CN105738527B - The method that Glucosamine is measured using amino-acid analyzer - Google Patents
The method that Glucosamine is measured using amino-acid analyzer Download PDFInfo
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- CN105738527B CN105738527B CN201610140753.1A CN201610140753A CN105738527B CN 105738527 B CN105738527 B CN 105738527B CN 201610140753 A CN201610140753 A CN 201610140753A CN 105738527 B CN105738527 B CN 105738527B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The present invention relates to a kind of method that Glucosamine is measured using amino-acid analyzer, this method can be used for measuring the qualitative, quantitative of the Glucosamine in food, health food, drug or its raw material and the like.This method comprises the following steps：Amino-acid analyzer, chromatographic column cation exchange resin column and chromatography eluant mobile phase are provided, using defined chromatographiccondition, test solution and reference substance solution etc. is prepared, dependence test object is tested.By the implementation of the present invention, efficient, quick, the sensitive and accurate qualitative and quantitative analysis method of Glucosamine is completely established, good basis has been established in the application and detection of food, health food and drug for Glucosamine.
Description
Technical field
The present invention relates to the Glucosamines in a kind of food, health food, drug or its raw material and the like
Qualitativing quantitative measuring method.
Background technology
Glucosamine is a kind of can be the important nutrient to form cartilage cell, be strong with the substance synthesized in human body
The natural tissues composition of health articular cartilage is also that cartilage cell carries out the essential substrate of biosynthesis and metabolism.With year
The growth in age, the shortage of the Glucosamine in human body is increasingly severe, so that causing bone and joint diseases and osteoarthritis.Largely
Medical research shows：Glucosamine can help to repair and safeguard cartilage, and can stimulate the growth of cartilage cell, improve joint
Arthralgia is alleviated in activity.It has the effect of good to treatment rheumatism joint inflammation, is synthetic antibiotic and anticancer drug
Primary raw material, applies also for food, in cosmetics and feed addictive.
At present there are many detection method for the measure of aminoglucose sugared content in food, health food and drug, such as
Be measured in GB/T20365-2006 using liquid chromatography, in addition with spectrophotometry, liquid chromatogram derivatization method,
Gas-chromatography derivatization method, thin-layered chromatography, polarimetry, capillary electrophoresis and liquid matter analytic approach etc..When food, health food
And in drug compound component it is more when, the above method have a variety of bottleneck problems, such as it is complicated for operation, influence factor is more, specificity
It is not strong, sensitivity is low or is not easy to promote.
As Glucosamine is more and more widely used in health food and drug, establish a set of quick, sensitive, simple
Just, accurate analysis method is extremely urgent.
Invention content
Present invention aims at the Glucosamine provided in a kind of food, health food, drug or its raw material and its
The qualitativing quantitative measuring method of analog, and expect that this method is with quick, sensitive, easy, accurate etc. one or more
The advantages of a aspect.It has been unexpectedly discovered that said one or multiple mesh can be effectively realized using the method for the present invention
's.It finds and is accomplished the present invention is based on this.
For this purpose, first aspect present invention provides a kind of Glucosamine measured in product or the method for its analog,
This method is measured using amino-acid analyzer, is included the following steps：
(1) analytical instrument amino-acid analyzer is provided；
(2) chromatographic column cation exchange resin column, the length 10-30cm of chromatographic column, diameter 3-10mm are provided；
(3) chromatography eluant mobile phase is provided, which is selected from：PH2-7 citric acid/sodium citrates buffer solution, other
Acid/corresponding salt buffer or pH7-14 sodium hydrate buffer solutions；
(4) chromatographiccondition：Amino-acid analyzer flow rate pump 10-100ml/h；30-100 DEG C of chromatographic column column temperature；Reaction
30-150 DEG C of temperature；Chromatography time 3-60min；Detection wavelength 200-800nm；Sample size 2-50 μ l；
(5) preparation of test solution：The test sample for being equivalent to the Glucosamine containing 0.5mg-250mg is taken, it is accurately weighed,
It is placed in 250ml volumetric flasks, water 150ml is added to dilute, be ultrasonically treated 15min, scale be settled to after cooling, through 0.45 μm of micropore
Membrane filtration, subsequent filtrate is as test solution；
(6) preparation of reference substance solution：Aminoglucose hydrochloride reference substance 1-200mg is taken, it is accurately weighed, it is placed in 10ml appearances
In measuring bottle, it is diluted with water and is settled to scale as reference substance storing solution, take reference substance storing solution appropriate, be diluted to a concentration of 1-
1000 μ g/ml reference substance solutions；(it should be noted that can be used for using the reference substance solution that aminoglucose hydrochloride is prepared
The Glucosamine of other forms is measured for example for measuring Glucosamine Sulphate, as long as making simply to change in last calculate
Calculation).
(7) preparation of auxiliary material solution：If it is weighed in the product there are auxiliary material and its known composition in prescription ratio
Test sample considerable amount of auxiliary material is taken when being prepared with test solution, it is accurately weighed, it is placed in 250ml volumetric flasks, is diluted with water
And it is settled to scale；
(8) it under above-mentioned chromatographiccondition, is analyzed using amino-acid analyzer, respectively by reference substance solution, test sample
Solution and auxiliary material solution injecting chromatograph record chromatogram；Read amino Portugal in reference substance solution and test solution chromatogram
The retention time and peak area of grape sugar or its analog peak；Use reference substance solution concentration and its linear regression side of calculated by peak area
Journey in the case where the related coefficient of the equation of linear regression is more than 0.999%, carries out test sample using sample retention time
It is qualitative, test sample is quantified using equation of linear regression.
According to method of the first aspect of the present invention, wherein the product is selected from：Comprising Glucosamine or its analog
Food, health food, drug or prepare they Glucosamine or its similar to raw material.
According to method of the first aspect of the present invention, wherein the Glucosamine analog is selected from：Aminoglucose hydrochloride,
Glucosamines acylate and the ammonia such as Glucosamine Sulphate, Glucosamine sulfate potassium chloride, Glucosamine calcium citrate
Base glucose mineral hydrochlorate etc..
According to method of the first aspect of the present invention, wherein should not occur and reference substance solution chromatography in the auxiliary material chromatogram
The consistent chromatographic peak of figure main peak retention time.In other words, if it does, reference substance should not be interfered by occurring chromatographic peak in auxiliary material chromatogram
The main peak of solution chromatogram.
According to method of the first aspect of the present invention, wherein the length 20cm of the chromatographic column of the cation exchange resin column,
Diameter 5mm.
According to method of the first aspect of the present invention, wherein the citric acid/sodium citrate that the mobile phase is pH3~pH7 is delayed
Fliud flushing.
According to method of the first aspect of the present invention, wherein in the mobile phase of citric acid/sodium citrate composition, citric acid
A concentration of 0.01M~0.1M of root.A concentration of 0.025M~0.05M of optimization citric acid root.
According to method of the first aspect of the present invention, wherein the mobile phase is selected from：PH3.20 citric acid/sodium citrates buffer
Liquid, pH4.25 citric acid/sodium citrates buffer solution, pH6.45 citric acid/sodium citrates buffer solution, and combinations thereof.The flowing
The pH value of phase is obtained by adjusting both citric acid and sodium citrate relative quantity.
According to method of the first aspect of the present invention, wherein the amino-acid analyzer flow rate pump is 25~50ml/h.
According to method of the first aspect of the present invention, wherein 40-80 DEG C of the chromatographic column column temperature.
According to method of the first aspect of the present invention, wherein 120-140 DEG C of the reaction temperature.
According to method of the first aspect of the present invention, wherein the chromatography time 30-50min.
According to method of the first aspect of the present invention, wherein the Detection wavelength 530-570nm.
According to method of the first aspect of the present invention, wherein sample size 10-30 μ l.
According to method of the first aspect of the present invention, it wherein in step (5), takes and is equivalent to 20~100mg Glucosamines
Test sample.
According to method of the first aspect of the present invention, wherein in step (6), a concentration of 1-500 μ g/ml reference substance solutions are made
(such as being tested for linear dependence).
It has been found that the product or reference substance that are detected in Examples below 1-11 of the present invention, have known without hard
Resin acid or magnesium stearate, and measured through each embodiment, main composition chromatography in the test sample of total Test and reference substance chromatogram
The tailing factor (T) at peak meets the requirement of pharmacopeia in the range of 0.95~1.05.But generally, it is auxiliary for being added to
The product of material, such as the food comprising Glucosamine or its analog, health food, drug, it will usually add fraction of hard
Resin acid or magnesium stearate, to improve the physical property of product.The present inventor has found in the experiment of supplement, when respectively to embodiment
In 1-9, the 11 inspection product (including test sample and reference substance) during 0.5% magnesium stearate of addition, it has been found that, in gained chromatogram
The tailing factor (T) of main composition chromatographic peak is clearly to expect to overcome in the range of 1.21~1.37, the defects of in this method
, although this defect is acceptable reluctantly in the assay.The present inventor has found in the experiment further supplemented, divides
0.5% magnesium stearate of addition not into embodiment 1-9, the 11 inspection product (including test sample and reference substance), and in each implementation
0.02% (w/v) triethylamine is additionally added in mobile phase used in example, and (citric acid radical still keeps raw water to put down in mobile phase, and still logical
Amount adjusting pH value to the raw water for crossing citric acid or its salt is put down；Similarly hereinafter), it has been found that, main composition chromatographic peak in gained chromatogram
Tailing factor (T) is restored in the range of 0.95~1.05.The present inventor has found in the experiment further supplemented, from market
Food, health food or drug of 20 batches obtained comprising Glucosamine or its analog, find through the detection of other methods
Wherein contain stearic acid or magnesium stearate；When the method for using embodiment 1 measures this 20 batches inspection product, test solution chromatogram
The tailing factor (T) of middle main composition chromatographic peak is in the range of 1.23~1.32；And it uses instead and is additionally added in mobile phase
During 0.02% (w/v) triethylamine, when similarly measuring this 20 batches inspection product, main composition chromatographic peak drags in test solution chromatogram
The tail factor (T) is restored in the range of 0.95~1.05.But in complementary testing of the above using triethylamine, as general
When triethylamine is changed to ethylenediamine, the purpose of above-mentioned improvement tailing factor is but can not achieve, these are the result is that at all can not be from existing
Technology obtains what is explained.Therefore, in one embodiment of the invention, triethylamine is also added in the mobile phase.One
In a embodiment, concentration of the triethylamine in mobile phase is 0.01~0.05% (w/v), and preferred concentration is 0.02% (w/v).
Any technical characteristic possessed by any embodiment of either side or the either side of the present invention is equally applicable
Any embodiment of other any embodiments or other either sides, as long as they will not be conflicting, certainly mutual
Between where applicable, if necessary can individual features be made with appropriate modification.Make to various aspects of the present invention with feature into one below
The description of step.All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary
When offering expressed meaning and the inconsistent present invention, it is subject to the statement of the present invention.In addition, the various terms that use of the present invention and
Phrase has well known to a person skilled in the art general sense, nonetheless, the present invention remain desirable at this to these terms and
Phrase is described in more detail and explains, the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention
Subject to the meaning stated.
Description of the drawings
Fig. 1, Fig. 2 and Fig. 3 are typical reference substance spectrogram, test sample spectrogram and the moon obtained in a specific example respectively
Property auxiliary material spectrogram.
Specific embodiment
The present invention can be further described by the following examples, however, the scope of the present invention and unlimited
In following embodiments.One of skill in the art, can be with it is understood that under the premise of without departing substantially from the spirit and scope of the present invention
Various change and modification are carried out to the present invention.The present invention carries out the material and test method that are arrived used in experiment general
And/or specific description.Although to realize the present invention many materials used in purpose and operating method be it is known in the art that
But the present invention is still described in detail as far as possible herein.
Embodiment 1 measures the Glucosamine in product using amino-acid analyzer
Mobile phase for pH3.20 citric acid/sodium citrates buffer solution (citrate concentration 0.05M, in following article example not
0.05M is referred both to when its concentration is provided), amino-acid analyzer flow rate pump is 35ml/h, and chromatographic column column temperature is 45 DEG C, and reaction temperature is
135 DEG C, analysis time 50min, Detection wavelength 570nm, sample size is 20 μ l.Cation exchange resin column, chromatographic column
Length 20cm, diameter 5mm, if not otherwise indicated, chromatographic column specification is identical with this following instance.
The preparation of test solution：The test sample for being equivalent to 20mg Glucosamines is taken, it is accurately weighed, it is placed in 250ml appearances
In measuring bottle, water 150ml is added to dilute, ultrasonic extraction 15min is settled to scale after cooling, through 0.45 μm of filtering with microporous membrane, continues
Filtrate is as test solution.
It is prepared by reference substance solution：Aminoglucose hydrochloride reference substance 20mg is taken, it is accurately weighed, it is placed in 10ml volumetric flasks,
It is diluted with water and is settled to scale as reference substance storing solution, take reference substance storing solution appropriate, be diluted to a concentration of 1-1000 μ g/
(it is respectively 1,5,10,50,100,500 μ g/ml reference substance solutions to be actually diluted to concentration to ml reference substance solutions, and following instance is such as
It is not indicated otherwise, also same with this).
The preparation of negative auxiliary material solution：Auxiliary material is weighed in prescription ratio, it is accurately weighed, it is placed in 250ml volumetric flasks, adds water
It dilutes and is settled to scale.(this example test sample is the street drug that principal component is aminoglucose hydrochloride, and auxiliary material composition has obtained
Know, and carry out the preparation of negative auxiliary material solution).
Under above-mentioned chromatographic condition, analyzed using amino-acid analyzer and measure reference substance, test sample and auxiliary material, record chromatography
Figure, calculates the equation of linear regression of standard items mass concentration and peak area, and related coefficient is more than 0.999%, is retained using sample
Time is qualitative, and equation of linear regression quantifies.Do not occur chromatographic peak consistent with reference substance retention time in negative auxiliary material chromatogram.
Embodiment 2 measures the Glucosamine in product using amino-acid analyzer
Mobile phase be pH4.25 citric acid/sodium citrates buffer solution (citrate concentration 0.025M), amino-acid analyzer
Flow rate pump is 35ml/h, and chromatographic column column temperature is 60 DEG C, and reaction temperature is 120 DEG C, analysis time 40min, and Detection wavelength is
570nm, sample size are 20 μ l.
The preparation of test solution：The test sample for being equivalent to 50mg Glucosamines is taken, it is accurately weighed, it is placed in 250ml appearances
In measuring bottle, water 150ml is added to dilute, ultrasonic extraction 15min is settled to scale after cooling, through 0.45 μm of filtering with microporous membrane, continues
Filtrate is as test solution.
It is prepared by reference substance solution：Aminoglucose hydrochloride reference substance 20mg is taken, it is accurately weighed, it is placed in 10ml volumetric flasks,
It is diluted with water and is settled to scale as reference substance storing solution, take reference substance storing solution appropriate, be diluted to a concentration of 1-1000 μ g/
Ml reference substance solutions.
The preparation of negative auxiliary material solution：Auxiliary material is weighed in prescription ratio, it is accurately weighed, it is placed in 250ml volumetric flasks, adds water
It dilutes and is settled to scale.(this example test sample is the delicatessen food that principal component is Glucosamine Sulphate, and auxiliary material composition has obtained
Know, and carry out the preparation of negative auxiliary material solution).
Under above-mentioned chromatographic condition, analyzed using amino-acid analyzer and measure reference substance, test sample and auxiliary material, record chromatography
Figure, calculates the equation of linear regression of standard items mass concentration and peak area, and related coefficient is more than 0.999%, is retained using sample
Time is qualitative, and equation of linear regression quantifies.Do not occur chromatographic peak consistent with reference substance retention time in negative auxiliary material chromatogram.
Embodiment 3 measures the Glucosamine in product using amino-acid analyzer
Mobile phase is pH6.45 citric acid/sodium citrates buffer solution (citrate concentration 0.03M), and amino-acid analyzer pumps
Flow velocity is 35ml/h, and chromatographic column column temperature is 80 DEG C, and reaction temperature is 130 DEG C, analysis time 30min, and Detection wavelength is
570nm, sample size are 20 μ l.
The preparation of test solution：The test sample for being equivalent to 100mg Glucosamines is taken, it is accurately weighed, it is placed in 250ml appearances
In measuring bottle, water 150ml is added to dilute, ultrasonic extraction 15min is settled to scale after cooling, through 0.45 μm of filtering with microporous membrane, continues
Filtrate is as test solution.
It is prepared by reference substance solution：Aminoglucose hydrochloride reference substance 20mg is taken, it is accurately weighed, it is placed in 10ml volumetric flasks,
It is diluted with water and is settled to scale as reference substance storing solution, take reference substance storing solution appropriate, be diluted to a concentration of 1-1000 μ g/
Ml reference substance solutions.
The preparation of negative auxiliary material solution：Auxiliary material is weighed in prescription ratio, it is accurately weighed, it is placed in 250ml volumetric flasks, adds water
It dilutes and is settled to scale.(this example test sample be principal component be Glucosamine sulfate potassium chloride analysis of functional food sold, auxiliary material group
Into having known, and carry out the preparation of negative auxiliary material solution).
Under above-mentioned chromatographic condition, analyzed using amino-acid analyzer and measure reference substance, test sample and auxiliary material, record chromatography
Figure, calculates the equation of linear regression of standard items mass concentration and peak area, and related coefficient is more than 0.999%, is retained using sample
Time is qualitative, and equation of linear regression quantifies.Do not occur chromatographic peak consistent with reference substance retention time in negative auxiliary material chromatogram.
Embodiment 4 measures the Glucosamine in product using amino-acid analyzer
(two kinds of buffer solutions are with volume ratio 1 for pH3.20 and pH6.45 citric acid/sodium citrates buffer solution for mobile phase:1 ratio
Mixing, citrate concentration 0.04M), amino-acid analyzer flow rate pump is 40ml/h, and chromatographic column column temperature is 80 DEG C, reaction temperature
It it is 135 DEG C, analysis time 30min, Detection wavelength 570nm, sample size is 20 μ l.
The preparation of test solution：The test sample for being equivalent to 100mg Glucosamines is taken, it is accurately weighed, it is placed in 250ml appearances
In measuring bottle, water 150ml is added to dilute, ultrasonic extraction 15min is settled to scale after cooling, through 0.45 μm of filtering with microporous membrane, continues
Filtrate is as test solution.
It is prepared by reference substance solution：Aminoglucose hydrochloride reference substance 20mg is taken, it is accurately weighed, it is placed in 10ml volumetric flasks,
It is diluted with water and is settled to scale as reference substance storing solution, take reference substance storing solution appropriate, be diluted to a concentration of 1-1000 μ g/
Ml reference substance solutions.
The preparation of negative auxiliary material solution：Auxiliary material is weighed in prescription ratio, it is accurately weighed, it is placed in 250ml volumetric flasks, adds water
It dilutes and is settled to scale.(this example test sample be principal component be Glucosamine calcium citrate analysis of functional food sold, auxiliary material group
Into having known, and carry out the preparation of negative auxiliary material solution).
Under above-mentioned chromatographic condition, analyzed using amino-acid analyzer and measure reference substance, test sample and auxiliary material, record chromatography
Figure, calculates the equation of linear regression of standard items mass concentration and peak area, and related coefficient is more than 0.999%, is retained using sample
Time is qualitative, and equation of linear regression quantifies.Do not occur chromatographic peak consistent with reference substance retention time in negative auxiliary material chromatogram.
Embodiment 5 measures the Glucosamine in product using amino-acid analyzer
Mobile phase is pH6.45 citric acid/sodium citrates buffer solution (citrate concentration 0.05M), and amino-acid analyzer pumps
Flow velocity is 30ml/h, and chromatographic column column temperature is 80 DEG C, and reaction temperature is 135 DEG C, analysis time 30min, and Detection wavelength is
550nm, sample size are 20 μ l.
The preparation of test solution：The test sample for being equivalent to 100mg Glucosamines is taken, it is accurately weighed, it is placed in 250ml appearances
In measuring bottle, water 150ml is added to dilute, ultrasonic extraction 15min is settled to scale after cooling, through 0.45 μm of filtering with microporous membrane, continues
Filtrate is as test solution.
It is prepared by reference substance solution：Aminoglucose hydrochloride reference substance 20mg is taken, it is accurately weighed, it is placed in 10ml volumetric flasks,
It is diluted with water and is settled to scale as reference substance storing solution, take reference substance storing solution appropriate, be diluted to a concentration of 1-1000 μ g/
Ml reference substance solutions.
The preparation of negative auxiliary material solution：Auxiliary material is weighed in prescription ratio, it is accurately weighed, it is placed in 250ml volumetric flasks, adds water
It dilutes and is settled to scale.(this example test sample is the delicatessen food that principal component is aminoglucose hydrochloride, and auxiliary material composition does not obtain
Know, the preparation without negative auxiliary material solution).
Under above-mentioned chromatographic condition, analyzed using amino-acid analyzer and measure reference substance, test sample and auxiliary material, record chromatography
Figure, calculates the equation of linear regression of standard items mass concentration and peak area, and related coefficient is more than 0.999%, is retained using sample
Time is qualitative, and equation of linear regression quantifies.Do not occur chromatographic peak consistent with reference substance retention time in negative auxiliary material chromatogram.
Embodiment 6 measures the Glucosamine in product using amino-acid analyzer
(two kinds of buffer solutions are with volume ratio 1 for pH4.25 and pH6.45 citric acid/sodium citrates buffer solution for mobile phase:1 ratio
Mixing), amino-acid analyzer flow rate pump is 50ml/h, and chromatographic column column temperature is 40 DEG C, and reaction temperature is 135 DEG C, and analysis time is
30min, Detection wavelength 570nm, sample size are 30 μ l.
The preparation of test solution：The test sample for being equivalent to 100mg Glucosamines is taken, it is accurately weighed, it is placed in 250ml appearances
In measuring bottle, water 150ml is added to dilute, ultrasonic extraction 15min is settled to scale after cooling, through 0.45 μm of filtering with microporous membrane, continues
Filtrate is as test solution.
It is prepared by reference substance solution：Aminoglucose hydrochloride reference substance 20mg is taken, it is accurately weighed, it is placed in 10ml volumetric flasks,
It is diluted with water and is settled to scale as reference substance storing solution, take reference substance storing solution appropriate, be diluted to a concentration of 1-1000 μ g/
Ml reference substance solutions.
The preparation of negative auxiliary material solution：Auxiliary material is weighed in prescription ratio, it is accurately weighed, it is placed in 250ml volumetric flasks, adds water
It dilutes and is settled to scale.(this example test sample is the analysis of functional food sold that principal component is aminoglucose hydrochloride, and auxiliary material forms
Know, and carry out the preparation of negative auxiliary material solution).
The result shows that：Occur the chromatographic peak consistent with reference substance retention time, and the peak peak in test solution chromatogram
Type is symmetrical, and theoretical cam curve is more than 2000, in negative auxiliary material sample solution chromatogram, nothing and aminoglucose hydrochloride reference substance
The consistent chromatographic peak of retention time occurs, i.e., without matrix interference.Reference substance spectrogram, sample spectrogram and negative auxiliary material spectrogram see Fig. 1,
Fig. 2 and Fig. 3.
Embodiment 7 measures the Glucosamine in product using amino-acid analyzer
(two kinds of buffer solutions are with volume ratio 2 for pH4.25 and pH6.45 citric acid/sodium citrates buffer solution for mobile phase:1 ratio
Mixing), amino-acid analyzer flow rate pump is 25ml/h, and chromatographic column column temperature is 80 DEG C, and reaction temperature is 140 DEG C, and analysis time is
30min, Detection wavelength 530nm, sample size are 10 μ l.
The preparation of test solution：Aminoglucose hydrochloride bulk pharmaceutical chemicals 100mg is taken as test sample, it is accurately weighed, it is placed in
In 250ml volumetric flasks, water 150ml is added to dilute, ultrasonic extraction 15min is settled to scale after cooling, through 0.45 μm of miillpore filter
Filtering, subsequent filtrate is as test solution.
It is prepared by reference substance solution：Aminoglucose hydrochloride reference substance 20mg is taken, it is accurately weighed, it is placed in 10ml volumetric flasks,
It is diluted with water and is settled to scale as reference substance storing solution, take reference substance storing solution appropriate, be diluted to a concentration of 1-1000 μ g/
Ml reference substance solutions.
(this example test sample is bulk pharmaceutical chemicals, no auxiliary material, does not in fact carry out the preparation of negative auxiliary material solution).
It under above-mentioned chromatographic condition, is analyzed using amino-acid analyzer and measures reference substance, test sample records chromatogram, meter
The equation of linear regression of standard items mass concentration and peak area is calculated, related coefficient is more than 0.999%, is determined using sample retention time
Property, equation of linear regression quantify.Do not occur chromatographic peak consistent with reference substance retention time in negative auxiliary material chromatogram.
Above example 1-7, the retention time by main composition peak in reference substance solution and test solution chromatogram are (each
Two retention times are consistent in example), can qualitative analysis easily be carried out to test sample.In addition, pass through reference substance solution
Gained equation of linear regression can be easily computed the content of target component in test sample, measure the confession in 7 examples
Test product object content is consistent with its product indicia content (in the range of the 98~102% of product indicia content；This field leads to
Often requirement is in the range of 90~110%).
By the implementation of the present invention, the efficient of Glucosamine is established perfectly, it is quickly, sensitive and accurately qualitative fixed
Analysis method has established good basis for application and detection of the Glucosamine in food, health food and drug.
Embodiment 8, linear dependence experiment
Aminoglucose hydrochloride reference substance 20mg is taken, it is accurately weighed, it is placed in 10ml volumetric flasks, is diluted with water and is settled to
Scale takes reference substance storing solution appropriate, it is respectively 1,5,10,50,100,500 μ g/ to be diluted to concentration as reference substance storing solution
Ml reference substance solutions, respectively 20 μ l of sample introduction analyses.It the results are shown in Table 1
Table 1：Linear dependence tests measurement result
| Number | 1 | 2 | 3 | 4 | 5 | 6 |
| Concentration (μ g/ml) | 1 | 5 | 10 | 50 | 100 | 500 |
| Peak area | 33.303 | 158.574 | 327.894 | 1711.366 | 3493.956 | 16904.270 |
Equation of linear regression：Y=33.80681x+19.00468, coefficient R2=0.99997
Result of the test shows that in 1-500 μ g/ml concentration ranges solution concentration value and peak area value linear relationship are good.
Embodiment 9, precision test
10 μ g/ml reference substance solutions of Example 8,6 needle of continuous sample introduction calculate its precision, the results are shown in Table 2.
Table 2：Precision test measurement result
Result of the test shows that precision is very high, can meet measure requirement completely.
10 detection limit of embodiment is tested
Precision measures 10 μ g/ml reference substance solution 0.5ml of embodiment 8, is diluted with water and is settled to 10ml, obtains a concentration of
0.5 μ g/ml reference substance solutions, 20 μ l of sample introduction analyses calculate quantitative limit (LOQ) according to S/N=10, calculate and detect according to S/N=3
It limits (LOD), the quantitative limit (LOQ) of method is 0.52 μ g/ml, and the detection limit (LOD) of method is 0.16 μ g/ml.
11 TIANZHU XINGNAO Capsul of embodiment is tested
By low middle and high concentration, it is appropriate to weigh aminoglucose hydrochloride for precision respectively, is added to corresponding blank auxiliary (embodiment
Used in 1) in, it is placed in 250ml volumetric flasks, water 150ml is added to dilute, ultrasonic extraction 15min is settled to scale after cooling, pass through
0.45 μm of filtering with microporous membrane, subsequent filtrate is as determination of recovery rates solution.Result of calculation is shown in Table 3.
Table 3：TIANZHU XINGNAO Capsul measurement result
By the present invention implementation, establish perfectly the efficient, quick, sensitive of Glucosamine and accurately it is qualitative fixed
Analysis method has established good basis for application and detection of the Glucosamine in food, health food and drug.
Claims (11)
1. the method for the Glucosamine or its analog in measure product, this method are measured using amino-acid analyzer,
Include the following steps：
(1) analytical instrument amino-acid analyzer is provided；
(2) chromatographic column cation exchange resin column, the length 10-30cm of chromatographic column, diameter 3-10mm are provided；
(3) chromatography eluant mobile phase is provided, which is selected from：PH3.20 citric acid/sodium citrates buffer solution, pH4.25 lemons
Lemon acid/sodium citrate buffer solution, pH6.45 citric acid/sodium citrates buffer solution, and combinations thereof, a concentration of 0.025M of citrate
~0.05M, and also added with triethylamine in the mobile phase, the concentration in mobile phase is 0.02% (w/v)；
(4) chromatographiccondition：Amino-acid analyzer flow rate pump 10-100ml/h；30-100 DEG C of chromatographic column column temperature；Reaction temperature
30-150℃；Chromatography time 3-60min；Detection wavelength 200-800nm；Sample size 2-50 μ l；
(5) preparation of test solution：The test sample for being equivalent to the Glucosamine containing 0.5mg-250mg is taken, it is accurately weighed, it is placed in
In 250ml volumetric flasks, water 150ml is added to dilute, be ultrasonically treated 15min, scale be settled to after cooling, through 0.45 μm of miillpore filter
Filtering, subsequent filtrate is as test solution；
(6) preparation of reference substance solution：Aminoglucose hydrochloride reference substance 1-200mg is taken, it is accurately weighed, it is placed in 10ml volumetric flasks
In, it is diluted with water and is settled to scale as reference substance storing solution, take reference substance storing solution appropriate, be diluted to a concentration of 1-1000
μ g/ml reference substance solutions；
(7) preparation of auxiliary material solution：If it is weighed and is supplied in prescription ratio there are auxiliary material and its known composition in the product
Test sample solution takes the considerable amount of auxiliary material of test sample when preparing, accurately weighed, is placed in 250ml volumetric flasks, is diluted with water and fixed
Hold to scale；
(8) it under above-mentioned chromatographiccondition, is analyzed using amino-acid analyzer, respectively by reference substance solution, test solution
And auxiliary material solution injecting chromatograph, record chromatogram；Read Glucosamine in reference substance solution and test solution chromatogram
Or the retention time and peak area at its analog peak；Using reference substance solution concentration and calculated by peak area its equation of linear regression,
In the case where the related coefficient of the equation of linear regression is more than 0.999%, test sample is determined using sample retention time
Property, test sample is quantified using equation of linear regression,
Wherein,
The product is selected from：Food, health food or drug comprising Glucosamine or its analog, containing hard in the product
Resin acid or magnesium stearate, the aminoglucose sugar analogue are selected from：Aminoglucose hydrochloride, Glucosamine Sulphate, amino Portugal
Grape sugar potassium sulfate salt, Glucosamine calcium citrate.
2. the method according to claim 1, should not occur in the auxiliary material chromatogram and reference substance solution chromatogram main peak retains
Time consistency chromatographic peak.
3. the method according to claim 1, the length 20cm of the chromatographic column of the cation exchange resin column, diameter 5mm.
4. the method according to claim 1, wherein the amino-acid analyzer flow rate pump is 25~50ml/h.
5. the method according to claim 1, wherein the chromatographic column column temperature is 40-80 DEG C.
6. the method according to claim 1, wherein the reaction temperature is 120-140 DEG C.
7. the method according to claim 1, wherein the chromatography time is 30-50min.
8. the method according to claim 1, the Detection wavelength is 530-570nm.
9. the method according to claim 1, the sample size 10-30 μ l.
10. in the method according to claim 1, wherein step (5), the test sample for being equivalent to 20~100mg Glucosamines is taken.
11. in the method according to claim 1, wherein step (6), a concentration of 1-500 μ g/ml reference substance solutions are made.
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