CN105738490A - Method for determining content of nucleotides in milk powder through adopting mixed acid as pH adjusting agent - Google Patents

Abstract

The invention relates to a method for determining the content of nucleotides in milk powder through adopting a mixed acid as a pH adjusting agent, and belongs to the technical field of food additive detection. The high performance liquid chromatography determination method of the nucleotides in the milk powder comprises the following steps: using a nucleotide standard substance to prepare a contrast substance solution; weighing a sample, adding distilled water, carrying out ultrasonic vibration shaking to make the nucleotides in the milk powder sample be dissolved in the distilled water; adjusting the pH value of the test solution to 3.2-3.4 by using nitric acid and hydrochloric acid to make proteins form a precipitate, filtering the solution to remove the precipitate, and adding water to the obtained filtrate to a constant volume in order to form a sample solution; determining the chromatographic peak areas of the nucleotides in the sample solution and the contrast substance solution through using high performance liquid chromatography; and calculating the content of the nucleotides in the sample by using an external standard technology according to the concentration and chromatographic peak areas of the nucleotides in the contrast substance solution, the chromatographic peak areas of the nucleotides of the sample solution and the mass of the sample. The method has the advantages of simplicity, convenient operation and high accuracy.

Description

Adopt the method that mixed acid measures milk powder nucleotide content as pH adjusting agent

Technical field

The present invention relates to a kind of method adopting mixed acid to measure milk powder nucleotide content as pH adjusting agent, belong to food additive detection technique field.

Background technology

Nucleotide is a kind of nutrient, infant growth is played an important role, so nucleotide can be used for dairy product additive.But, the demand of nucleotide is had certain limit by human body, not The more the better, and the content of milk nucleotide must be controlled by.In the milk powder standard of milk product manufacturing enterprise, the content of nucleotide being classified as important indicator, so controlling particularly important to the content of milk nucleotide, this is accomplished by corresponding technical support.The detection method of report mainly has microbial method, high performance capillary electrophoresis, high performance liquid chromatography etc. at present.Wherein high-efficient liquid phase chromatogram technique analysis speed is fast, efficiency is high, highly sensitive, it is most widely used.

High performance liquid chromatography detection milk powder nucleotide, must process sample before measuring, and wherein in sample, the separation of protein is most important.Related data is reported, during sample pretreating, it is common to employing adds a certain amount of acetic acid in sample solution or perchloric acid makes protein precipitation, then is separated off, and the method is unsatisfactory through test of many times proving effect.Sample solution adds acetic acid or perchloric acid makes protein precipitation, but protein precipitation thickness, solution layering is unclear, emulsion layer is had to occur, it is difficult to filter and washing, having a strong impact on the high performance liquid chromatography mensuration to nucleotide, this separation method not only disturbs the separation of chromatographic peak in nucleotide component, and has a strong impact on nucleotide accuracy of measurement.When therefore adopting high effective liquid chromatography for measuring milk nucleotide, how to make the protein in milk efficiently separate and do not affect the accuracy that nucleotide content measures, becoming the technical barrier that must solve.

Summary of the invention

It is an object of the invention to provide a kind of method adopting mixed acid to measure milk powder nucleotide content as pH adjusting agent, its method is simple, easy to operate, degree of accuracy is high, Separation of Proteins is complete, eliminates the interference that nucleotide is measured, and the mensuration for sample nucleotide provides good condition.

The method that employing mixed acid of the present invention measures milk powder nucleotide content as pH adjusting agent, comprises the steps:

(1) by nucleotide standard substance preparation reference substance solution；

(2) weigh milk powder sample, add distilled water in ultrasonic device, shake dissolution process, make sample nucleotide be dissolved in distilled water；

(3) being 3.2-3.4 by nitric acid and hydrochloric acid adjusting sample solution ph, make protein in sample solution form precipitation, be filtered to remove, filtrate water constant volume, as need testing solution；Nitric acid and hydrochloric acid volume ratio are 1:1；

(4) with high-performance liquid chromatogram determination need testing solution and reference substance solution nucleotide chromatographic peak area；

(5) according to reference substance solution nucleotide concentration and chromatographic peak area thereof, need testing solution nucleotide chromatographic peak area and sample mass, the content of sample nucleotide is calculated by external standard method.

Nucleotide content computing formula is as follows:

$\mathrm{Xi}=\frac{\mathrm{Ai}\×\mathrm{Cs}\×\mathrm{Vi}}{\mathrm{As}\×\mathrm{Mi}}\×100---\left(1\right)$

In formula:

X_iCertain nucleotide content, mg/100g in sample；

A_iNeed testing solution nucleotide chromatographic peak area；

A_sReference substance solution nucleotide chromatographic peak area；

C_sReference substance solution nucleotide concentration, mg/ml；

M_iWeigh sample mass, g.

Sample nucleotide total content (mg/100g) computing formula is as follows:

X=∑ X_i————————————(2)

Beneficial effects of the present invention is as follows:

Milk powder nucleotide high-performance liquid chromatogram determination method provided by the invention, in sample pretreating, it is 3.2-3.5 by nitric acid adjusting sample solution ph, make that method for protein isolation is simple, separate thoroughly, eliminate the interference that nucleotide is measured, in milk powder, 5 kinds of nucleotide all can obtain good must separation and accurate quantitative analysis, and its accuracy is high, precision is high.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is described further.

Embodiment 1

Milk powder nucleotide content detection:

1, the preparation of nucleotide standard reference material solution:

The preparation of 1.1 reference substance storing solutions: weigh Sigma company nucleotide aggregate sample (containing cytidylic acid, uridine monophosphate disodium, guanosine monophosphate disodium, adenylic acid, inosine monophosphate) standard substance 200mg (being accurate to 0.1mg) in 50ml volumetric flask, dissolves and constant volume with distilled water.

The preparation of 1.2 reference substance solution: draw reference substance storing solution 2.00ml in 25ml volumetric flask, use distilled water constant volume.

2, sample dissolution:

Weigh the commercially available milk powder sample 2.00g containing 5 kinds of nucleotide (cytidylic acid, uridine monophosphate disodium, guanosine monophosphate disodium, adenylic acid, inosine monophosphate) in 100ml beaker, add 64～66 DEG C of distilled water 30ml, fully shake up, in ultrasonic device ultrasonic 10 minutes.

3, the separation of sample protein:

PH meter indicates, and regulates pH value to 3.2-3.4 with nitric acid and hydrochloric acid solution (1+1), makes protein form precipitation, stand 15 minutes, with filter paper filtering in 50ml volumetric flask, a small amount of distilled water wash beaker and precipitation, merging filtrate and washing liquid, distilled water constant volume is standby as need testing solution.

4, the setting of chromatographic condition:

4.1 mobile phases: 0.01mol/L potassium phosphate buffer (pH5.0 includes 1.4mmol/L tetrabutyl Ammonium hydrogen sulfate): methanol=1000:40；

4.2 chromatographic columns: C18,4.6 × 250mm, HypersilODS2,5 μ；

4.3 detectors: UV-detector；

4.4 detection methods: chromatographic peak area outer marking quantitative method；

4.5 sampling volumes: 20 μ l；

4.6 chromatogram column temperatures: room temperature.

5, need testing solution and reference substance solution are measured, it is thus achieved that nucleotide chromatographic peak area.Measure 3 times respectively, take its meansigma methods by formula (1), formula (2) result of calculation.

The reliability demonstration of the inventive method:

1, determination of recovery rates:

Weigh a certain amount of milk powder sample being not added with nucleotide, add certain nucleotide a certain amount of, measure this kind of nucleotide total amount in sample, parallel laboratory test twice according to the method for embodiment 1, average.Measured quantity obtains this kind of nucleotide response rate compared with addition.Measurement result is as shown in table 1 below:

Table 1 determination of recovery rates table

Nucleotide title	Addition (μ g)	Measured quantity (μ g)	Response rate %
				Cytidylic acid	1870	1855	99.20
Uridine monophosphate disodium	1925	1893	98.34
				Guanosine monophosphate disodium	2014	1975	98.06
Adenylic acid	2103	2068	98.34
				Inosine monophosphate	1835	1791	97.60

2, the mensuration of precision:

The commercially available milk powder sample containing 5 kinds of nucleotide in Example 1, according to 5 nucleotide total contents of embodiment 1 method parallel assay, calculates precision.Measurement result is as shown in table 2 below:

Table 2 precision measures table

3, conclusion:

Milk powder nucleotide high-performance liquid chromatogram determination method provided by the invention, in sample pretreating, method for protein isolation is simple, separate thoroughly, eliminate the interference that nucleotide is measured, the efficient liquid-phase chromatography method of the present invention, 5 kinds of nucleotide in milk powder: adenylic acid, uridylic acid, cytidylic acid, inosinic acid and guanyl all can obtain good must separation and accurate quantitative analysis.The efficient liquid-phase chromatography method accuracy of the present invention is high, and precision is high.5 kinds of nucleotide response rate meansigma methodss are 98.31%, meet the requirement of quantitative analysis method, it is possible to for the mensuration of milk powder nucleotide content.

Claims (1)

1. one kind adopts the method that mixed acid measures milk powder nucleotide content as pH adjusting agent, it is characterised in that comprise the steps:

(1) by nucleotide standard substance preparation reference substance solution；

(2) weigh milk powder sample, add distilled water in ultrasonic device, shake dissolution process, make sample nucleotide be dissolved in distilled water；

(3) being 3.2-3.4 by nitric acid and hydrochloric acid adjusting sample solution ph, make protein in sample solution form precipitation, be filtered to remove, filtrate water constant volume, as need testing solution；Nitric acid and hydrochloric acid volume ratio are 1:1；

(4) with high-performance liquid chromatogram determination need testing solution and reference substance solution nucleotide chromatographic peak area；

(5) according to reference substance solution nucleotide concentration and chromatographic peak area thereof, need testing solution nucleotide chromatographic peak area and sample mass, the content of sample nucleotide is calculated by external standard method.