CN104391051A - Method for measuring nucleotide content in dairy product - Google Patents
Method for measuring nucleotide content in dairy product Download PDFInfo
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- CN104391051A CN104391051A CN201410576919.5A CN201410576919A CN104391051A CN 104391051 A CN104391051 A CN 104391051A CN 201410576919 A CN201410576919 A CN 201410576919A CN 104391051 A CN104391051 A CN 104391051A
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Abstract
The invention discloses a method for measuring nucleotide content in a dairy product and belongs to the field of food additives and detection methods thereof. The method comprises the following steps: (1), preparing stock solutions of GMP, UMP, CMP, IMP, AMP, wherein the concentrations of the stock solutions are 1 mg/ml, and preparing into solutions with the series normal concentrations of 10.0 [mu]g/ml, 20.0 [mu]g/ml, 30.0 [mu]g/ml, 40.0 [mu]g/ml and 50.0 [mu]g/ml, wherein the sample size is 10 [mu]l, measuring the peak heights of the solutions through a liquid chromatograph and calculating a regression equation; (2), weighing 10.0 g of a sample, dissolving the sample with 20 ml of warm water, putting into an ultrasonic bath for oscillation for 10 min, then taking out, transferring into a 50-ml volumetric flask, adding acetic acid for protein precipitation, after diluting, uniformly mixing, filtering through filtering paper, filtering through a filtering membrane, and measuring the peak height of the sample through the high performance liquid chromatograph; (3), according to a standard curve equation and the peak height value of the sample, obtaining the nucleotide content in the dairy product. According to the invention, the liquid chromatography is adopted to measure the nucleotide content in an infant formula milk powder, the method is simple and convenient to operate, the measurement precision and accuracy can meet analysis requirements of the dairy product, and the method can be used as a normal inspection method.
Description
Technical field
The present invention relates to a kind of assay method of dairy products nucleotide content, belong to food additives and detection method field thereof.
Background technology
Nucleotide is the composition forming nucleic acid, is mainly used in baby formula milk powder at present in dairy products.Five kinds are mainly contained as additive nucleotide---cytidine (CMP), urea pyrimidine nucleoside (UMP), adenosine (AMP), guanosine (GMP), inosine (IMP).Can also join in addition in beverage, bakery product and various major and minor food and drink.The main Physiological Function of nucleotide is energetic supersession, the protein synthesis of wide participation human body, has that the activity, activating cell function, the enhancing antibody that improve various enzyme are formed, the effect of enhancing immunologic function.Concerning human body, have good health-care effect, they can promote growing of neurocyte, be with " nerve growth factor " about or can as the nutritional labeling of nerve growth matter utilization.Promote that irony absorbs and affects lipid-metabolism, change Intestinal flora and provide intestinal mucosa complete required nutrition, reach the effect promoting immunologic function.Some researchs clinically find, add the babies ' formula milk powder of nucleotide, can reduce the generation of diarrhoea and promote growing compared with microsomia fetalis.
General not containing nucleotide in milk, in the baby formula milk powder national standard implemented at present, do not specify this index; But according to the special efficacy of nucleotide pair infant, all as important indicator in the standard of many enterprises particularly external individual proprietorship or joint and Chinese large-sized Dairy Enterprise, the detection method being applicable to dairy products must be studied and defined as early as possible for this reason.The detection method of current report mainly contains microbial method, ultraviolet spectrophotometry, high performance liquid chromatography etc., but major part is all the method formulated for nucleotide adjuvant, there is no suitable method for the sample as Dairy products complicated component.
Summary of the invention
The present invention aims to provide a kind of assay method that is easy, dairy products nucleotide content fast and accurately.
The assay method of dairy products nucleotide content of the present invention, comprises the following steps:
(1) standard curve making:
The preparation of standard solution: preparation GMP, UMP, GMP, IMP, AMP concentration is respectively the storing solution of 1mg/ml; Then be mixed with the solution that concentration is respectively the series standard concentration of 10.0,20.0,30.0,40.0,50.0 μ g/ml, sample size is respectively 10 μ l, measures its peak height and calculate regression equation and related coefficient by high performance liquid chromatograph.
(2) Specimen Determination:
Accurately take the sample of 10.0g, put into ultrasonic bath after dissolving with 20ml warm water and shake 10min taking-up, proceed in 50ml volumetric flask, add the acetic acid protein precipitation of 5ml 10%, pass through 0.45 μm of membrane filtration again after mixing Filter paper filtering after constant volume, measure its peak height by high performance liquid chromatograph.
(3) calculate:
According to the peak height value of typical curve equation and sample, the content of various nucleotide in dairy products can be obtained.
Preferably, the chromatographic condition of high performance liquid chromatograph of the present invention is: enlightening horse C18 post, 5 μm of 4.6*200mm; Column temperature 25 DEG C; Flow rate of mobile phase is 1.0ml/min; UV-detector wavelength 254nm.
Preferred, mobile phase of the present invention comprises two kinds, the first is the mixed liquor of the dipotassium hydrogen phosphate 40mL of the potassium dihydrogen phosphate 960ml+0.1mol/L of 0.01mol/L, and the second is the C of the dipotassium hydrogen phosphate+1mmol/L of the potassium dihydrogen phosphate+0.005mol/L of acetonitrile: 0.01mol/L
16h
36brN mixed liquor is the mixed liquor of 5:95.
The first mobile phase of the present invention is mainly used in measuring GMP, IMP, AMP; The second mobile phase is mainly used in measuring CMP, UMP.If measure nucleotide adjuvant, due to the interference not by dairy component, can only adopt the first mobile phase, get final product the nucleotide content that single-time measurement goes out five kinds of components.
After measured, assay method the method typical curve good linearity of the present invention, be proportionate between concentration and peak height, correlativity is good, can be used for external standard method and carries out quantitative measurement.Better, the recovery is at 92-97%, and average out to 94.6%, the coefficient of variation that sample replication is 8 times is 1.79% for the method recovery and Precision test result.
Assay method of the present invention adopts high effective liquid chromatography for measuring baby formula milk powder nucleotide content to be a comparatively ideal method, and this method is easy and simple to handle, is easy to grasp, and it measures the requirement that preci-sion and accuracy can meet dairy products analysis.Therefore, can be used as common detection methods application.
Embodiment
Embodiment one:
(1) preparation of standard solution: preparation GMP, UMP, GMP, IMP, AMP concentration is respectively the storing solution of 1mg/ml; Then the solution that concentration is respectively the series standard concentration of 10.0,20.0,30.0,40.0,50.0 μ g/ml is mixed with, sample size is respectively 10 μ l, be 1.0ml/min by high performance liquid chromatograph at enlightening horse C18 post, 5 μm of 4.6*200mm, column temperature 25 DEG C, flow rate of mobile phase, under UV-detector wavelength 254nm, mobile phase be the condition of the mixed liquor of the dipotassium hydrogen phosphate 40mL of the potassium dihydrogen phosphate 960ml+0.1mol/L of 0.01mol/L, record relation curve and the related coefficient of peak height and concentration.
Thus the relation curve obtaining peak height-concentration as shown in Table-1.
Table-1 typical curve
According to the regression equation that table-1 can calculate five kinds of nucleotide typical curves be:
CMP:Y=0.1754+0.0022X, related coefficient is R=0.9868;
UMP:Y=0.04868+0.0023X, related coefficient is R=0.9879;
GMP:Y=0.4380+0.0031X, related coefficient is R=0.9792;
IMP:Y=-0.1507+0.0025X, related coefficient is R=0.9799;
AMP:Y=-0.8532+0.0064X, related coefficient is R=0.9553;
Be proportionate between concentration and peak height, correlativity is good, can be used for external standard method and carries out quantitative measurement.
(2) Specimen Determination:
Choose the baby milk of five commercially available brands, accurately take the sample of 10.0g, put into ultrasonic bath after dissolving with 20ml warm water and shake 10min taking-up, proceed in 50ml volumetric flask, add the acetic acid protein precipitation of 5ml 10%, 0.45 μm of membrane filtration is passed through again after mixing Filter paper filtering after constant volume, by high performance liquid chromatograph at enlightening horse C18 post, 5 μm of 4.6*200mm, column temperature 25 DEG C, flow rate of mobile phase is 1.0ml/min, UV-detector wavelength 254nm, the first mobile phase is the mixed liquor of the dipotassium hydrogen phosphate 40mL of the potassium dihydrogen phosphate 960ml+0.1mol/L of 0.01mol/L, the second mobile phase is the C of the dipotassium hydrogen phosphate+1mmol/L of the potassium dihydrogen phosphate+0.005mol/L of acetonitrile: 0.01mol/L
16h
36brN mixed liquor is measure its peak height under the condition of the mixed liquor of 5:95.
(3) calculate:
According to the peak height value of typical curve equation and sample, the content of dairy products nucleotide can be obtained.As shown in table-2.
Table-2 nucleotide contents
From 5 sample determination results, twice parallel relative deviation all 5%/within.
Embodiment two: the recovery and reappearance are tested
(1) assay method of the present invention carries out 6 replications, and the result recovery is between 92%-97%, and average recovery rate is 94.6%, and its accuracy can meet the requirement measuring baby formula milk powder.
(2) assay method of the present invention carries out 8 parallel sample mensuration, and result statistics shows, the repeatability of measurement result is better, and the coefficient of variation 1.79% can meet the requirement adopting and measure this thuja acid content in baby formula milk powder.
Claims (3)
1. the assay method of dairy products nucleotide content, comprises the following steps:
(1) standard curve making:
The preparation of standard solution: preparation GMP, UMP, GMP, IMP, AMP concentration is respectively the storing solution of 1mg/ml; Then be mixed with the solution that concentration is respectively the series standard concentration of 10.0,20.0,30.0,40.0,50.0 μ g/ml, sample size is respectively 10 μ l, measures its peak height and calculate regression equation and related coefficient by high performance liquid chromatograph;
(2) Specimen Determination:
Accurately take the sample of 10.0g, put into ultrasonic bath after dissolving with 20ml warm water and shake 10min taking-up, proceed in 50ml volumetric flask, add the acetic acid protein precipitation of 5ml 10%, pass through 0.45 μm of membrane filtration again after mixing Filter paper filtering after constant volume, measure its peak height by high performance liquid chromatograph;
(3) calculate:
According to the peak height value of typical curve equation and sample, the content of various nucleotide in dairy products can be obtained.
2. the assay method of dairy products nucleotide content as claimed in claim 1, is characterized in that the chromatographic condition of described high performance liquid chromatograph is: enlightening horse C18 post, 5 μm of 4.6*200mm; Column temperature 25 DEG C; Flow rate of mobile phase is 1.0ml/min; UV-detector wavelength 254nm.
3. the assay method of dairy products nucleotide content as claimed in claim 1 or 2, it is characterized in that described mobile phase comprises two kinds, the first is the mixed liquor of the dipotassium hydrogen phosphate 40mL of the potassium dihydrogen phosphate 960ml+0.1mol/L of 0.01mol/L, and the second is the C of the dipotassium hydrogen phosphate+1mmol/L of the potassium dihydrogen phosphate+0.005mol/L of acetonitrile: 0.01mol/L
16h
36brN mixed liquor is the mixed liquor of 5:95.
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| CN201410576919.5A CN104391051A (en) | 2014-10-24 | 2014-10-24 | Method for measuring nucleotide content in dairy product |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593807A (en) * | 2018-06-12 | 2018-09-28 | 大连民族大学 | A method of purine content in marine product is detected based on high performance liquid chromatography |
| CN114935623A (en) * | 2022-05-18 | 2022-08-23 | 华谱科仪(大连)科技有限公司 | Method for detecting nucleotide in food |
-
2014
- 2014-10-24 CN CN201410576919.5A patent/CN104391051A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593807A (en) * | 2018-06-12 | 2018-09-28 | 大连民族大学 | A method of purine content in marine product is detected based on high performance liquid chromatography |
| CN114935623A (en) * | 2022-05-18 | 2022-08-23 | 华谱科仪(大连)科技有限公司 | Method for detecting nucleotide in food |
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Application publication date: 20150304 |