Detecting method of carboxymethyl lysine and application thereof

A carboxymethyllysine and detection method technology, which is applied in the field of detection of carboxymethyllysine in food, can solve the problems of low accuracy, complex sample processing, shortened service life of the instrument, etc., and achieve short detection time , high accuracy and good sensitivity

Inactive Publication Date: 2014-03-05
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the strong polarity of CML, most of the existing HPLC-MS / MS methods use C18 columns combined with ion-pairing reagents for separation, causing damage to the chromatographic column and mass spectrometry ion source, and shortening the service life of the instrument
In view of the difficulties in the selection of specific antibodies, complex sample processing, and low accuracy in the existing detection methods for CML in food, research and development of a new detection method that is simple, accurate, and fast is important for improving existing cooking methods and controlling food sources. The intake of sexual CML and the promotion of human health diet are of great significance

Method used

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  • Detecting method of carboxymethyl lysine and application thereof
  • Detecting method of carboxymethyl lysine and application thereof
  • Detecting method of carboxymethyl lysine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Accurately weigh a certain amount of milk powder, reduce it with sodium borohydride in sodium borate buffer solution; then use chloroform-methanol mixture (volume ratio 2:1) to remove fat and precipitate protein; after the precipitate is hydrolyzed with hydrochloric acid, Add a certain concentration of D4-CML solution, and extract and purify it through the MCX cation exchange column; the extract is concentrated by purging with nitrogen, and then redissolved in a certain volume of methanol-water solution (volume ratio is 4:1) to obtain the test solution; The test solution was eluted in a HILIC chromatographic column using methanol-water (both containing 2mM ammonium acetate and 0.1% formic acid) as the mobile phase for gradient elution, and CML was retained and separated from the sample matrix. Electrospray ionization source (ESI), positive ion scanning, and multiple reaction monitoring mode (MRM) were selected for tandem mass spectrometry analysis.

[0018] The CML cont...

Embodiment 2

[0020] Cut the fried dough sticks into pieces and pulverize them, and then reduce them with sodium borohydride in a sodium borate buffer solution; then use a chloroform-methanol mixture (2:1 by volume) to remove fat and precipitate proteins; after the precipitates are hydrolyzed with hydrochloric acid, add A certain concentration of D4-CML solution was extracted and purified by MCX cation exchange column; the extract was concentrated by nitrogen purging and then redissolved in a certain volume of methanol-water solution (volume ratio: 4:1) to obtain the solution to be tested; The solution was gradiently eluted in a HILIC chromatographic column with methanol-water (both containing 2mM ammonium acetate and 0.1% formic acid) as the mobile phase, and CML was retained and separated from the sample matrix. Electrospray ionization source (ESI), positive ion scanning, and multiple reaction monitoring mode (MRM) were selected for tandem mass spectrometry analysis. image 3 It is the mu...

Embodiment 3

[0023] Cut the French fries into pieces and pulverize them, and then reduce them with sodium borohydride in a sodium borate buffer solution; then use a chloroform-methanol mixture (2:1 by volume) to remove fat and precipitate proteins; after the precipitates are hydrolyzed with hydrochloric acid, Add a certain concentration of D4-CML solution, and extract and purify it through the MCX cation exchange column; the extract is concentrated by purging with nitrogen, and then redissolved in a certain volume of methanol-water solution (volume ratio is 4:1) to obtain the test solution; The test solution was eluted in a HII.IC chromatographic column using methanol-water (both containing 2mM ammonium acetate and 0.1% formic acid) as the mobile phase for gradient elution, and CML was retained and separated from the sample matrix. The electrospray ionization source (ESI), positive ion scanning, and multiple reaction monitoring mode (MRM) were selected for tandem mass spectrometry analysis....

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Abstract

The invention discloses a detecting method of carboxymethyl lysine (CML). The method is characterized by measuring the content of the CML in food adopting high performance liquid chromatography-tandem mass spectrometry in combination with an HILIC (Hydrophilic Interaction Liquid Chromatography) column for strong-polarity CML separation under a higher organic phase proportion. Crushed food passes through an MCX (Mixed-Mode Cation-Exchange) small column for being extracted and purified after being reduced by sodium borohydride and hydrolyzed by hydrochloric acid; sample liquor is subjected to gradient elution in the HILIC column by taking methanol-water (containing 2 mM ammonium acetate and 0.1% formic acid) as a mobile phase, so that CML is separated from a sample substrate; tandem mass spectrum analysis is carried out by using an electrospray ion source, positive ion scanning and a multi-reaction monitoring mode; quantitative analysis is carried out by using D4-CML as an internal standard substance, wherein analysis time is 12 minutes, and CML has an absorption peak at the retention time of 3.47 minutes. The detecting method disclosed by the invention is high in sensitivity, high in accuracy and good in precision, does not need to carry out derivatization onto samples, simplifies sample pre-treatment, shortens detecting time and saves detecting cost.

Description

technical field [0001] The invention relates to the technical field of detection of carboxymethyl lysine in food, specifically a kind of separation of carboxymethyl lysine combined with HILIC hydrophilic interaction liquid chromatography column, using high performance liquid chromatography-tandem mass spectrometry technology Method for the detection of carboxymethyllysine in food. Background technique [0002] Cooking methods that use the Maillard reaction to impart attractive colors and flavors to food have been admired for many years. However, in recent years, more and more studies have shown that some Maillard reaction products are harmful to the human body, and studies have shown that food-borne advanced glycation end products (AGEs) are associated with human diabetes, uremia, atherosclerosis The occurrence of diseases such as sclerosis and aging are closely related. Carboxymethyllysine (CML) is a kind of AGEs with high content and found earlier in food, and it is usua...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88
Inventor 黄轶群赖克强孙晓华牛丽红王靖
Owner SHANGHAI OCEAN UNIV
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