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A high-performance liquid chromatography detection method for nucleotides in food

A high-performance liquid chromatography and nucleotide technology, applied in the field of food detection, can solve the problems of expensive instruments, large matrix interference, poor reproducibility of measurement results, etc., and achieve the effect of wide application range and good linear relationship.

Inactive Publication Date: 2018-08-07
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Poor reproducibility of ion exchange chromatography and capillary electrophoresis
Liquid chromatography-mass spectrometry has the disadvantages of expensive instruments, large matrix interference, and high detection costs

Method used

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  • A high-performance liquid chromatography detection method for nucleotides in food
  • A high-performance liquid chromatography detection method for nucleotides in food
  • A high-performance liquid chromatography detection method for nucleotides in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 detection method

[0069] 1. Sample pretreatment

[0070] Weigh 0.5g of the sample into a 50mL polytetrafluoroethylene centrifuge tube, add 10mL of aqueous solution, adjust the pH between 4 and 5 with glacial acetic acid, vortex for 5 minutes, add 40mL of ethanol solution, and centrifuge at 10000r / min after vortexing for 5min Wash with ethanol solution for 10 minutes, spin dry at 60°C, dilute to volume with 5-10mL water, take 1mL and pass through a 0.22μm filter membrane in a sample bottle for analysis on the machine.

[0071] 2. Perform high performance liquid chromatography detection

[0072] (1) Chromatographic column: SB-AQ column.

[0073] (2) Mobile phase: tetrabutylammonium bisulfate.

[0074] (3) The detection wavelength of liquid chromatography is 254nm.

[0075] (4) The column temperature is between 60 and 70°C.

[0076] (5) The time for the sample to be put on the machine is 60 to 240 hours after the sample is prepared, and the sample needs ...

Embodiment 2

[0078] The selection and optimization of the sample pretreatment method of embodiment 2

[0079] The selection and optimization of the influencing factors of specific pretreatment methods are as follows:

[0080] 1. Selection of Precipitating Acid

[0081] Comparing different acids as protein precipitating agents, it is found that after different acids are precipitated, the impurities that appear are not the same (as attached Figures 11 to 14 shown). Oxalic acid has strong interference at the peak position of CMP, followed by hydrochloric acid. The peak time of formic acid and acetic acid impurities is basically the same, but formic acid has more impurities near CMP. It shows that it is better to use acetic acid as precipitant.

[0082] 2. The influence of ethanol and acetic acid (acetic acid) coprecipitation

[0083] as attached figure 1 and 2 As shown, comparing the direct application of acetic acid precipitation to constant volume and the co-precipitation of acetic a...

Embodiment 3

[0088] The optimization of embodiment 3 assay conditions

[0089] 1. Selection of chromatographic column: After comparing SB-AQ, TC, XDB, T3, amide and other C18 chromatographic columns, it was found that SB-AQ has better separation and stability of nucleotides, so SB-AQ column was selected for Determination.

[0090] 2. Selection of mobile phase

[0091] Adding the ion-pair reagent tetrabutylammonium bisulfate to the mobile phase can better separate the five nucleotides and impurities. The effect of different concentrations of ion-pairing reagents on the retention time of UMP, AMP and IMP is within ±2min, while the effect on the retention time of CMP and AMP is within ±1min, so it can be effective by adjusting the ion-pairing reagent concentration or other conditions. Separation of five nucleotides as well as impurities. When the concentration of tetrabutylammonium bisulfate was 0.6g / L, the resolution of 5 kinds of nucleotides was the best, and the resolution of IMP and th...

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Abstract

The invention discloses an HPLC (high performance liquid chromatography) detection method for nucleotide in food. The method comprises steps as follows: firstly, pretreatment of a sample: a to-be-detected sample is weighed, water is added, the pH is adjusted to 4-5 with acetic acid, vortex treatment is performed for 5-10 min, an ethanol solution is added, centrifugation is performed at 8,000-12,000 r / min for 5-15 min after vortex treatment for 5-10 min, the ethanol solution is used for washing, spin-drying is performed at 55-70 DEG C, volume metering is performed with water, and 0.5-2 mL of the solution is filtered by a 0.22 mu m filter film to enter a sample injection flask for analysis; then HPLC detection is performed. Acetic acid and ethyl alcohol are used as coprecipitators, an SB-AQ column and tetrabutyl ammonium bisulfate are used for separating and detecting nucleotide, the average recovery rate of the method is 82.44%-98.92%, the relative standard deviation is 0.60%-2.27%, and the method can be used for detecting nucleotide in milk, rice flour and milk rice flour simultaneously, is suitable for various products and wide in application range and has bright application prospect.

Description

technical field [0001] The invention belongs to the technical field of food detection. More specifically, it relates to a high-performance liquid chromatography detection method for nucleotides in food. Background technique [0002] Nucleotide is a component of nucleic acid, which is composed of nitrogen-containing bases, ribose or deoxyribose, and phosphoric acid. Nucleotides are widely distributed in the human body and have various biological functions. The physiological functions of nucleotides are inseparable from the important structure of five-carbon sugars, namely nucleosides. Due to the large difference in the concentration of nucleotides in milk and breast milk, breast milk contains a variety of nucleosides and nucleotides, but milk generally does not contain them, so the addition of nucleotides to dairy products is currently mainly used for infant formula In milk powder. There are five main types of nucleotides added to infant formula: cytosine nucleotide (cyti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
CPCG01N30/02G01N2030/027
Inventor 易蓉韦晓群翁文川李荀王志元郑璇陈文锐吴志航张玉文黄维龙杜斯航
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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