CN105738527A - Method for glucosamine determination by amino acid analyzer - Google Patents
Method for glucosamine determination by amino acid analyzer Download PDFInfo
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- CN105738527A CN105738527A CN201610140753.1A CN201610140753A CN105738527A CN 105738527 A CN105738527 A CN 105738527A CN 201610140753 A CN201610140753 A CN 201610140753A CN 105738527 A CN105738527 A CN 105738527A
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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
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Abstract
The invention relates to a method for glucosamine determination by an amino acid analyzer.The method can be used for qualitative and quantitative determination of glucosamine and analogues thereof in foods, healthcare foods, medicines or raw materials thereof.The method includes steps: providing the amino acid analyzer, a cation exchange resin chromatographic column and a mobile phase for elution chromatography, preparing a test solution, a reference solution and the like according to specified chromatographic analysis conditions, and testing related test objects.By implementation of the method for glucosamine determination by the amino acid analyzer, a glucosamine qualitative and quantitative analysis method which is efficient, rapid, sensitive and accurate is established completely to lay a good foundation for detection and application of the glucosamine to foods, healthcare foods and medicines.
Description
Technical field
The present invention relates to the qualitativing quantitative measuring method of glucosamine in a kind of food, health food, medicine or its raw material and the like.
Background technology
Glucosamine be a kind of can the material of synthesis in human body, be the important nutrient forming chondrocyte, be the natural tissues composition of healthy articular cartilage, be also that chondrocyte carries out the requisite substrate of biosynthesis and metabolism.With advancing age, the shortage of the glucosamine in human body is increasingly severe, so that causing joint disease and osteoarthritis.A large amount of medical researches show: glucosamine can help reparation and safeguard cartilage, and can stimulate the growth of chondrocyte, improves joint motion, alleviates arthralgia.Treatment rheumatism joint inflammation is had good curative effect by it, is the primary raw material of synthetic antibiotic and cancer therapy drug, applies also for food, cosmetics and in feed additive.
There is multiple detection method at present for the mensuration of glucosamine content in food, health food and medicine, liquid chromatography is such as adopted to be measured in GB/T20365-2006, in addition with spectrophotography, liquid chromatograph derivatization method, gas chromatogram derivatization method, thin layer chromatography, polarimetry, high performance capillary electrophoresis and liquid matter analytic process etc..When component composite in food, health food and medicine is more, said method has multiple bottleneck problem, as many in complicated operation, influence factor, specificity is strong, sensitivity is low or not easily popularization etc..
Along with glucosamine is increasingly widely applied in health food and medicine, set up a set of quick, sensitive, easy, accurately analyze method extremely urgent.
Summary of the invention
Present invention aim at providing the qualitativing quantitative measuring method of glucosamine in a kind of food, health food, medicine or its raw material and the like, and expect the advantage that this method has one or more aspect such as quick, sensitive, easy, accurate.It has been unexpectedly discovered that use the inventive method can be effectively realized said one or multiple purpose.The present invention finds based on this and is accomplished.
For this, first aspect present invention provides a kind of method of glucosamine measured in goods or its analog, and the method uses amino-acid analyzer to be measured, and comprises the steps:
(1) analytical tool 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) providing chromatography eluant mobile phase, this mobile phase is selected from: pH2-7 citric acid/sodium citrate buffer, other acid/corresponding salt buffer or pH7-14 sodium hydrate buffer solution；
(4) chromatographiccondition: amino-acid analyzer flow rate pump 10-100ml/h；Chromatographic column column temperature 30-100 DEG C；Reaction temperature 30-150 DEG C；Chromatography time 3-60min；Detection wavelength 200-800nm；Sample size 2-50 μ l；
(5) preparation of need testing solution: take the test sample be equivalent to containing 0.5mg-250mg glucosamine, accurately weighed, it is placed in 250ml volumetric flask, the 150ml that adds water dilutes, supersound process 15min, being settled to scale after cooling, through the filtering with microporous membrane of 0.45 μm, subsequent filtrate is as need testing solution；
(6) preparation of reference substance solution: take glucosamine hydrochloride reference substance 1-200mg, accurately weighed, it is placed in 10ml volumetric flask, dilute with water is also settled to scale as reference substance storing solution, taking reference substance storing solution appropriate, being diluted to concentration is 1-1000 μ g/ml reference substance solution；(it should be noted that use the reference substance solution of glucosamine hydrochloride preparation to may be used for the glucosamine measuring other form such as measuring glucosamine sulfate, as long as simply converting when in the end calculating).
(7) preparation of adjuvant solution: if there is adjuvant and its composition known in described goods, taken test sample considerable amount of adjuvant when preparing then is weighed with need testing solution in prescription ratio, accurately weighed, it is placed in 250ml volumetric flask, dilute is also settled to scale；
(8) under above-mentioned chromatographiccondition, adopt amino-acid analyzer analysis, respectively by reference substance solution, need testing solution and adjuvant solution injecting chromatograph, record chromatogram；Read retention time and the peak area at glucosamine or its analog peak in reference substance solution and need testing solution chromatogram；Use reference substance solution concentration and its equation of linear regression of calculated by peak area, when the correlation coefficient of this equation of linear regression is more than 0.999%, utilize sample retention time to carry out qualitative to test sample, utilize equation of linear regression that test sample is carried out quantitatively.
Method according to a first aspect of the present invention, wherein said goods are selected from: comprise glucosamine or the food of its analog, health food, medicine, or prepare their glucosamine or its analog raw material.
Method according to a first aspect of the present invention, wherein said aminoglucose sugar analogue is selected from: glucosamine acylate and the glucosamine inorganic acid salts etc. such as glucosamine hydrochloride, glucosamine sulfate, Glucosamine sulfate potassium chloride, glucosamine Citric acid calcium.
, should there is not chromatographic peak consistent with reference substance solution chromatogram main peak retention time in method according to a first aspect of the present invention in wherein said adjuvant chromatogram.In other words, if it does, adjuvant chromatogram occurring, chromatographic peak should not disturb the main peak of reference substance solution chromatogram.
Method according to a first aspect of the present invention, the length 20cm of the chromatographic column of wherein said cation exchange resin column, diameter 5mm.
Method according to a first aspect of the present invention, wherein said mobile phase is the citric acid/sodium citrate buffer of pH3～pH7.
Method according to a first aspect of the present invention, in the mobile phase of wherein said citric acid/sodium citrate composition, citrate concentration is 0.01M～0.1M.Optimization citric acid root concentration is 0.025M～0.05M.
Method according to a first aspect of the present invention, wherein said mobile phase is selected from: pH3.20 citric acid/sodium citrate buffer, pH4.25 citric acid/sodium citrate buffer, pH6.45 citric acid/sodium citrate buffer and combination thereof.The pH value of described mobile phase obtains by regulating citric acid and sodium citrate relative quantity.
Method according to a first aspect of the present invention, wherein said amino-acid analyzer flow rate pump is 25～50ml/h.
Method according to a first aspect of the present invention, wherein said chromatographic column column temperature 40-80 DEG C.
Method according to a first aspect of the present invention, wherein said reaction temperature 120-140 DEG C.
Method according to a first aspect of the present invention, wherein said chromatography time 30-50min.
Method according to a first aspect of the present invention, wherein said detection wavelength 530-570nm.
Method according to a first aspect of the present invention, wherein sample size 10-30 μ l.
Method according to a first aspect of the present invention, wherein in step (5), takes the test sample being equivalent to 20～100mg glucosamine.
Method according to a first aspect of the present invention, wherein in step (6), making concentration is 1-500 μ g/ml reference substance solution (such as linear dependence test).
Have been found that, the goods detected in Examples below 1-11 of the present invention or reference substance, know all without stearic acid or magnesium stearate, and measure through each embodiment, in the test sample of total Test and reference substance chromatogram, the tailing factor (T) of main composition chromatographic peak is all in 0.95～1.05 scope, meets the requirement of pharmacopeia.But, generally, for the addition of the goods of adjuvant, for instance comprise glucosamine or the food of its analog, health food, medicine, it will usually add fraction of stearic acid or magnesium stearate, to improve the physical property of product.The present inventor finds in supplementary test, when respectively to when 0.5% magnesium stearate is added in embodiment 1-9, inspection product (including test sample and reference substance) described in 11, have been found that, in gained chromatogram, the tailing factor (T) of main composition chromatographic peak is all in 1.21～1.37 scopes, defect in this method is clearly what expectation overcame, although this defect is acceptable reluctantly in mensuration.The present inventor finds in the test further supplemented, 0.5% magnesium stearate is added respectively in embodiment 1-9, inspection product (including test sample and reference substance) described in 11, and (in mobile phase, citric acid radical still keeps former level to extra interpolation 0.02% (w/v) triethylamine, and the amount yet by citric acid or its salt regulates pH value extremely former level in mobile phase used by each embodiment；Lower same), it has been found that, in gained chromatogram, the tailing factor (T) of main composition chromatographic peak all returns in 0.95～1.05 scope.The present inventor finds in the test further supplemented, and 20 batches obtained from market comprise glucosamine or the food of its analog, health food or medicine, all finds wherein to contain stearic acid or magnesium stearate through the detection of other method；When the method using embodiment 1 measures these 20 batches inspection product, in need testing solution chromatogram, the tailing factor (T) of main composition chromatographic peak is all in 1.23～1.32 scopes；And when using extra interpolation 0.02% (w/v) triethylamine in mobile phase instead, when similarly measuring these 20 batches inspection product, in need testing solution chromatogram, the tailing factor (T) of main composition chromatographic peak all returns in 0.95～1.05 scope.But, these use in the complementary testing of triethylamine herein above, when changing triethylamine into ethylenediamine, but can not realize the above-mentioned purpose improving tailing factor, and these results are cannot to obtain explanation from prior art at all.Therefore, in one embodiment of the invention, described mobile phase has been also added with triethylamine.In one embodiment, triethylamine concentration in mobile phase is 0.01～0.05% (w/v), it is preferable that concentration is 0.02% (w/v).
Any technology feature other any embodiment equally applicable that any embodiment of either side of the present invention or this either side has or any embodiment of other either side, as long as they will not be conflicting, certainly at where applicable each other, individual features can be made suitably to modify by necessary words.It is further described with feature to various aspects of the present invention below.All documents that the present invention is recited, their full content is incorporated herein by, and if when implication expressed by these documents is inconsistent with the present invention, it is as the criterion with the statement of the present invention.In addition, various terms and phrase that the present invention uses have and well known to a person skilled in the art general sense, nonetheless, the present invention remains desirable at this, these terms and phrase are described in more detail and explained, the term mentioned and phrase, if any inconsistent with common art-recognized meanings, are as the criterion with the implication that the present invention states.
Accompanying drawing explanation
Fig. 1, Fig. 2 and Fig. 3 are the typical reference substance spectrogram, test sample spectrogram and the negative adjuvant spectrogram that obtain in an instantiation respectively.
Detailed description of the invention
The present invention can be conducted further description by the following examples, but, the scope of the present invention is not limited to following embodiment.One of skill in the art is it is understood that under the premise without departing substantially from the spirit and scope of the present invention, it is possible to the present invention carries out various change and modification.The material that used and test method in test are carried out generality and/or concrete description by the present invention.Although for realize many materials that the object of the invention uses and operational approach is to it is known in the art that but the present invention remains in this to be described in detail as far as possible.
Embodiment 1, use amino-acid analyzer measure the glucosamine in goods
Mobile phase is pH3.20 citric acid/sodium citrate buffer (citrate concentration 0.05M, do not there is provided in Examples below and refer both to 0.05M during its concentration), amino-acid analyzer flow rate pump is 35ml/h, chromatographic column column temperature is 45 DEG C, reaction temperature is 135 DEG C, analysis time is 50min, and detection wavelength is 570nm, and sample size is 20 μ l.Cation exchange resin column, the length 20cm of chromatographic column, diameter 5mm, if not otherwise indicated, chromatographic column specification is identical with this for following instance.
The preparation of need testing solution: take the test sample being equivalent to 20mg glucosamine, accurately weighed, it is placed in 250ml volumetric flask, the 150ml that adds water dilutes, and supersound extraction 15min is settled to scale after cooling, through the filtering with microporous membrane of 0.45 μm, subsequent filtrate is as need testing solution.
Prepared by reference substance solution: take glucosamine hydrochloride reference substance 20mg, accurately weighed, it is placed in 10ml volumetric flask, dilute with water is also settled to scale as reference substance storing solution, take reference substance storing solution appropriate, be diluted to concentration be 1-1000 μ g/ml reference substance solution (actual be diluted to concentration respectively 1,5,10,50,100,500 μ g/ml reference substance solution, following instance if not otherwise indicated, also with this with).
The preparation of negative adjuvant solution: weigh adjuvant in prescription ratio, accurately weighed, it is placed in 250ml volumetric flask, dilute is also settled to scale.(this example test sample is main constituent is the street drug of glucosamine hydrochloride, and adjuvant composition is known, and carries out the preparation of negative adjuvant solution).
Under above-mentioned chromatographic condition, amino-acid analyzer analysis is adopted to measure reference substance, test sample and adjuvant, record chromatogram, calculates the equation of linear regression of standard substance mass concentration and peak area, and correlation coefficient is more than 0.999%, utilizing sample retention time qualitative, equation of linear regression comes quantitatively.There is not chromatographic peak consistent with reference substance retention time in negative adjuvant chromatogram.
Embodiment 2, use amino-acid analyzer measure the glucosamine in goods
Mobile phase is pH4.25 citric acid/sodium citrate buffer (citrate concentration 0.025M), and amino-acid analyzer flow rate pump is 35ml/h, and chromatographic column column temperature is 60 DEG C, reaction temperature is 120 DEG C, analysis time is 40min, and detection wavelength is 570nm, and sample size is 20 μ l.
The preparation of need testing solution: take the test sample being equivalent to 50mg glucosamine, accurately weighed, it is placed in 250ml volumetric flask, the 150ml that adds water dilutes, and supersound extraction 15min is settled to scale after cooling, through the filtering with microporous membrane of 0.45 μm, subsequent filtrate is as need testing solution.
Prepared by reference substance solution: take glucosamine hydrochloride reference substance 20mg, accurately weighed, is placed in 10ml volumetric flask, and dilute with water is also settled to scale as reference substance storing solution, takes reference substance storing solution appropriate, and being diluted to concentration is 1-1000 μ g/ml reference substance solution.
The preparation of negative adjuvant solution: weigh adjuvant in prescription ratio, accurately weighed, it is placed in 250ml volumetric flask, dilute is also settled to scale.(this example test sample is main constituent is the delicatessen food of glucosamine sulfate, and adjuvant composition is known, and carries out the preparation of negative adjuvant solution).
Under above-mentioned chromatographic condition, amino-acid analyzer analysis is adopted to measure reference substance, test sample and adjuvant, record chromatogram, calculates the equation of linear regression of standard substance mass concentration and peak area, and correlation coefficient is more than 0.999%, utilizing sample retention time qualitative, equation of linear regression comes quantitatively.There is not chromatographic peak consistent with reference substance retention time in negative adjuvant chromatogram.
Embodiment 3, use amino-acid analyzer measure the glucosamine in goods
Mobile phase is pH6.45 citric acid/sodium citrate buffer (citrate concentration 0.03M), and amino-acid analyzer flow rate pump is 35ml/h, and chromatographic column column temperature is 80 DEG C, reaction temperature is 130 DEG C, analysis time is 30min, and detection wavelength is 570nm, and sample size is 20 μ l.
The preparation of need testing solution: take the test sample being equivalent to 100mg glucosamine, accurately weighed, it is placed in 250ml volumetric flask, the 150ml that adds water dilutes, and supersound extraction 15min is settled to scale after cooling, through the filtering with microporous membrane of 0.45 μm, subsequent filtrate is as need testing solution.
Prepared by reference substance solution: take glucosamine hydrochloride reference substance 20mg, accurately weighed, is placed in 10ml volumetric flask, and dilute with water is also settled to scale as reference substance storing solution, takes reference substance storing solution appropriate, and being diluted to concentration is 1-1000 μ g/ml reference substance solution.
The preparation of negative adjuvant solution: weigh adjuvant in prescription ratio, accurately weighed, it is placed in 250ml volumetric flask, dilute is also settled to scale.(this example test sample is main constituent is the analysis of functional food sold of Glucosamine sulfate potassium chloride, and adjuvant composition is known, and carries out the preparation of negative adjuvant solution).
Under above-mentioned chromatographic condition, amino-acid analyzer analysis is adopted to measure reference substance, test sample and adjuvant, record chromatogram, calculates the equation of linear regression of standard substance mass concentration and peak area, and correlation coefficient is more than 0.999%, utilizing sample retention time qualitative, equation of linear regression comes quantitatively.There is not chromatographic peak consistent with reference substance retention time in negative adjuvant chromatogram.
Embodiment 4, use amino-acid analyzer measure the glucosamine in goods
Mobile phase is that (two kinds of buffer mix pH3.20 and pH6.45 citric acid/sodium citrate buffer with volume ratio 1:1 ratio, citrate concentration 0.04M), amino-acid analyzer flow rate pump is 40ml/h, chromatographic column column temperature is 80 DEG C, reaction temperature is 135 DEG C, analysis time is 30min, and detection wavelength is 570nm, and sample size is 20 μ l.
The preparation of need testing solution: take the test sample being equivalent to 100mg glucosamine, accurately weighed, it is placed in 250ml volumetric flask, the 150ml that adds water dilutes, and supersound extraction 15min is settled to scale after cooling, through the filtering with microporous membrane of 0.45 μm, subsequent filtrate is as need testing solution.
Prepared by reference substance solution: take glucosamine hydrochloride reference substance 20mg, accurately weighed, is placed in 10ml volumetric flask, and dilute with water is also settled to scale as reference substance storing solution, takes reference substance storing solution appropriate, and being diluted to concentration is 1-1000 μ g/ml reference substance solution.
The preparation of negative adjuvant solution: weigh adjuvant in prescription ratio, accurately weighed, it is placed in 250ml volumetric flask, dilute is also settled to scale.(this example test sample is main constituent is the analysis of functional food sold of glucosamine Citric acid calcium, and adjuvant composition is known, and carries out the preparation of negative adjuvant solution).
Under above-mentioned chromatographic condition, amino-acid analyzer analysis is adopted to measure reference substance, test sample and adjuvant, record chromatogram, calculates the equation of linear regression of standard substance mass concentration and peak area, and correlation coefficient is more than 0.999%, utilizing sample retention time qualitative, equation of linear regression comes quantitatively.There is not chromatographic peak consistent with reference substance retention time in negative adjuvant chromatogram.
Embodiment 5, use amino-acid analyzer measure the glucosamine in goods
Mobile phase is pH6.45 citric acid/sodium citrate buffer (citrate concentration 0.05M), and amino-acid analyzer flow rate pump is 30ml/h, and chromatographic column column temperature is 80 DEG C, reaction temperature is 135 DEG C, analysis time is 30min, and detection wavelength is 550nm, and sample size is 20 μ l.
The preparation of need testing solution: take the test sample being equivalent to 100mg glucosamine, accurately weighed, it is placed in 250ml volumetric flask, the 150ml that adds water dilutes, and supersound extraction 15min is settled to scale after cooling, through the filtering with microporous membrane of 0.45 μm, subsequent filtrate is as need testing solution.
Prepared by reference substance solution: take glucosamine hydrochloride reference substance 20mg, accurately weighed, is placed in 10ml volumetric flask, and dilute with water is also settled to scale as reference substance storing solution, takes reference substance storing solution appropriate, and being diluted to concentration is 1-1000 μ g/ml reference substance solution.
The preparation of negative adjuvant solution: weigh adjuvant in prescription ratio, accurately weighed, it is placed in 250ml volumetric flask, dilute is also settled to scale.(this example test sample is main constituent is the delicatessen food of glucosamine hydrochloride, and adjuvant composition is not known by, and does not carry out the preparation of negative adjuvant solution).
Under above-mentioned chromatographic condition, amino-acid analyzer analysis is adopted to measure reference substance, test sample and adjuvant, record chromatogram, calculates the equation of linear regression of standard substance mass concentration and peak area, and correlation coefficient is more than 0.999%, utilizing sample retention time qualitative, equation of linear regression comes quantitatively.There is not chromatographic peak consistent with reference substance retention time in negative adjuvant chromatogram.
Embodiment 6, use amino-acid analyzer measure the glucosamine in goods
Mobile phase is pH4.25 and pH6.45 citric acid/sodium citrate buffer (two kinds of buffer mix with volume ratio 1:1 ratio), amino-acid analyzer flow rate pump is 50ml/h, chromatographic column column temperature is 40 DEG C, reaction temperature is 135 DEG C, analysis time is 30min, detection wavelength is 570nm, and sample size is 30 μ l.
The preparation of need testing solution: take the test sample being equivalent to 100mg glucosamine, accurately weighed, it is placed in 250ml volumetric flask, the 150ml that adds water dilutes, and supersound extraction 15min is settled to scale after cooling, through the filtering with microporous membrane of 0.45 μm, subsequent filtrate is as need testing solution.
Prepared by reference substance solution: take glucosamine hydrochloride reference substance 20mg, accurately weighed, is placed in 10ml volumetric flask, and dilute with water is also settled to scale as reference substance storing solution, takes reference substance storing solution appropriate, and being diluted to concentration is 1-1000 μ g/ml reference substance solution.
The preparation of negative adjuvant solution: weigh adjuvant in prescription ratio, accurately weighed, it is placed in 250ml volumetric flask, dilute is also settled to scale.(this example test sample is main constituent is the analysis of functional food sold of glucosamine hydrochloride, and adjuvant composition is known, and carries out the preparation of negative adjuvant solution).
Result shows: occur the chromatographic peak consistent with reference substance retention time in need testing solution chromatogram, and this peak-to-peak type is symmetrical, theoretical cam curve is more than 2000, in negative adjuvant sample solution chromatogram, occur without the chromatographic peak consistent with glucosamine hydrochloride reference substance retention time, namely without matrix interference.Reference substance spectrogram, sample spectrogram and negative adjuvant spectrogram are shown in Fig. 1, Fig. 2 and Fig. 3.
Embodiment 7, use amino-acid analyzer measure the glucosamine in goods
Mobile phase is pH4.25 and pH6.45 citric acid/sodium citrate buffer (two kinds of buffer mix with volume ratio 2:1 ratio), amino-acid analyzer flow rate pump is 25ml/h, chromatographic column column temperature is 80 DEG C, reaction temperature is 140 DEG C, analysis time is 30min, detection wavelength is 530nm, and sample size is 10 μ l.
The preparation of need testing solution: take glucosamine hydrochloride crude drug 100mg as test sample, accurately weighed, it is placed in 250ml volumetric flask, the 150ml that adds water dilutes, and supersound extraction 15min is settled to scale after cooling, through the filtering with microporous membrane of 0.45 μm, subsequent filtrate is as need testing solution.
Prepared by reference substance solution: take glucosamine hydrochloride reference substance 20mg, accurately weighed, is placed in 10ml volumetric flask, and dilute with water is also settled to scale as reference substance storing solution, takes reference substance storing solution appropriate, and being diluted to concentration is 1-1000 μ g/ml reference substance solution.
(this example test sample is crude drug, without adjuvant, does not in fact carry out the preparation of negative adjuvant solution).
Under above-mentioned chromatographic condition, amino-acid analyzer analysis is adopted to measure reference substance, test sample, record chromatogram, calculates the equation of linear regression of standard substance mass concentration and peak area, and correlation coefficient is more than 0.999%, utilizing sample retention time qualitative, equation of linear regression comes quantitatively.There is not chromatographic peak consistent with reference substance retention time in negative adjuvant chromatogram.
Above example 1-7, by the retention time (in each example, two retention times are all consistent) at reference substance solution composition peak main with need testing solution chromatogram, it is possible to easily test sample is carried out qualitative analysis.Additionally, by reference substance solution gained equation of linear regression, all can readily calculate out the content of target component in test sample, record the test sample object content in 7 examples consistent with its product indicia content (all in 98～102% scopes of product indicia content；This area usually requires that in 90～110% scopes).
By the enforcement of the present invention, establish the efficient of glucosamine perfectly, quickly, sensitive and qualitative and quantitative analysis method accurately, establish good basis for glucosamine in food, health food and the application in medicine and detection.
Embodiment 8, linear dependence are tested
Take glucosamine hydrochloride reference substance 20mg, accurately weighed, it is placed in 10ml volumetric flask, dilute with water is also settled to scale as reference substance storing solution, take reference substance storing solution appropriate, being diluted to concentration respectively 1,5,10,50,100,500 μ g/ml reference substance solution, sample introduction 20 μ l analyzes respectively.Result is in Table 1
Table 1: linear dependence test determination result
| Numbering | 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, in 1-500 μ g/ml concentration range, solution concentration value is good with peak area value linear relationship.
Embodiment 9, precision test
10 μ g/ml reference substance solution of Example 8, continuous sample introduction 6 pin, calculate its precision, result is in Table 2.
Table 2: precision test measurement result
Result of the test shows, precision is very high, can meet mensuration requirement completely.
Embodiment 10 detection limit is tested
Precision measures 10 μ g/ml reference substance solution 0.5ml of embodiment 8, dilute is also settled to 10ml, obtaining concentration is 0.5 μ g/ml reference substance solution, sample introduction 20 μ l analyzes, quantitative limit (LOQ) is calculated according to S/N=10, calculating detection limit (LOD) according to S/N=3, 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.
Embodiment 11 TIANZHU XINGNAO Capsul is tested
By low middle and high concentration, to weigh glucosamine hydrochloride appropriate for precision respectively, join in corresponding blank auxiliary (embodiment 1 is used), it is placed in 250ml volumetric flask, the 150ml that adds water dilutes, and supersound extraction 15min is settled to scale after cooling, through the filtering with microporous membrane of 0.45 μm, subsequent filtrate is as determination of recovery rates solution.Result of calculation is in Table 3.
Table 3: TIANZHU XINGNAO Capsul measurement result
By the enforcement of the present invention, establish the efficient, quick, sensitive of glucosamine and qualitative and quantitative analysis method accurately perfectly, established good basis for glucosamine in food, health food and the application in medicine and detection.
Claims (10)
1. the method measuring the glucosamine in goods or its analog, the method uses amino-acid analyzer to be measured, and comprises the steps:
(1) analytical tool 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) providing chromatography eluant mobile phase, this mobile phase is selected from: pH2-7 citric acid/sodium citrate buffer, other acid/corresponding salt buffer or pH7-14 sodium hydrate buffer solution；
(4) chromatographiccondition: amino-acid analyzer flow rate pump 10-100ml/h；Chromatographic column column temperature 30-100 DEG C；Reaction temperature 30-150 DEG C；Chromatography time 3-60min；Detection wavelength 200-800nm；Sample size 2-50 μ l；
(5) preparation of need testing solution: take the test sample be equivalent to containing 0.5mg-250mg glucosamine, accurately weighed, it is placed in 250ml volumetric flask, the 150ml that adds water dilutes, supersound process 15min, being settled to scale after cooling, through the filtering with microporous membrane of 0.45 μm, subsequent filtrate is as need testing solution；
(6) preparation of reference substance solution: take glucosamine hydrochloride reference substance 1-200mg, accurately weighed, it is placed in 10ml volumetric flask, dilute with water is also settled to scale as reference substance storing solution, taking reference substance storing solution appropriate, being diluted to concentration is 1-1000 μ g/ml reference substance solution；
(7) preparation of adjuvant solution: if there is adjuvant and its composition known in described goods, taken test sample considerable amount of adjuvant when preparing then is weighed with need testing solution in prescription ratio, accurately weighed, it is placed in 250ml volumetric flask, dilute is also settled to scale；
(8) under above-mentioned chromatographiccondition, adopt amino-acid analyzer analysis, respectively by reference substance solution, need testing solution and adjuvant solution injecting chromatograph, record chromatogram；Read retention time and the peak area at glucosamine or its analog peak in reference substance solution and need testing solution chromatogram；Use reference substance solution concentration and its equation of linear regression of calculated by peak area, when the correlation coefficient of this equation of linear regression is more than 0.999%, utilize sample retention time to carry out qualitative to test sample, utilize equation of linear regression that test sample is carried out quantitatively.
2. method according to claim 1, wherein said goods are selected from: comprise glucosamine or the food of its analog, health food, medicine, or prepare their glucosamine or its analog raw material.
3. the method according to any one of claim 1 to 2, wherein:
Described aminoglucose sugar analogue is selected from: glucosamine acylate and the glucosamine inorganic acid salts etc. such as glucosamine hydrochloride, glucosamine sulfate, Glucosamine sulfate potassium chloride, glucosamine Citric acid calcium；
Described adjuvant chromatogram should not occur chromatographic peak consistent with reference substance solution chromatogram main peak retention time；
The length 20cm of the chromatographic column of described cation exchange resin column, diameter 5mm；
Described mobile phase is the citric acid/sodium citrate buffer of pH3～pH7；
Described mobile phase is also added with triethylamine, for instance its concentration in mobile phase is 0.01～0.05% (w/v), for instance be 0.02% (w/v)；
In the mobile phase of described citric acid/sodium citrate composition, citrate concentration is 0.01M～0.1M；Optimization citric acid root concentration is 0.025M～0.05M；And/or
Described mobile phase is selected from: pH3.20 citric acid/sodium citrate buffer, pH4.25 citric acid/sodium citrate buffer, pH6.45 citric acid/sodium citrate buffer and combination thereof.
4. the method according to any one of claims 1 to 3, wherein said amino-acid analyzer flow rate pump is 25～50ml/h.
5. the method according to any one of Claims 1-4, wherein said chromatographic column column temperature 40-80 DEG C.
6. the method according to any one of claim 1 to 5, wherein said reaction temperature 120-140 DEG C.
7. the method according to any one of claim 1 to 6, wherein said chromatography time 30-50min.
8. the method according to any one of claim 1 to 7, wherein: described detection wavelength 530-570nm；And/or, described sample size 10-30 μ l.
9. the method according to any one of claim 1 to 8, wherein in step (5), takes the test sample being equivalent to 20～100mg glucosamine.
10. the method according to any one of claim 1 to 9, wherein in step (6), making concentration is 1-500 μ g/ml reference substance solution.
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