualitative/quantitative detection of a compound of interest according to one embodiment of the present invention;
FIG. 2 shows a schematic diagram of an apparatus for open electrospray mass spectrometry loaded with an extraction plate according to an embodiment of the present invention;
FIG. 3 shows a schematic diagram of the results of different extraction solvent types on the extraction of 5 quinolone antibiotics according to one embodiment of the present invention;
FIG. 4 shows a graphical representation of the results of different formic acid content in extraction solvents on the effect of 5 quinolone antibiotics according to one embodiment of the present invention;
FIG. 5 shows a graphical representation of the results of the effect of extraction time on 5 quinolone antibiotics according to one embodiment of the present invention;
FIG. 6 is a graph showing the results of the effect of loading on extraction of 5 quinolone antibiotics according to one embodiment of the present invention;
FIG. 7 shows a graphical representation of the results of different spray voltages on 5 quinolone antibiotics according to one embodiment of the present invention;
figure 8 shows a graphical representation of the results of different spray ionized solvents' effect on 5 quinolone antibiotics according to 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.
In the description of the present invention, the terms "longitudinal", "lateral", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, which are for convenience of description of the present invention only and do not require that the present invention must be constructed and operated in a specific orientation, and thus, should not 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, there is provided a method for the qualitative/quantitative detection of a compound of interest. According to the detection method provided by the embodiment of the invention, the extraction plate is utilized to have specific adsorption and enrichment effects on the compound to be extracted and the structural analogue thereof, the specificity of extraction and enrichment is strong, the detection steps are simplified, and the detected background noise is low and the specificity is high.
It is noted that the method for qualitative/quantitative detection of target compounds according to the embodiments of the present invention has strong detection specificity and simple method, and is particularly suitable for detection of complex substrate samples, such as specific compounds in food, and quinolone antibiotics (FQs) are widely used in livestock and poultry breeding industry, which may cause residual quinolone antibiotics in animal-derived food, and food with overproof antibiotic residues for human being, which may cause imbalance of in vivo flora, generation of drug-resistant strains, toxicity of central nervous system, and even cause allergy. Therefore, the rapid detection of the trace FQs residue in the food is of great significance to guarantee the food safety and assist the food safety supervision. Therefore, we used the above detection method for detecting trace amounts of quinolone antibiotics in food samples.
Referring to fig. 1, the method for qualitatively/quantitatively detecting a target compound according to an embodiment of the present invention is explained, which includes:
s100 sample processing
According to an embodiment of the present invention, a test solution is provided. The sample solution can be obtained by pretreatment such as extraction of a sample.
The method of impurity removal and extraction will vary from sample to sample and will be readily selected by the skilled person based on his own experience, and as a rule, a solution of formic acid in acetonitrile will be used to remove proteins and then dry the deproteinised sample, for example, milk, where acetonitrile is used to remove proteins, low temperature centrifugation, supernatant collection and nitrogen blow drying, and an extraction solvent taken as the test solution after reconstitution according to some embodiments of the invention, the volume of the milk sample is 3-20m L, preferably 8m L, the extraction solvent being an acetonitrile solution containing formic acid (0, 0.05%, 0.1%, 0.5%, 1%) in various proportions, preferably 0.05% formic acid.
According to some embodiments of the present invention, when the solvent of the solution to be tested is methanol or acetone, the adsorption amount is small, and acetonitrile is used as the sample solvent, the adsorption amount is largest, and preferably, the sample solvent to be tested is acetonitrile.
According to the embodiment of the invention, the volume of the solution to be detected is 6-10ml, the adsorption amount of the extraction plate is large, preferably, the volume of the solution to be detected is 8ml, and the adsorption amount of the extraction plate is better.
S200 enrichment extraction
According to the embodiment of the invention, the extraction plate is contacted with the solution to be detected, so that the extraction plate is enriched with the target compound, and the enriched extraction plate is obtained. Further, according to an embodiment of the present invention, the extraction plate includes: the adsorption layer is formed by crosslinking polymers, is porous and has a compound embedding space to be extracted.
According to an embodiment of the present invention, the method of preparing the extraction plate comprises: acidizing the substrate to obtain an acidized substrate; silanizing the acidified substrate to silanize at least part of the surface of the acidified substrate to obtain a silanized substrate; performing double bond modification treatment on at least part of the surface of the silanized substrate to obtain a modified substrate; dissolving a compound to be extracted, a monomer compound and a cross-linking agent in a pore-foaming agent so as to obtain a pre-polymerization liquid; contacting at least part of the surface of the modified substrate with a prepolymerization solution to carry out a crosslinking reaction so as to obtain a crosslinked polymer embedded with a compound to be extracted; and (3) eluting the cross-linked polymer embedded with the compound to be extracted, and removing the compound to be extracted embedded on the cross-linked polymer to obtain the compound extraction plate.
Specifically, according to the embodiment of the invention, the method for preparing the compound extraction plate can comprise the steps of immersing the substrate in a 1.5-2.5 mol/L sulfuric acid solution, carrying out ultrasonic treatment for 0.5-1.5 hours, washing the surface of the substrate with water to be neutral, washing with acetone, and drying with nitrogen for later use so as to obtain an acidified substrate, immersing the acidified substrate in an alkaline solution containing tetraethyl orthosilicate for shaking reaction at room temperature so as to silanize at least part of the surface of the acidified substrate, washing with ultrapure water and ethanol, and drying with nitrogen so as to obtain the silanized substrate, immersing the silanized substrate in a methanol solution containing 3- (methacryloxy) propyl trimethoxy silane for shaking reaction at room temperature so as to modify double bonds on the surface of the substrate, repeatedly washing with ethanol, drying with nitrogen so as to obtain the modified substrate, dissolving the compound to be extracted in a pore-forming agent, adding a monomer compound, carrying out reaction at room temperature for 3-6 hours, adding a cross-linking agent and an initiator for 5 minutes so as to obtain a pre-modified solution, immersing the substrate in a pre-polymerization solution, carrying out a pre-polymerization treatment at a temperature of 60 ℃, and carrying out a pre-polymerization treatment under a pre-polymerization condition of 60 ℃, and carrying out elution by using nitrogen so as to obtain the pre-polymer extraction solution, and carrying out a pre-polymerization treatment under a pre-polymerization condition of the pre-polymerization condition.
According to an embodiment of the invention, the substrate is a stainless steel plate or a bamboo plate. Thus, the coating of the crosslinked polymer is easy and hardly deformed. The inventors have found that when using an inert material as the solid substrate, a fixation and conduction action by a copper clamp is usually required to apply high voltage to the solid substrate, which increases the risk of the solid substrate moving to some extent and affects the parallelism of the experiment. Moreover, through previous experiments, the porous membrane is found to have high background interference when being used as a solid phase substrate, and the stainless steel sheet shows low background noise. In addition, because the stainless steel sheet is conductive, high voltage can be directly applied to the stainless steel sheet, so that the copper clamp can be omitted in the experiment, and the experiment operation can be further simplified. Furthermore, in accordance with a preferred embodiment of the present invention, the substrate is a stainless steel sheet.
According to an embodiment of the invention, the monomer compound is methacrylic acid and the cross-linking agent is ethylene glycol dimethacrylate.
According to an embodiment of the invention, the crosslinking reaction further comprises: the initiator is Azodiisobutyronitrile (AIBN), the catalytic crosslinking reaction is carried out, the pore-forming agent is a chloroform-methanol mixed solution, the pore-forming agent can effectively dissolve template molecules, so that the template molecules and functional monomers are stably combined, the function of the pore-forming agent can be fully exerted, and the crosslinked object is promoted to form a three-dimensional cavity which is matched with the size and the shape of the molecules and contains the compound to be detected. According to a preferred embodiment of the invention, the porogen has a volume ratio of chloroform to methanol of 4-6: 1.
According to the embodiment of the invention, the modified substrate is immersed in the cross-linked polymer, nitrogen is filled, prepolymerization is carried out at 55-65 ℃, and then the substrate is taken out and polymerized at 60-70 ℃. Thus, immersion polymerization was repeated. According to the embodiment of the present invention, the stainless steel sheet is taken out after pre-polymerizing at 60-70 ℃ for 1.5-2.5 hours, polymerized at 60-70 ℃ for 10-15 hours, and repeatedly immersion polymerized 3-10 times, preferably 7 times.
According to the embodiment of the invention, the elution treatment is carried out by using an eluent, specifically, the eluent is a methanol-acetic acid mixed solution, and preferably, the eluent is a methanol solution containing 8-12% of acetic acid.
According to the embodiment of the invention, the elution treatment can be carried out by soaking the coated substrate with an eluent for 30min, repeatedly eluting for 3-8 times, preferably 6 times, washing with ultrapure water to neutrality after elution, washing with acetone and volatilizing to be used. Thereby, sufficient removal of the compound to be extracted from the crosslinked polymer is facilitated.
According to an embodiment of the invention, the molar ratio of the compound to be extracted to the monomer compound and the cross-linking agent is from 1: 4 to 8: 25 to 35, preferably the molar ratio is 1: 6: 30. Therefore, the proportion of the monomer compound grafted to the cross-linking agent is appropriate, and under the proportion, the combination of the cross-linking agent and the initiator can effectively initiate polymerization reaction, so that the formed molecularly imprinted polymer has a good imprinting effect.
The inventor researches and discovers that after the extraction plate is contacted with the solution to be tested, the binding rate of the target compound and the extraction plate is obviously increased within 5-30min, and the binding rate is close to equilibrium within 30-60 min. Furthermore, according to the embodiment of the present invention, the inventor synthesizes the flow of experiment, the contact is to immerse at least part of the extraction plate in the solution to be tested, and the contact is carried out for 10-60min under the condition of shaking at 180rpm, preferably: it is 30 min. Therefore, the compound is favorably and fully combined with the extraction plate, and the detection accuracy and the recovery rate are higher.
S300 analytical detection
According to an embodiment of the invention, qualitative/quantitative analytical detection is performed on the enriched extraction plates.
According to an embodiment of the invention, the analytical detection is an open electrospray mass spectrometry detection. In the open electrospray mass spectrometry detection, the extraction plate has at least one angular end, and preferably, the substrate is in an isosceles triangle shape. The inventors have found that the extraction plate has an angled tip where electrospray can form when a high voltage is applied and then enter the mass spectrometer for analysis. When the base plate is isosceles triangle, the three-dimensional moving platform of being convenient for carries out distance control, conveniently adjusts the distance of base plate point and mass spectrum introduction port. The tip of the extraction plate is 3-8mm, preferably 5mm, from the mass spectrometer inlet. The inventors have found that when the spray voltage applied to the extraction plate is fixed, the distance from the tip of the extraction plate to the mass spectrometer inlet has a significant effect on the response of the mass spectrometer, and that too far or too close a distance can reduce the mass spectrometer response signal.
According to the present invention, the electrospray ionization conditions of the electrospray mass spectrometry are such that the spray voltage is 2.5-5Kv, preferably 3.5Kv, and the inventors have studied that the signal intensity of the quinolone compound increases with increasing voltage when the high voltage power supply voltage is 0-3.5Kv or 4Kv, and the signal intensity of 5 analytes to be detected decreases with increasing voltage when the voltage is 4-6Kv, preferably the high voltage power supply voltage is selected to be 3.5 Kv. spray desorption solvent, methanol solution containing (0-1)% acetic acid, the volume of the desorption solvent being 10-30 μ L, preferably 20 μ L, and the inventors have found that when the spray solvent is methanol solvent containing different proportions (0-1%) of acetic acid, the amount of the desorption solvent adsorbed to the extraction plate does not increase with the addition of acetic acid, and further preferably the desorption solvent is pure methanol, it may be due to the methanol's suitable ability to make it block the extraction plate from hydrogen bonding with the extraction plate, thereby making it more effective for the extraction of the compounds to be eluted from the extraction plate.
According to the embodiment of the invention, the detection conditions of the electrospray ionization mass spectrum are as follows: the detection mode is as follows: multiple Reaction Monitoring (MRM); atomizing gas pressure: 55 psi; auxiliary gas pressure: 50 psi; air curtain pressure: 20 psi; ion source temperature: 550 ℃; residence time: 100 ms. Therefore, the target object can be detected more stably, and the detection accuracy and sensitivity are high.
According to another aspect of the present invention, there is provided a kit for qualitative/quantitative detection of a target compound, according to an embodiment of the present invention, the kit comprises an extraction plate and a desorption solvent, wherein the extraction plate is prepared by the method described above, the extraction plate comprises a substrate and an adsorption layer covering at least a part of a surface of the substrate, the adsorption layer is formed of a cross-linked polymer, is porous, and has a space in which a compound to be extracted is embedded, and the desorption solvent is a methanol solution of 20 μ L (0-1)% acetic acid.
The kit for qualitative/quantitative detection of the target compound provided by the embodiment of the invention has strong detection specificity and simple detection process, is particularly suitable for detection of complex matrix samples, such as detection of specific compounds in food, and the carbostyril antibiotic (FQs) is widely used in livestock and poultry breeding industry, so that part of carbostyril antibiotic remained in animal-derived food and food with overproof antibiotic residue eaten by human can cause in vivo flora imbalance, generation of drug-resistant strains, toxicity of the central nervous system and even more serious allergy. Therefore, the rapid detection of the trace FQs residue in the food is of great significance to guarantee the food safety and assist the food safety supervision. Therefore, we used the above detection method for detecting trace amounts of quinolone antibiotics in food samples.
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.
The materials and reagents used in the examples of the invention are shown in Table 1.
TABLE 1
Example 1
By utilizing the method provided by the embodiment of the invention, the enrofloxacin is taken as a compound to be extracted, and the extraction plate is prepared, which comprises the following specific steps:
(a) the stainless steel sheet is cut into isosceles triangles with the bottom of 1cm and the waist of 2 cm.
(b) The stainless steel sheet was subjected to ultrasonic treatment with 2 mol/L sulfuric acid for 1 hour, then repeatedly washed with ultrapure water to neutrality, rinsed with acetone, and then rapidly dried by nitrogen blow.
(c) And putting the treated stainless steel sheet into a conical flask containing 4m L ultrapure water and 50m L ethanol, dropwise adding 5m L concentrated ammonia water and 2m L tetraethyl orthosilicate (TEOS), putting the mixture on a shaker, reacting at 180rpm at room temperature for 12 hours, washing the mixture by using the ultrapure water and the ethanol in sequence, and drying the mixture by using nitrogen to obtain the stainless steel sheet (SSS @ SiO2) with the surface modified with silicon-oxygen bonds.
(d) SSS @ SiO2Immersing in 60m L methanol solution, dropwise adding 2m L3- (methacryloxy) propyl trimethoxy silane (MPS), reacting at 180rpm for 24 hours at room temperature, repeatedly washing with ethanol for 3-5 times, and drying with nitrogen gas to obtain SSS @ SiO with surface modified double bonds2@MPS。
(e) Dissolving 1mmol enrofloxacin (template molecule) and 6mmol methacrylic acid (MAA) (functional monomer) in 40m L chloroform: methanol (5: 1v/v) mixed solution (pore-foaming agent), prepolymerizing at room temperature for 4h, adding 30mmol Ethylene Glycol Dimethacrylate (EGDMA) and 2mmol Azobisisobutyronitrile (AIBN) into the solution, completely dissolving, adding the treated SSS @ SiO2@ MPS, ultrasonic removalOxygen is filled for 10min, nitrogen is filled for 5min, prepolymerization is carried out for 2 hours in an oven at 60 ℃, SSS @ SiO is taken out2@ MPS, it was observed that a thin MIP layer formed on the surface, polymerizing for 12h at 65 ℃. Then, the polymerized SSS @ SiO2@ MPS @ MIP was repeatedly immersed in the freshly prepared pre-polymerization solution and polymerization was repeated 7 times.
(f) Repeatedly soaking and eluting with methanol and acetic acid (9: 1v/v) to remove template molecules. After elution, the column was washed with pure water to neutrality, then with methanol, and finally dried with nitrogen flow to obtain the extraction plate.
Example 2
In this example, mass spectrometric detection parameter optimization of quinolone compounds in milk samples was performed using the extraction plate of example 1, as follows:
1. experimental methods
(1) Adding commercially available milk 8m L into 50m L polypropylene centrifuge tube, adding 16m L acetonitrile and 3g sodium chloride, vortex and shaking for 5min, centrifuging at 4 deg.C at 8000r/min for 10min, collecting supernatant, blow-drying with nitrogen, dissolving 8m L acetonitrile solution containing 0.05% formic acid, and using as test solution
(2) The carbostyril compound is enriched to obtain an enriched substance, and the specific operation is as follows: and (3) adding the test solution into an extraction plate, extracting at 150rpm for 30min, taking out, quickly cleaning the surface by using acetonitrile, and removing non-specific co-extract.
(3) The method comprises the steps of placing the extraction plate on a three-dimensional moving platform set up in a laboratory, adjusting the tip of the extraction plate to be in the same straight line with a mass spectrum inlet, adjusting the tip of the extraction plate to be located 5mm away from the mass spectrum sample inlet, then dropwise adding 20u L methanol on the surface of the extraction plate, standing for 10s, applying a high voltage of +3.5Kv to the tail of the extraction plate, enabling a solution to be detected to move to the tip of the extraction plate, generating analyte ions at the tip and forming spray, and enabling the analyte ions to enter mass spectrum analysis, wherein an analysis schematic diagram and a food diagram are shown in FIGS. 3-4.
2. Results of the experiment
(1) The MRM mass spectrum parameters of the 5 optimized quinolone analytes are shown in table 2, and the peak areas of the quantitative ions are used as the measurement indexes.
Table 2:
note: is a quantitative ion
Example 3
In this example, the enrichment efficiency OF 5 quinolone antibiotic extraction plates in milk detected by an open electrospray mass spectrometry is verified, the enrichment efficiency OF the synthesized extraction plate on a molecule to be detected is considered, 5 quinolone antibiotics in a simulated biological sample are analyzed as an example, 5 quinolone antibiotic mother solutions are added into a blank milk substrate to prepare a 100ng/m L concentration, and the milk sample is detected by using the method OF example 2.
Example 4
In this example, taking 5 quinolone antibiotics in a simulated biological sample as an example, 5 quinolone antibiotic standard mother solutions to be detected of 100ng/m L are added into a blank milk substrate, and the mass spectrometry detection conditions of example 2 are used for parallel analysis three times to study the influence of extraction condition parameters such as sample solvent, extraction time, sample volume and the like on the detection result.
1. Sample solvent study
The type of sample solvent will affect the nature of the analyte groups and thus the force with which the analyte binds to the extraction plate. Thus, a suitable sample solvent has a large influence on the extraction capacity of the extraction plate.
Considering that 5 kinds OF quinolone antibiotics (ENR, OF L, CIP, NOR, PEF) each have two ionizable groups, one carboxyl group (pKa 12.4-6.81) and one heterocyclic group (pKa 28.04-8.8), since each compound has a specific pKa value, different pH values affect the compounds to assume different molecular ionic states, the effect OF the pH value OF the aqueous solution on the results was first studied, and when the pH value was 4, a more excellent compound signal was obtained.
The effect of different formic acid contents (0.05%, 0.1%, 0.5%, 1%) in the acetonitrile system on the extraction results was examined using the same conditions and the results are shown in fig. 4, which shows that higher signal intensity can be obtained when acetonitrile contains 0.05% formic acid as the sample solvent.
2. Study of adsorption time
The extraction time is an important factor in the extraction process, and in order to obtain better extraction time, the influence of different extraction times of 0, 10, 20, 30, 40, 50 and 60min on the extraction amount is studied, and the extraction amount is detected in the detection steps which are performed in parallel for three times. As shown in FIG. 5, the signal intensity of 5FQs increased with the increase of extraction time within 0-30 min; then, when the extraction time exceeded 30 minutes, the signal intensity of the analyte remained almost unchanged without significant change, mainly due to the dynamic equilibrium between the analyte in the sample solution and the analyte adsorbed on the extraction plate, demonstrating that the extraction process of the extraction plate can be completed in a very short time, which indicates that the extraction plate has the potential to extract the analyte rapidly, and in conclusion, the extraction time was taken to be 30min to obtain a larger adsorption capacity.
3. Sample volume study
The effect of different sample volumes 3, 5, 8, 10, 15, 20m L on the amount extracted was studied in triplicate and the results are shown in FIG. 6, where the signal intensity increases with increasing sample volume in the range of 3-8ml and decreases with increasing sample volume as sample volume continues to increase.
4. Study of spray ionization conditions
Spray ionization conditions determine how much target is eluted from the extraction plate and the signal of the target that can be detected, playing an important role in ambient mass spectrometry. Two experimental parameters, spray voltage and spray solvent, were studied and the signal intensities of 5FQs analytes were compared to find better spray ionization conditions.
A. Spray voltage optimization
The spray voltage has a great influence on the signal of the analyte in the open mass spectrometry experiment, and a comparison study of different voltages is carried out, wherein when the voltage is too low, the analyte eluted from the eluent cannot be ionized, so that the signal cannot be detected in the detection time, however, when the voltage is too high, the spray moving speed is too fast, so that the instrument cannot collect the whole effective signal, therefore, a 5FQs methanol solution of 50ng/m L is dripped on the surface of an extraction plate, the signal intensity of 5 quinolone antibiotics is detected by applying different voltages, each voltage is detected three times, and as shown in fig. 7, the signal intensity of 5FQs is increased along with the increase of the applied voltage, when the high voltage is increased to 3.5kV or 4kV, the highest signal intensity of most analytes can be obtained, and then the signal intensity of 5 analytes is decreased along with the increase of the voltage, therefore, in order to obtain the stable and higher signal intensity of most compounds, +3.5kV is used as the preferred voltage.
B. Spray solvent optimization
The spray solvent is required to not only maximally elute the analyte adsorbed on the extraction plate, but also to have higher ionization efficiency for the analyte. To obtain higher signal intensity, 5 quinolone antibiotic standards (100 ng/g) were added to the blank milk base, and the effect of methanol (MeOH), MeOH with 0.1% acetic acid, MeOH with 1% acetic acid as spray solvents on 5FQs signal intensity was examined, analyzed in parallel three times, and the amount of extraction was determined. The results are shown in fig. 8 and show that the elution capacity of 5FQs did not increase with the addition of acetic acid (HOAc). Therefore, the effect of using methanol as the eluent is better.
5. Methodology validation of mass spectrometric detection
The open electrospray mass spectrometry method is considered to detect 5 carbostyril antibiotics to be detected with different concentrations in a blank milk substrate to obtain the linearity OF a standard curve, the L OD and L OQ. results are shown in Table 3, and the correlation coefficients (r) OF the 5 compounds to be detected are respectively in the linear ranges OF 1-500ng/m L (ENR), 1-1000ng/m L (OF L), 5-1000ng/m L (CIP), 2-1000ng/m L (NOR) and 2-1000ng/m L (PEF)2) 0.999, 0.9965, 0.9976, 0.9932, 0.9985.
L ODs of 5 to-be-detected quinolone antibiotics range from 0.1 ng/m L, and L OQs ranges from 1 ng/m L, the detection precision of the 5 to-be-detected substances is considered, the result is shown in table 3, the recovery rates of different adding concentrations in an actual sample are considered, the result is shown in table 4, the ESI-MS of the embodiment of the invention has good parallelism in daily precision and daytime precision, RSD% is respectively 7.00% -10.40% and 4.46% -11.44%, the method can obtain good recovery rates, the recovery rate is 81.43% -98.40%, the SD% value is 2.04% -9.79%, and the method has good quantification capability for trace compounds in an actual sample.
TABLE 4
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.
Claims (10)
1. A method for performing qualitative/quantitative detection of a target compound, comprising:
providing a solution to be detected;
contacting an extraction plate with the solution to be detected, and enriching the target compound in the extraction plate so as to obtain an enriched extraction plate; and
carrying out qualitative/quantitative analysis and detection on the enriched extraction plate,
wherein the extraction plate comprises:
a substrate; and
an adsorption layer which is coated on at least part of the surface of the substrate, is formed by crosslinked polymer, is porous and has a compound embedding space to be extracted.
2. The method according to claim 1, characterized in that the preparation method of the extraction plate comprises:
acidizing the substrate to obtain an acidized substrate;
subjecting the acidified substrate to silanization treatment to silanize at least part of the surface of the acidified substrate so as to obtain a silanized substrate;
performing double bond modification treatment on at least part of the surface of the silanized substrate so as to obtain a modified substrate;
dissolving a compound to be extracted, a monomer compound and a cross-linking agent in a pore-foaming agent so as to obtain a pre-polymerization liquid;
contacting at least part of the surface of the modified substrate with the pre-polymerization solution to carry out a crosslinking reaction so as to obtain a crosslinked polymer embedded with the compound to be extracted; and
subjecting the cross-linked polymer in which the compound to be extracted is embedded to elution treatment, and removing the compound to be extracted embedded on the cross-linked polymer, so as to obtain the compound extraction plate.
3. The method of claim 2, wherein the monomer compound is methacrylic acid, the crosslinking agent is ethylene glycol dimethacrylate,
optionally, the crosslinking reaction further comprises:
the initiator is azobisisobutyronitrile;
the pore-foaming agent is a chloroform-methanol mixed solution, and preferably, the volume ratio of chloroform to methanol of the pore-foaming agent is 4-6: 1.
4. The method according to claim 1, wherein the contacting is performed by immersing at least part of the extraction plate in the solution to be tested, while shaking at 180rpm, for 10-60min, preferably 30min,
optionally, the volume of the solution to be tested is 6-10ml, preferably 8 ml.
5. The method of claim 1, wherein the analytical detection is an open electrospray mass spectrometry detection,
optionally, the extraction plate has at least one angular end, preferably, the extraction plate is in the shape of an isosceles triangle,
optionally, the tip of the extraction plate is 3-8mm, preferably 5mm, from the mass spectrometry inlet.
6. The method of claim 5, wherein the spray ionization conditions of the ambient electrospray mass spectrometry are:
spraying voltage: 2.5-5Kv, preferably, 3.5 Kv;
spray desorption solvent-methanol solution containing (0-1)% acetic acid, the volume of desorption solvent is 10-30 μ L, preferably 20 μ L.
Optionally, the detection conditions of the open electrospray mass spectrometry are as follows:
the detection mode is as follows: multiple Reaction Monitoring (MRM);
atomizing gas pressure: 55 psi;
auxiliary gas pressure: 50 psi;
air curtain pressure: 20 psi;
ion source temperature: 550 ℃;
residence time: 100 ms.
7. The method according to claim 5, wherein the desorption solvent of the open electrospray mass spectrometry is a methanol solution containing (0-1)% acetic acid, and the volume of the desorption solvent is 10-30 μ L, preferably 20 μ L.
8. The method according to claim 1, wherein the target compound is a quinolone compound.
9. A kit for the qualitative/quantitative detection of a target compound, comprising:
an extraction plate prepared using the method of any one of claims 1-8, the extraction plate comprising:
a substrate; and
an adsorption layer which is coated on at least part of the surface of the substrate, is formed by crosslinked polymer, is porous and has a compound embedding space to be extracted; and
a desorption solvent which is a 20 μ L (0-1)% acetic acid in methanol.
10. The kit according to claim 9, wherein the target compound is a quinolone compound.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114636775A (en) * | 2022-03-02 | 2022-06-17 | 中国检验检疫科学研究院 | Separation and ionization integrated extraction device for carbamate compounds and application |
| CN115078598A (en) * | 2022-05-05 | 2022-09-20 | 天津国科医工科技发展有限公司 | Kit for directly sampling and testing blood concentration sample and application |
| CN116794155A (en) * | 2023-08-04 | 2023-09-22 | 苏州市食品检验检测中心(苏州市食品安全快检中心) | Method for determining quinolone compounds in animal-derived food |
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| CN101942056A (en) * | 2010-09-10 | 2011-01-12 | 中国检验检疫科学研究院 | Preparation method of molecularly imprinted polymer of methyltestosterone |
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Cited By (4)
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| CN114636775A (en) * | 2022-03-02 | 2022-06-17 | 中国检验检疫科学研究院 | Separation and ionization integrated extraction device for carbamate compounds and application |
| CN114636775B (en) * | 2022-03-02 | 2024-03-29 | 中国检验检疫科学研究院 | Separation and ionization integrated extraction device for carbamate compounds and application thereof |
| CN115078598A (en) * | 2022-05-05 | 2022-09-20 | 天津国科医工科技发展有限公司 | Kit for directly sampling and testing blood concentration sample and application |
| CN116794155A (en) * | 2023-08-04 | 2023-09-22 | 苏州市食品检验检测中心(苏州市食品安全快检中心) | Method for determining quinolone compounds in animal-derived food |
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