CN112881581B - Method for quantitatively detecting pyrrolin and methyl glyoxal hydrogen imidazolone in aquatic product - Google Patents
Method for quantitatively detecting pyrrolin and methyl glyoxal hydrogen imidazolone in aquatic product Download PDFInfo
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Abstract
The invention discloses a method for quantitatively detecting pyrrolin and methyl glyoxal hydrogen imidazolone in aquatic products, and belongs to the technical field of food safety detection. The method adopts an enzymatic hydrolysis method to extract the pyrrolin and the methyl glyoxal hydrogen imidazolone, and can overcome the defect that the pyrrolin and the methyl glyoxal hydrogen imidazolone are unstable when meeting acid; solid-phase extraction is utilized to realize impurity removal and enrichment of the pyrroline and the methylglyoxal hydrogen imidazolone, so that the matrix effect of quantitative detection is reduced; the method adopts an internal standard method to simultaneously quantify the pyrrolide and the methylglyoxal hydrogen imidazolone, and can correct the contents of the pyrrolide and the methylglyoxal hydrogen imidazolone; the method has the advantages that the Waters Atlantis T3 liquid chromatographic column is used, liquid phase-mass spectrometry is used for simultaneously and quantitatively detecting the pyrrolizium and the methylglyoxal hydrogen imidazolone, the pyrrolizium with weak polarity and the methylglyoxal hydrogen imidazolone with strong polarity in the aquatic products can be better reserved and quantitatively detected in a short time, the detection efficiency is improved, and the detection method is high in sensitivity.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a method for quantitatively detecting pyrrolizine and methyl glyoxaline hydrogen imidazolone in aquatic products.
Background
Food processing actually involves a series of complex reactions of biology, physics, chemistry and the like of each component of food, particularly the occurrence of Maillard reaction which is accompanied with the high-protein and high-fat food in the heat treatment process, and the Maillard reaction product, namely advanced glycation end products (AGEs) is formed. AGEs are formed from the carbonylamino reaction between the carbonyl group of reducing sugars and amino acids, proteins and nitrogen-containing compounds. A great deal of research shows that excessive intake of dietary AGEs can cause accumulation of AGEs in vivo, thereby causing various chronic diseases such as diabetes, nephropathy, Alzheimer's disease and the like. More than ten species of AGEs have been found in food products, including carboxymethyllysine, carboxyethyllysine, methylglyoxal benzimidazolone (MG-H1), pyrrolidones (pyrralines), and the like. Most researches mainly focus on two AGEs, namely carboxymethyl lysine and carboxyethyl lysine, and at present, the content of the pyrroline in the oil product is found to be high, while the researches on the detection method of the pyrroline in the aquatic product are not reported, and at present, an external standard method is mostly adopted for quantitative detection of the pyrroline, and no internal standard is added to correct the content of the pyrroline, so that the measurement result of the pyrroline is inaccurate. Aquatic products belong to high-protein high-fat foods and are important sources of AGEs, so that quantitative detection of AGEs in aquatic products has important practical significance for improving food safety level.
Food systems are complex and various, and the detection of AGEs in food has no unified standard, and the current methods for detecting AGEs mainly comprise an enzyme-linked immunosorbent assay and a liquid chromatography-mass spectrometry combined method. The enzyme-linked immunosorbent assay has higher requirements on antibodies and is greatly interfered; the liquid phase-mass spectrum combination is a relatively wide application method, and the method has the advantages of high separation, high sensitivity, high selectivity and the like on complex samples, and is a preferred method for detecting trace substances in food. However, the polarity of the pyrroline is weaker and is suitable for separating and retaining the pyrroline by using a reverse chromatographic column, while the polarity of the methylglyoxal hydrogen imidazolone is stronger, most of the research focuses on high-polarity AGEs such as carboxymethyl lysine and carboxyethyl lysine, and the simultaneous retention and separation of the polar AGEs can be realized by using a hydrophilic chromatographic column. Therefore, the currently used chromatographic column is difficult to simultaneously reserve and separate two AGEs, namely the pyrroline and the methylglyoxal hydrogen imidazolone, and although the reservation effect can be increased by using nonafluorovaleric acid plasma on the reagent, the ion pair reagent can shorten the service life of the chromatographic column, weaken the ionization effect of liquid substances, and remain and pollute the mass spectrum in the mass spectrum. Different AGEs pretreatment modes are different, the AGEs such as carboxymethyl lysine and carboxyethyl lysine are stable when meeting acid, an acidolysis method is mainly adopted for extraction, and the pyrroline and methyl glyoxal hydrogen imidazolone are unstable when meeting acid and are mainly extracted by an enzymolysis method.
Based on the differences between the properties of AGEs and pretreatment methods, many studies on pyrrolizim have been conducted by single detection. Therefore, no method for simultaneously and quantitatively detecting the pyrrolidon (Pyrraline) with weaker polarity and the methylglyoxal hydrogen imidazolone (MG-H1) with stronger polarity in the aquatic products exists at present.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for quantitatively detecting the pyrrolizine and the methylglyoxal hydrogen imidazolone in an aquatic product.
The structural formula of methylglyoxal benzimidazolone (MG-H1) is as follows:
the structural formula of the pyrroline (Pyrraline) is as follows:
the method adopts an enzymatic hydrolysis method to extract the pyrrolin and the methyl glyoxal hydrogen imidazolone, and can overcome the defect that the pyrrolin and the methyl glyoxal hydrogen imidazolone are unstable when meeting acid; solid phase extraction is utilized to realize impurity removal and enrichment of the pyrroline and the methylglyoxal hydrogen imidazolone, so that the matrix effect in quantitative detection is reduced; the method adopts an internal standard method to simultaneously quantify the pyrrolide and the methylglyoxal hydrogen imidazolone, and can correct the contents of the pyrrolide and the methylglyoxal hydrogen imidazolone; a Waters Atlantis T3(2.1 x 150mm,5 mu m) liquid chromatographic column is used, a mobile phase without ions added to a reagent is selected, the pollution of the ions to the reagent on a mass spectrum is avoided, then the liquid phase-mass spectrum combination is used for simultaneously and quantitatively detecting the pyrroline and the methylglyoxal hydrogen imidazolone, so that the pyrroline with weak polarity and the methylglyoxal hydrogen imidazolone with strong polarity in aquatic products can be better reserved and quantitatively detected in a short time, the detection efficiency is improved, and the detection method has high sensitivity.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for quantitatively detecting pyrrolin and methyl glyoxal hydrogen imidazolone in aquatic products comprises the following steps:
step 1: extraction of pyrrolins and methylglyoxal hydroimidazolidones from samples
1.1, extracting the pyrrolidon and the methyl glyoxal hydrogen imidazolone in a sample by adopting an enzymolysis method;
1.2 adding an internal standard into the sample solution after enzymolysis for correcting errors, and filtering by using a filter membrane to remove impurities;
1.3 extracting the sample solution after adding the standard through a solid phase extraction column, and collecting eluent;
step 2: carrying out quantitative detection on the eluent obtained in the step 1 by using liquid phase-mass spectrometry;
and step 3: drawing a standard curve: adding an internal standard into the standard, detecting by liquid phase-mass spectrometry, and drawing a standard curve by taking the ratio of the concentration of the standard to the concentration of the internal standard as a horizontal coordinate and the ratio of the peak area of the standard to the peak area of the internal standard as a vertical coordinate to obtain a equation;
and 4, step 4: and (3) comparing the detection data obtained in the step (2) with the standard curve and equation obtained in the step (3), and calculating to obtain the concentrations of the pyrroline and the methylglyoxal hydrogen imidazolone in the sample.
On the basis of the scheme, the specific operation of extracting the pyrroline and the methylglyoxal hydrogen imidazolone in the sample by the enzymolysis method is as follows:
weighing 20mg of sample into 1mL of 20mM HCl solution, respectively adding 40 mu L of 6mg/mL pepsin solution and 20 mu L of 2mg/mL thymol solution, filling nitrogen, quickly sealing, and shaking for 24 hours at 37 ℃;
② adjusting the pH value of the system after the reaction in the step I to 7.4 by 260mM KOH solution, adding 300 μ L of 0.5M potassium phosphate buffer and 1mL of 2mg/mL streptokinase (XIV type) solution, charging nitrogen, rapidly sealing, and then shaking for 24h at 37 ℃;
thirdly, adding 100 mu L of leucine aminopeptidase M (VI-S type) solution and 50 mu L of prolidase solution into the system after the reaction in the second step in sequence, then filling nitrogen, rapidly sealing, and shaking for 24 hours at 37 ℃.
The solvent of the pepsin solution is 20mM HCl solution; the enzyme activity is 2400U/mL;
the solvent of the thymol solution is 20mM HCl solution;
the solvent of the solution of the pronase (form XIV) is 10mM PBS buffer solution; the enzyme activity is 7U/mL;
the solvent of the leucine aminopeptidase M (VI-S type) solution is 10mM PBS buffer solution; enzyme activity is 6U/sample;
the prolidase solution solvent is 10mM PBS buffer solution; the enzyme activity is 8U/sample.
The thymol has broad-spectrum antibacterial and antiseptic functions, and the growth and reproduction of various microorganisms can be inhibited by adding the thymol solution into the system; potassium phosphate buffer solution is added to ensure that the pH value of the system is within a proper threshold value, and the enzyme solution can exert the activity.
Based on the scheme, the internal standard adopted in the step 1 is MG-H1-d3。
On the basis of the scheme, the solid phase extraction column extraction method comprises the following steps:
taking 3mL of methanol to activate the HLB Pro extraction column, then taking 3mL of ultrapure water to balance the HLB Pro extraction column, taking 1mL of the enzymolysis sample solution subjected to the labeling in the step 1.2, passing through the HLB Pro extraction column, taking 3mL of ultrapure water to remove impurities, then eluting with 3mL of 5% ammonia methanol solution, collecting the eluent, blowing and drying at 60 ℃ with nitrogen, and finally re-dissolving in 10% acetonitrile water solution.
On the basis of the scheme, the solid phase extraction column is CNW Poly-Sery HLB Pro, and the specification is 60mg/3 mL.
The HLB Pro extraction column adopted by the invention has the characteristics of extreme acid and alkali resistance, high flow rate and short column passing time, and is suitable for polar and non-polar compounds.
On the basis of the scheme, the liquid chromatogram condition of the liquid phase-mass spectrum combination is as follows:
a chromatographic column: waters Atlantis T3 (2.1X 150mm,5 μm); mobile phase: an aqueous solution containing 0.1% formic acid and 10% acetonitrile; flow rate: 0.2 mL/min; column temperature: 30 ℃; sample introduction volume: 10 mu L of the solution; gradient elution procedure: and (3) carrying out isocratic elution.
On the basis of the scheme, the mass spectrum conditions of the liquid phase-mass spectrum combination are as follows:
electrospray ion source (ESI); a positive ion scanning mode; multiple reaction monitoring mode (MRM); the drying temperature is 350 ℃; the drying airflow rate is 12L/min; atomizer pressure was 45 psi; the capillary voltage is 3500V; the fragmentation voltage was 135V.
On the basis of the scheme, the mass-to-charge ratio of the parent ion of the pyrrolin in the liquid phase-mass spectrum combined use is 255, and the mass-to-charge ratio of the ion of the characteristic fragment is detected quantitatively to be 175; qualitatively detecting the mass-to-charge ratio of the characteristic fragment ions to be 237; the mass-to-charge ratio of the methylglyoxal hydrogen imidazolone parent ion is 229, the mass-to-charge ratio of the quantitative detection characteristic fragment ion is 70, and the mass-to-charge ratio of the qualitative detection characteristic fragment ion is 114.
Based on the above protocol, the standard curve is plotted with the concentration of the standard solution in the range of 0.01-1. mu.g/mL.
On the basis of the scheme, the aquatic product is sturgeon.
The technical scheme of the invention has the advantages that:
1. the method adopts an enzymatic hydrolysis method to simultaneously extract the pyrrolin and the methyl glyoxal hydrogen imidazolone in the aquatic products, and can overcome the defect that the pyrrolin and the methyl glyoxal hydrogen imidazolone are unstable when meeting acid;
2. the method adopts an internal standard method to simultaneously quantify the pyrrolide and the methylglyoxal hydrogen imidazolone, and can correct the contents of the pyrrolide and the methylglyoxal hydrogen imidazolone;
3. the method can better retain and quantitatively detect the pyrroline with weaker polarity and the methyl glyoxal hydrogen imidazolone with stronger polarity in a shorter time, thereby improving the detection efficiency;
4. the invention has better separation effect, high sensitivity, good linear relation and R2Are all above 0.998;
5. the method realizes the simultaneous detection of the pyrrolin and the methyl glyoxal hydrogen imidazolone in the aquatic products.
6. The invention uses the mobile phase without adding ion pair reagent, thereby avoiding the pollution of mass spectrum.
Drawings
FIG. 1 is a standard curve for pyrrolin;
FIG. 2 is a methylglyoxal benzimidazolone standard curve;
FIG. 3 is a total ion chromatogram of a standard solution;
FIG. 4 is a mass spectrum of a target compound of a sample;
FIG. 5 is a total ion chromatogram of methylglyoxal hydroimidazolone and pyrrol;
FIG. 6 is a total ion chromatogram of methylglyoxal hydroimidazolone and pyrrol;
FIG. 7 is a total ion chromatogram of methylglyoxal hydroimidazolone and pyrrol.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
1. Reagent and apparatus
(1) Reagent
Standard product of pyrrolizin, Toronto Research Chemicals, Canada
Methylglyoxal benzimidazolone standard substance, American International Laboratory
Standard substance of BIOBERRY (American BIOBERRY) with interior label of methylglyoxal and hydrogen imidazolone
Pepsin, pronase (type XIV), leucine aminopeptidase M (type VI-S), prolidase Sigma-Aldrich
Chemical reagents of monopotassium phosphate, potassium hydroxide, PBS, hydrochloric acid, methanol, acetonitrile and ammonia water national medicine group
(2) Device
6410Triple Quad Mass spectrometer Agilent Technologies USA
1260 precision liquid chromatograph Agilent Technologies, USA
Analytical balance Beijing Sadolis scientific instruments Co., Ltd
XW-80A Mini vortex Mixer Shanghai Huxi Analyzer Co., Ltd
KQ-300VDE model double-frequency numerical control ultrasonic cleaner Qunshan City ultrasonic instrument Co Ltd
Data technology company Limited of Shandong cloud network of N-20 multifunctional Nitrogen blowing Instrument
CNW Poly-Sery HLB Pro solid phase extraction column Shanghai' an spectrum experiment science and technology Co., Ltd
LC-CQ-24F solid phase extraction device Shanghai Lichen West Instrument science and technology Co., Ltd
2. Extraction of pyrrolins and methylglyoxal hydroimidazolones
2.1 weighing 20mg of a frozen sturgeon sample which is cooked for 20min and dried in a freeze-dried manner into 1mL of 20mM HCl solution, respectively adding 40 mu L of 6mg/mL pepsin solution (the enzyme activity is 2400U/mL) and 20 mu L of 2mg/mL thymol solution (the solvents of the two are both 20mM HCL), filling nitrogen, quickly covering the cap, and then shaking for 24h at 37 ℃;
2.2 adjusting the pH of the system to 7.4 with 260mM KOH solution, adding 300. mu.L of 0.5M potassium phosphate buffer and 1mL of 2mg/mL streptokinase (type XIV) solution (solvent is 10mM PBS buffer, enzyme activity is 7U/mL), charging nitrogen, rapidly capping, and then shaking for 24h at 37 ℃;
2.3 adding 100 μ L leucine aminopeptidase M (VI-S type) solution (making each sample have 6U of enzyme activity and the solvent is 10mM PBS buffer) and 50 μ L proline peptidase solution (making each sample have 8U of enzyme activity and the solvent is 10mM PBS buffer) into the system respectively, then filling nitrogen, quickly capping, and shaking at 37 ℃ for 24 h;
3. solid phase extraction
Taking 3mL of methanol activated HLB Pro extraction column (CNW Poly-Sery HLB Pro, specification 60MG/3mL), then taking 3mL of ultrapure water to balance the HLB Pro extraction column, taking 1mL of step 2.3 enzymolysis liquid, adding 20 μ L of 5 μ g/mL methyl glyoxal hydrogen imidazolone internal standard (MG-H1-d)3) Filtering with 0.22 μm filter membrane, passing through HLB Pro extraction column, removing impurities with 3mL ultrapure water, vacuum drying, eluting with 3mL 5% ammonia water methanol solution, collecting eluate, drying at 60 deg.C under nitrogen blowing, and re-dissolving in 10% acetonitrile water solution.
4. Preparing a mixed standard solution (shown in Table 1), using MG-H1-d with a concentration of 5. mu.g/mL3MG-H1, Pyrraline standards were prepared as 1mL of 0, 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1. mu.g/mL standard solutions containing 0.1. mu.g/mL internal standard.
The concentration range of the mixed standard solution is 0.01-1 mug/mL, and the mixed standard solution and the solid-phase extraction liquid are subjected to quantitative detection by adopting liquid phase-mass spectrometry.
TABLE 1 preparation of standard solutions
5. Liquid phase-mass spectrometry combined detection
5.1 liquid chromatography conditions
A chromatographic column: waters Atlantis T3 (2.1X 150mm,5 μm);
mobile phase: an aqueous solution containing 0.1% formic acid and 10% acetonitrile;
flow rate: 0.2 mL/min;
column temperature: 30 ℃;
sample introduction volume: 10 mu L of the solution;
gradient elution procedure: and (3) carrying out isocratic elution.
5.2 Mass Spectrometry conditions
Electrospray ion source (ESI);
a positive ion scanning mode;
multiple reaction monitoring mode (MRM);
the drying temperature is 350 ℃;
the drying airflow rate is 12L/min;
atomizer pressure was 45 psi;
the capillary voltage is 3500V;
the fragmentation voltage was 135V.
5.3 Standard Curve is prepared
The abscissa is the ratio of the concentration of the pyrrolide (or the concentration of the methylglyoxal hydrogen imidazolone) to the concentration of the methylglyoxal hydrogen imidazolone internal standard, and the ordinate is the ratio of the area of the pyrrolide peak (or the area of the methylglyoxal hydrogen imidazolone) to the area of the methylglyoxal hydrogen imidazolone internal standard. The standard curve of the obtained pyrroline is Y-1.8021X +0.3551, R20.9985 (fig. 1); the standard curve of methylglyoxal hydrogen imidazolone is Y-0.5726X-0.0029, R20.9987 (as in fig. 2), wherein X is the concentration ratio and Y is the peak area ratio. FIG. 3 is a total ion chromatogram of a standard solution, wherein methylglyoxal-hydrogen imidazolone first-out peak is obtained, and the peak-out time is 1.91 min; the peak appears after the pyrrolin, and the peak appearing time is 4.8 min. The chromatographic condition proves that the separation effect is good, and the two AGEs can be separated.
5.4, carrying out quantitative detection on the enzymolysis liquid by adopting liquid phase-mass spectrometry, wherein, as shown in a mass spectrogram of a sample target compound, the mass-to-charge ratio of the pyrrolin parent ions is 255, and the mass-to-charge ratio of the characteristic fragment ions is 175; qualitatively detecting the mass-to-charge ratio of characteristic fragment ions to be 237; the mass-to-charge ratio of the methylglyoxal-hydrogen imidazolone parent ion is 229, the mass-to-charge ratio of the characteristic fragment ion is 70 in quantitative detection, and the mass-to-charge ratio of the characteristic fragment ion is 114 in qualitative detection. Mass spectrum data are obtained by a multi-reaction detection method, as shown in fig. 4, mass spectrograms with mass-to-charge ratios of 70, 114, 175 and 237 appear in the mass spectrograms, and respectively correspond to characteristic fragment ions of methylglyoxal hydrogen imidazolone and pyrroline, so that the mass spectrograms can be used for simultaneously detecting two AGEs of the pyrroline and the methylglyoxal hydrogen imidazolone. And the experimental result shows that when the concentration of the pyrrolin and the methylglyoxal hydrogen imidazolone is 0.01 mu g/mL, the signal to noise ratio (S/N) is more than 10, which indicates that the quantitative Limit (LOQ) of the method can reach 0.01 mu g/mL.
Comparative example 1
The HLB Pro extraction column in step 3 of example 1 was replaced with a Poly-Sery-MCX solid phase extraction column, with the other conditions being unchanged.
The detection result shows that the extraction effect of the pyrroline is poor after the HLB Pro extraction column is replaced.
Comparative example 2
The column in step 5.1 of example 1 was replaced with a Waters Symmetry C18 (2.1X 100mm,3.5 μm), and the other conditions were unchanged.
The detection result is shown in fig. 5, the pyrroline peaks at 1.77min, and the methylglyoxal hydrogen imidazolone does not peak, which indicates that the methylglyoxal hydrogen imidazolone is not retained on the chromatographic column.
Comparative example 3
The column in step 5.1 of example 1 was replaced with Agilent ZORBAX SB-C18 (4.6X 150mm), and the other conditions were unchanged.
The detection result is shown in fig. 6, no peak is generated on both the pyrroline and the methylglyoxal hydrogen imidazolone, and both are proved to be not retained on the chromatographic column.
Comparative example 4
The mobile phase in step 5.1 of example 1 was replaced with an aqueous solution containing 0.1% formic acid and 5% acetonitrile, the other conditions being unchanged.
As a result of the examination, when the mobile phase was an aqueous solution containing 0.1% formic acid and 10% acetonitrile, methylglyoxal-hydrogen imidazolone showed a peak at 1.91min and pyrroline showed a peak at 4.8 min. When the mobile phase is an aqueous solution containing 0.1% formic acid and 5% acetonitrile, as shown in fig. 7, the time of the methyl glyoxal hydrogen imidazolone peak is unchanged, while the time of the pyrrole peak is 5.13min, which increases the time cost.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (6)
1. A method for quantitatively detecting pyrrolin and methyl glyoxal hydrogen imidazolone in aquatic products is characterized by comprising the following steps:
step 1: extraction of pyrrolins and methylglyoxal hydroimidazolidones from samples
1.1, extracting the pyrrolidon and the methyl glyoxal hydrogen imidazolone in a sample by adopting an enzymolysis method;
1.2 adding an internal standard into the sample solution after enzymolysis for correcting errors, and filtering by using a filter membrane to remove impurities;
1.3 extracting the sample solution after adding the standard through a solid phase extraction column, and collecting eluent;
step 2: carrying out quantitative detection on the eluent obtained in the step 1 by using liquid phase-mass spectrometry;
and step 3: drawing a standard curve: adding an internal standard into the series of concentration gradient standard solutions, detecting by liquid phase-mass spectrometry, and drawing a standard curve by taking the ratio of the concentration of the standard solution to the concentration of the internal standard as a horizontal coordinate and the ratio of the peak area of the standard solution to the peak area of the internal standard as a vertical coordinate to obtain an equation;
and 4, step 4: comparing the detection data obtained in the step 2 with the standard curve and equation obtained in the step 3, and calculating to obtain the concentrations of the pyrroline and the methylglyoxal hydrogen imidazolone in the sample;
the specific operation of the enzymolysis method for extracting the pyrrolizium and the methylglyoxal hydrogen imidazolone in the sample is as follows:
weighing 20mg of sample into 1mL of 20mM HCl solution, respectively adding 40 mu L of 6mg/mL pepsin solution and 20 mu L of 2mg/mL thymol solution, filling nitrogen, quickly sealing, and shaking for 24 hours at 37 ℃;
② adjusting the pH value of the system after the reaction in the step I to 7.4 by 260mM KOH solution, adding 300 μ L of 0.5M potassium phosphate buffer solution and 1mL of 2mg/mL streptokinase-XIV solution, charging nitrogen, rapidly sealing, and then shaking for 24h at 37 ℃;
thirdly, sequentially adding 100 mu L of leucine aminopeptidase M-VI-S type solution and 50 mu L of proline peptidase solution into the system after the reaction in the second step, then filling nitrogen, quickly sealing the opening, and shaking for 24 hours at 37 ℃;
the solid phase extraction column is CNW Poly-Sery HLB Pro, and the specification is 60mg/3 mL;
the extraction method of the solid phase extraction column comprises the following steps:
taking 3mL of methanol to activate an HLB Pro extraction column, then taking 3mL of ultrapure water to balance the HLB Pro extraction column, taking 1mL of the enzymolysis sample solution subjected to the labeling in the step 1.2, passing through the HLB Pro extraction column, taking 3mL of ultrapure water to remove impurities, then eluting with 3mL of 5% ammonia water methanol solution, collecting eluent, drying by nitrogen blowing at 60 ℃, and finally re-dissolving in 10% acetonitrile water solution;
the liquid chromatogram condition of the liquid phase-mass spectrum combination is as follows:
a chromatographic column: waters Atlantis T3, 2.1X 150mm,5 μm; mobile phase: an aqueous solution containing 0.1% formic acid and 10% acetonitrile; flow rate: 0.2 mL/min; column temperature: 30 ℃; sample introduction volume: 10 mu L of the solution; gradient elution procedure: and (3) carrying out isocratic elution.
2. The method for quantitatively detecting the pyrrolidones and the methylglyoxal hydroimidazolidones in the aquatic products according to claim 1, wherein the internal standard used in the step 1 is MG-H1-d3。
3. The method for quantitatively detecting the pyrrolizine and the methylglyoxal hydrogen imidazolone in the aquatic products according to claim 1, wherein the mass spectrometry conditions of the liquid phase-mass spectrometry combination are as follows:
electrospray ion source (ESI); a positive ion scanning mode; multiple reaction monitoring mode (MRM); the drying temperature is 350 ℃; the drying airflow rate is 12L/min; atomizer pressure was 45 psi; the capillary voltage is 3500V; the fragmentation voltage was 135V.
4. The method for quantitatively detecting the pyrrolizium and the methylglyoxal hydrogen imidazolone in the aquatic products according to claim 3, wherein the mass-to-charge ratio of the parent ion of the pyrrolizium in the liquid phase-mass spectrometry is 255, and the mass-to-charge ratio of the ion of the characteristic fragment in the quantitative detection is 175; qualitatively detecting the mass-to-charge ratio of the characteristic fragment ions to be 237; the mass-to-charge ratio of the methylglyoxal hydrogen imidazolone parent ion is 229, the mass-to-charge ratio of the quantitative detection characteristic fragment ion is 70, and the mass-to-charge ratio of the qualitative detection characteristic fragment ion is 114.
5. The method for quantitatively detecting the pyrrolidones and the methylglyoxal hydroimidazolidones in the aquatic products according to claim 1, wherein the concentration range of the standard solution in the standard curve drawing is 0.01-1 μ g/mL.
6. The method for quantitatively detecting the pyrrolidones and the methylglyoxal hydroimidazolidones in the aquatic products according to any one of claims 1 to 5, wherein the aquatic products are sturgeons.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101268093A (en) * | 2005-03-31 | 2008-09-17 | 法马门斯公司 | Age inhibitors |
| CN102846598A (en) * | 2012-08-03 | 2013-01-02 | 广州康臣药物研究有限公司 | Application of coumarin in preparing formation inhibitors of advanced glycation end products (AGEs) |
| CN103884787A (en) * | 2014-02-21 | 2014-06-25 | 华南理工大学 | Detection method of peptide-advanced glycation end product |
| CN108956819A (en) * | 2018-07-28 | 2018-12-07 | 广东食品药品职业学院 | A kind of method that gas phase-mass spectrometry detects food-borne pyrroles's element |
| CN110988193A (en) * | 2019-12-24 | 2020-04-10 | 中国海洋大学 | Method for detecting advanced glycosylation end products in aquatic products |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102014216854A1 (en) * | 2014-08-25 | 2016-02-25 | Technische Universität Dresden | Method for the detection of a species-appropriate animal feed and for distinguishing "biologically" and "conventionally" produced dairy products |
| JP2018063208A (en) * | 2016-10-14 | 2018-04-19 | Ｂｌｏｏｍ Ｔｅｃｈｎｏｌｏｇｙ 株式会社 | Concentration method of advanced glycation endproduct |
-
2021
- 2021-01-15 CN CN202110053268.1A patent/CN112881581B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101268093A (en) * | 2005-03-31 | 2008-09-17 | 法马门斯公司 | Age inhibitors |
| CN102846598A (en) * | 2012-08-03 | 2013-01-02 | 广州康臣药物研究有限公司 | Application of coumarin in preparing formation inhibitors of advanced glycation end products (AGEs) |
| CN103884787A (en) * | 2014-02-21 | 2014-06-25 | 华南理工大学 | Detection method of peptide-advanced glycation end product |
| CN108956819A (en) * | 2018-07-28 | 2018-12-07 | 广东食品药品职业学院 | A kind of method that gas phase-mass spectrometry detects food-borne pyrroles's element |
| CN110988193A (en) * | 2019-12-24 | 2020-04-10 | 中国海洋大学 | Method for detecting advanced glycosylation end products in aquatic products |
Non-Patent Citations (4)
| Title |
|---|
| Application of preheating treatment in up- and down-regulating the glycation process of dietary proteins;Di Zhao 等;《Food Hydrocolloids》;20190810;第98卷;第1-8页 * |
| Free Maillard Reaction Products in Milk Reflect Nutritional Intake of Glycated Proteins and Can Be Used to Distinguish "Organic" and "Conventionally" Produced Milk;Uwe Schwarzenbolz 等;《Journal of Agricultural and Food Chemistry》;20160523;第64卷;第5071-5078页 * |
| Green Tea Extract Decreases Arg-Derived Advanced Glycation Endproducts but Not Lys-Derived AGEs in UHT Milk during 1‑Year Storage;Mahesha M. Poojary 等;《Journal of Agricultural and Food Chemistry》;20201117;第68卷;第14261-14273页 * |
| 固相萃取高效液相色谱法检测食品中吡咯素的研究;刘慧琳 等;《食品工业科技》;20161231;第33卷(第22期);第90-94页 * |
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