CN111272911A - Sample pretreatment method for determining Amadori compound in food - Google Patents
Sample pretreatment method for determining Amadori compound in food Download PDFInfo
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Abstract
A sample pretreatment method for measuring an Amadori compound in a food, characterized by: firstly, the food extract is adjusted to be weakly acidic and then passes through a strongly acidic cation exchange solid phase extraction column, then ammonia water solution is used for elution, the eluent passes through a phenylboronic acid affinity chromatography solid phase extraction column, and finally the Amadori compound is eluted by formic acid solution, so that the extraction and purification in the process of measuring the Amadori compound in the food sample are realized. The method is simple, rapid, highly sensitive and high-flux, particularly shows high selectivity in the aspect of removing interferents in a food matrix, and improves the sensitivity and accuracy of the Amadori compound determination.
Description
Technical Field
The invention relates to a sample pretreatment technology in an Amadori compound determination process for determining Maillard reaction, in particular to a sample pretreatment method for determining an Amadori compound in food, which is applicable to various heat-processed products such as tobacco, tea, soy sauce and the like and belongs to the field of analytical chemistry.
Background
The maillard reaction is a series of complex reactions generated by heating between amino acid or peptide containing free amino group, protein and reducing sugar, generally, the maillard reaction is firstly carried out the carbonyl ammonia condensation reaction between sugar and primary amino substance to generate Schiff base, then the Schiff base is converted into stable cyclic structure product glucosamine through intramolecular cyclization, the glucosamine is converted into cyclic fructosamine through Amadori rearrangement (the Heyns compound with glucosamine structure is generated through the maillard reaction between fructose and amino acid), and the intermediates are degraded into aldehydes, pyrazines and other compounds with special fragrance volatility through different ways. The composition difference of amino acid and reducing sugar contained in various foods is also large under the influence of gene type, maturity, position, climate and heat processing process conditions. By monitoring the content of the Amadori compound in the food and the tobacco, the degree of Maillard reaction in the food and tobacco processing and storing processes can be known in time, the occurrence of the Maillard reaction which is not beneficial to the quality of the food and the tobacco is inhibited, meanwhile, a theoretical basis is provided for the influence of the Amadori compound on food aroma components, the deep research of the formation mechanism of part of aroma components is facilitated, and a foundation is laid for the research of the functionality of the Amadori compound.
As the Amadori compound has the characteristics of large polarity, low volatility, no strong chromophoric group and the like, the separation and detection of the Amadori compound are concerned. Although the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has high sensitivity and strong selectivity, the method not only can carry out chromatographic separation on a complex mixed system, but also can obtain the structural information of the analyzed component through mass selection of mass spectrometry and multistage tandem mass spectrometry. However, most food matrices are very complex and the sensitivity and accuracy of the assay cannot be guaranteed without specific purification and enrichment of the extract to eliminate matrix effects. The methods for measuring Amadori compounds in tobacco disclosed in patent CN201610917998.0 and patent CN201710554270.0, which include the steps of crushing, extracting, diluting, measuring and the like, cannot effectively remove interfering impurities in the samples, and are not suitable for other food samples such as soy sauce, tea leaves and the like. The invention patent CN201310487876.9 discloses a method for measuring Amadori compounds in tobacco, which comprises sample treatment processes such as methanol extraction and n-hexane pigment removal. However, this method only removes non-polar chromatograms and is not effective for removing polar compounds such as sugars, alkaloids, etc., which interfere with the assay. Therefore, a sample pretreatment technology aiming at the analysis of the Amadori compound in a food complex system needs to be developed, and the extract is subjected to specific purification and enrichment to overcome the phenomenon of mass spectrum signal attenuation caused by a sample matrix.
Disclosure of Invention
The invention aims at the problems that the prior complex system related to the Maillard reaction contains a plurality of non-Amadori compound components, before the determination is carried out by the liquid chromatography-mass spectrometry technology, the extract needs to be specifically purified and enriched to overcome the phenomenon of mass spectrum signal attenuation caused by a sample matrix or co-elution substances, according to the structural characteristics of the Amadori compound that the polarity is stronger and the Amadori compound contains a sugar unit and an amino acid, a two-step solid phase extraction technology is provided to extract the Amadori compound, firstly, the Amadori and Heyns compounds and other alkaline compounds are enriched by using a strong acid cation exchange resin, the sugar substances are removed at the same time, an ammonia water eluent directly passes through a phenyl boronic acid affinity chromatography solid phase extraction column, the enrichment of the Amadori compound is realized by utilizing the affinity recognition function between boric acid and a cis-dihydroxy compound under the alkaline condition, other impurities are removed at the same time, according to the characteristic that the, and eluting the Amadori compound by formic acid solution, thereby simultaneously realizing the extraction and purification of the Amadori compound in a Maillard reaction system and establishing an analysis method with both sensitivity and accuracy.
The purpose of the invention is realized by the following technical scheme:
a sample pretreatment method for determining Amadori compounds in food comprises the steps of firstly adjusting a food extracting solution to be weakly acidic, then passing the food extracting solution through a strong-acid cation exchange solid phase extraction column, then eluting the food extracting solution with an ammonia water solution, passing an eluent through a phenylboronic acid affinity chromatography solid phase extraction column, and finally eluting the Amadori compounds with a formic acid water solution to realize extraction and purification of the Amadori compounds in the food sample. The method specifically comprises the following steps:
(1) extracting 100mg of food sample with 10mL of water under ultrasonic condition, filtering the extractive solution, and adjusting pH to 4;
(2) after the extracting solution passes through a strong acid cation exchange solid phase extraction column, a phenylboronic acid affinity chromatography solid phase extraction column is connected in series with the extracting solution, 3mL of ammonia water solution is used for elution, and the eluent can directly flow through the phenylboronic acid affinity chromatography solid phase extraction column.
(3) Separating two solid phase extraction columns connected in series, eluting phenylboronic acid affinity chromatography solid phase extraction column with 3mL formic acid aqueous solution。
(4) And (3) carrying out HPLC-MS/MS analysis on the eluent, carrying out positive ion ionization mode, carrying out multi-reaction monitoring scanning, and determining the content of various Amadori compounds.
Grinding the tobacco leaf and tea leaf samples in the step (1) into powder, and carrying out ultrasonic extraction after drying the powder by a 40-mesh sieve; liquid samples such as soy sauce can be diluted 5-20 times to obtain samples, and other samples can be processed by the method.
The ultrasonic power in the step (1) is 500-2000W, and the ultrasonic time is 20-40 minutes.
The pH value of the sample is adjusted in the above step (1) to be hydrochloric acid or phosphoric acid of 0.1 mol/l.
The filler of the strong-acid cation exchange solid-phase extraction column is sulfonated polystyrene resin or similar products, such as Oasis MCX (Waters company) or similar products.
The filler of the phenylboronic acid affinity chromatography solid phase extraction column is Bond Elut PBA (Agilent company) or the like.
The pH value of the ammonia water solution for elution is 9-10, and the ammonia water concentration is 1 mol/L.
Finally, the pH value of the formic acid aqueous solution for elution is between 3 and 5, and the concentration of the formic acid is 1 mol/L.
The HPLC-MS/MS instrument can be a high performance liquid chromatography-triple quadrupole mass spectrometer, and conditions of a chromatographic column and a chromatographic mobile phase and mass spectrometry parameters can be adjusted according to actual analysis conditions.
The method has the outstanding advantages that two-step solid phase extraction is adopted before chromatographic mass spectrometry, so that interfering substances in food or tobacco matrixes can be effectively removed, and the sensitivity of analysis and the accuracy of the method are improved.
Drawings
FIG. 1 is a schematic diagram of the operation steps of the method of the present invention.
Detailed Description
For better understanding of the present invention, the following examples are provided to further illustrate the key technologies in the technical solutions of the present invention, but the present invention is not limited to the following examples.
Example 1
Crushing cut tobacco, sieving with a 40-mesh sieve, adding 100mg into a conical flask with a plug, adding 20mL of water, carrying out ultrasonic extraction for 30 minutes, filtering, adjusting the pH of the filtrate to 4 by using 0.1 mol/L hydrochloric acid, passing the solution through an Oasis MCX solid phase extraction (6 mL) small column, connecting the small column with a Bond Elut PBA solid phase extraction series, eluting the Oasis MCX solid phase extraction small column by using an ammonia water solution with the pH of 9, and passing the eluent through the Bond Elut PBA small column. The two columns in series were disassembled and the content of Amadori compound was determined by HPLC-MS/MS by eluting the BondElut PBA column with 3ml of aqueous formic acid at pH 4. FIG. 1 is a schematic diagram of the whole sample pretreatment process.
Chromatographic conditions are as follows: atlantis T3 column (2.1X 250 mm, 5 μm), mobile phase: 0.2% aqueous formic acid (phase a) and acetonitrile (phase B), gradient elution procedure: 0-0.1 min, 89.5% A-78% A, 0.1-10 min, 78% A. Flow rate 600. mu.L/min, sample introduction: 5 μ L, equilibrate with initial mobile phase for 8 min between each two samples. Mass spectrum conditions: electrospray ion source (ESI), multiple reaction monitoring positive ion mode (MRM); spraying voltage: 5500V, ion source temperature: 550 ℃; air curtain air: 20 psi, 60 psi for the atomizing air, 75 psi for the drying air, and 9 psi for the impinging air.
Example 2
Diluting 1mL of soy sauce sample to 20mL, adjusting pH to 4 with 0.1 mol/L hydrochloric acid, passing the solution through an Oasis MCX solid phase extraction (6 mL) small column, connecting the small column with Bond Elut PBA solid phase extraction in series, eluting the Oasis MCX solid phase extraction small column with an ammonia solution with pH of 9, and passing the eluent through the Bond Elut PBA small column. The two columns in series were disassembled and the Bond Elut PBA column was eluted with 3ml of aqueous formic acid at pH4 and the Amadori compound content was determined by HPLC-MS/MS.
Claims (10)
1. A sample pretreatment method for measuring an Amadori compound in a food, characterized by: firstly, the food extract is adjusted to be weakly acidic and then passes through a strongly acidic cation exchange solid phase extraction column, then ammonia water solution is used for elution, the eluent passes through a phenylboronic acid affinity chromatography solid phase extraction column, and finally the Amadori compound is eluted by formic acid water solution, so that the extraction and purification of the Amadori compound in the food sample are realized.
2. The sample pretreatment method according to claim 1, characterized in that: the method comprises the following specific steps:
(1) extracting 100mg of food sample with 10mL of water under ultrasonic condition, filtering the extractive solution, and adjusting pH to 4;
(2) after the extracting solution passes through a strong acid cation exchange solid phase resin small column, connecting a phenylboronic acid affinity chromatography solid phase extraction small column with the strong acid cation exchange solid phase resin small column in series, eluting by using 3mL of ammonia water solution, and directly flowing eluent through the phenylboronic acid affinity chromatography solid phase extraction small column;
(3) separating two solid phase extraction small columns connected in series, eluting the phenylboronic acid affinity chromatography solid phase extraction small columns with 3mL of formic acid aqueous solution;
(4) and (3) carrying out HPLC-MS/MS analysis on the eluent, carrying out positive ion ionization mode, carrying out multi-reaction monitoring scanning, and determining the content of various Amadori compounds.
3. The sample pretreatment method according to claim 1 or 2, characterized in that: the food extract is prepared from tobacco, tea leaf and soy sauce。
4. The sample pretreatment method according to claim 1 or 2, characterized in that: the filler of the strong-acid cation exchange solid-phase extraction column is sulfonated polystyrene resin or the like.
5. The sample pretreatment method according to claim 1 or 2, characterized in that: the pH value of the ammonia water solution for elution is between 9 and 10.
6. The sample pretreatment method according to claim 1 or 2, characterized in that: the filler of the phenylboronic acid affinity chromatography solid phase extraction column is Bond Elut PBA or the like.
7. The sample pretreatment method according to claim 1 or 2, characterized in that: finally, the pH value of the formic acid aqueous solution for elution is between 3 and 5.
8. The sample pretreatment method according to claim 3, characterized in that: grinding the tobacco and tea samples into powder, sieving the powder with a 40-mesh sieve, performing ultrasonic extraction, and diluting the soy sauce liquid sample by 5-20 times, and performing ultrasonic extraction to obtain an extracting solution.
9. The sample pretreatment method according to claim 1, characterized in that: the ultrasonic power is 500-2000w, and the time is 20-40 min.
10. The sample pretreatment method according to claim 2, characterized in that: the acid used for adjusting the pH value in the step (1) is hydrochloric acid or phosphoric acid of 0.1 mol/l.
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| CN102059103A (en) * | 2010-12-08 | 2011-05-18 | 南京大学 | Hydrophylic phenylboric acid functional porous integral material, preparation method and application thereof |
| CN103278574A (en) * | 2013-04-26 | 2013-09-04 | 首都医科大学附属北京朝阳医院 | Method for detecting hemoglobin by liquid chromatogram-triple tandem quadrupole mass spectrometer |
| CN104549182A (en) * | 2014-12-29 | 2015-04-29 | 上海烟草集团有限责任公司 | Branched boric acid functionalized monolithic column and preparation method and application thereof |
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| US4629692A (en) * | 1982-12-06 | 1986-12-16 | Miles Laboratories, Inc. | Immunoassay for nonenzymatically glucosylated proteins and protein fragments an index of glycemia |
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| CN103278574A (en) * | 2013-04-26 | 2013-09-04 | 首都医科大学附属北京朝阳医院 | Method for detecting hemoglobin by liquid chromatogram-triple tandem quadrupole mass spectrometer |
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Application publication date: 20200612 |