CN104880530B - A kind of method of glucosamine content in rp-hplc analysis lactalbumin glycation product - Google Patents
A kind of method of glucosamine content in rp-hplc analysis lactalbumin glycation product Download PDFInfo
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- CN104880530B CN104880530B CN201510243576.5A CN201510243576A CN104880530B CN 104880530 B CN104880530 B CN 104880530B CN 201510243576 A CN201510243576 A CN 201510243576A CN 104880530 B CN104880530 B CN 104880530B
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Abstract
The present invention relates to the quantitative detecting method of glucosamine content in a kind of lactalbumin glycation product, belong to detection analysis technical field；This method uses hydrochloric acid to be hydrolyzed by the glucosamine in glycated milk albumin, and carry out column front derivation, the content of glucosamine in recycling rp-hplc determination protolysate, method is fast and convenient, highly sensitive, it is suitable for the mensuration of glucosamine content in various protein amino glucose glycation products.
Description
Technical field
The invention belongs to detect analytical technology, relate generally to glucosamine content in a kind of lactalbumin glycation product
Quantitative detecting method.
Background technology
Lactalbumin is that the protein stayed is referred to as in raw milk in addition to the casein of precipitation at pH isoelectric point, IP
For lactalbumin, account for the 18%～20% of lactoprotein.Lactalbumin is carried out glycosylation modification, owing to glycosyl gives glycosyl
Change the important functional properties of albumen, therefore can improve a lot of functional character, including emulsibility, retentiveness, oil absorption and sensitization
Property.But release and the detection by quantitative problem of glucosamine are to develop the key of glycosylation modified technology in glycosylation albumen.
Analyze glucosamine and can use colorimetry, by anti-with developer again after the primary amino radical acetylation of glucosamine
Should, carrying out colorimetric analysis afterwards, the method complex operation is time-consuming, and sensitivity and degree of accuracy are bad.High performance liquid chromatography
It is a kind of analysis method rapidly and efficiently, reversed-phase column nh 2 column Composition distribution can be used to detect, or after pre-column derivatization
Ultraviolet or fluorescence detector is utilized to carry out assay.The derivative reagent measuring glucosamine has a lot, some derivative reagent
Sample pretreatment process comparatively laborious, select suitable derivative reagent and exploitation pre-treating method be determine analysis method pass
Key.By hydrochloric acid, the glucosamine in glycated milk albumin is hydrolyzed, and carry out column front derivation, recycle reversed phase high efficiency
The content of glucosamine in liquid chromatography for measuring protolysate, method is fast and convenient, highly sensitive, for glycosylation albumen
The detection of middle glucosamine provides to be supported.
Summary of the invention
It is an object of the invention to, by hydrochloric acid, the glucosamine in glycated milk albumin is hydrolyzed, and carry out post
Front derivative, the content of glucosamine in recycling rp-hplc determination protolysate, method is fast and convenient,
Highly sensitive, provide for the detection of amino sugar in glycosylation albumen and support.
The method of the present invention is as follows:
A kind of utilize the method for glucosamine content in rp-hplc analysis lactalbumin glycation product,
The method comprises the following steps: (1) accurately weighs lactalbumin glucosamine glycation product 60mg, is added to hydrolyze in bottle,
Add the hydrochloric acid 5ml of 6mol/L, seal, hydrolyze 3-5h at 100 DEG C, take hydrolyzed solution 1ml, regulate pH to 7.0, use ultra-pure water constant volume
To 25ml；(2) take raw milk albumin 60mg, be added to hydrolyze in bottle, add the hydrochloric acid 5ml of 6mol/L, seal, water at 100 DEG C
Solve 3-5h, take hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water；(3) obtained by step (1) and step (2)
Sample and space management liquid, add 0.7ml borate buffer solution and the mixing of 0.2-0.4ml derivative reagent, and derivative reagent is 50mg
O-phthalaldehyde(OPA) is dissolved in 1ml methanol, 10ml borate buffer and 0.1mL3-mercaptopropionic acid, and 25 DEG C of dark places derive 12-18min；
(4) the sample liquid after deriving carries out rp-hplc analysis, and chromatographic column is Elite C18, and chromatographic column parameter is 4.6mm
× 250mm, flowing includes A liquid and B liquid mutually, and A liquid is methanol, and B liquid is 0.025mol/L Acetic acid-sodium acetate buffer, the pH of B liquid
3.4-3.8, in elution process, the volume ratio of A liquid and B liquid is 60:40-76:24, and flow velocity is 1ml/min, and column temperature is 35 DEG C, sample introduction
Amount is 10 μ l, and high performance liquid chromatography detector is fluorescence detector, and excitation wavelength 337nm launches wavelength 454nm.
Described hydrolysising condition is: hydrolyze 4h at 100 DEG C.
The liquid-derived condition of described sample is: take sample and blank space that 0.3ml is obtained by step (1) and step (2) respectively
Reason liquid, adds 0.7ml borate buffer solution and the mixing of 0.3ml derivative reagent, and 25 DEG C of dark places derive 15min.
Described analysis chromatographic condition is: chromatographic column is Elite C18, and chromatographic column parameter is 4.6mm × 250mm, described
Flowing include A liquid and B liquid mutually, A liquid is methanol, and B liquid is 0.025mol/L Acetic acid-sodium acetate buffer, the pH 3.6 of B liquid,
In elution process, the volume ratio of A liquid and B liquid is 68:32, and sample size is 10 μ l.
The feature of the inventive method: easy to operate simply, highly sensitive favorable reproducibility, confirmation ability are strong, can be used for various
The mensuration of glucosamine content in protein-glucosamine glycation product.
Accompanying drawing explanation
Fig. 1 is the techniqueflow chart of the present invention；
Fig. 2 is standard glucosamine (20 μ g/mL) chromatogram；
Fig. 3 is sample chromatogram figure；
Fig. 4 is glucosamine standard curve.
Detailed description of the invention
Come below in conjunction with the accompanying drawings specific embodiment is further described.
A kind of utilize the method for glucosamine content in rp-hplc analysis lactalbumin glycation product,
The method comprises the following steps: (1) accurately weighs lactalbumin glucosamine glycation product 60mg, is added to hydrolyze in bottle,
Add the hydrochloric acid 5ml of 6mol/L, seal, hydrolyze 3-5h at 100 DEG C, take hydrolyzed solution 1ml, regulate pH to 7.0, use ultra-pure water constant volume
To 25ml；(2) take raw milk albumin 60mg, be added to hydrolyze in bottle, add the hydrochloric acid 5ml of 6mol/L, seal, water at 100 DEG C
Solve 3-5h, take hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water；(3) obtained by step (1) and step (2)
Sample and space management liquid, add 0.7ml borate buffer solution and the mixing of 0.2-0.4ml derivative reagent, and derivative reagent is 50mg
O-phthalaldehyde(OPA) is dissolved in 1ml methanol, 10ml borate buffer and 0.1mL3-mercaptopropionic acid, and 25 DEG C of dark places derive 12-18min；
(4) the sample liquid after deriving carries out rp-hplc analysis, and chromatographic column is Elite C18, and chromatographic column parameter is 4.6mm
× 250mm, flowing includes A liquid and B liquid mutually, and A liquid is methanol, and B liquid is 0.025mol/L Acetic acid-sodium acetate buffer, the pH of B liquid
3.4-3.8, in elution process, the volume ratio of A liquid and B liquid is 60:40-76:24, and flow velocity is 1ml/min, and column temperature is 35 DEG C, sample introduction
Amount is 10 μ l, and high performance liquid chromatography detector is fluorescence detector, and excitation wavelength 337nm launches wavelength 454nm.
Described hydrolysising condition is: hydrolyze 4h at 100 DEG C.
The liquid-derived condition of described sample is: take sample and blank space that 0.3ml is obtained by step (1) and step (2) respectively
Reason liquid, adds 0.7ml borate buffer solution and the mixing of 0.3ml derivative reagent, and 25 DEG C of dark places derive 15min.
Described analysis chromatographic condition is: chromatographic column is Elite C18, and chromatographic column parameter is 4.6mm × 250mm, described
Flowing include A liquid and B liquid mutually, A liquid is methanol, and B liquid is 0.025mol/L Acetic acid-sodium acetate buffer, the pH 3.6 of B liquid,
In elution process, the volume ratio of A liquid and B liquid is 68:32, and sample size is 10 μ l.
Embodiment 1
(1) accurately weigh lactalbumin glucosamine glycation product 60mg, be added to hydrolyze in bottle, add 6mol/L's
Hydrochloric acid 5ml seals, and hydrolyzes 4h, takes hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water at 100 DEG C.
(2) taking raw milk albumin 60mg, be added to hydrolyze in bottle, the hydrochloric acid 5ml adding 6mol/L seals, water at 100 DEG C
Solve 4h, take hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water.
(3) sample obtained by step (1) and step (2) and space management liquid, add 0.7ml borate buffer solution and
0.3ml derivative reagent mixes, and derivative reagent is that 50mg o-phthalaldehyde(OPA) is dissolved in 1ml methanol, 10ml borate buffer and 0.1ml
3-mercaptopropionic acid, 25 DEG C of dark places derive 15min.
(4) the sample liquid after deriving carries out rp-hplc analysis, and chromatographic column is Elite C18, and chromatographic column is joined
Number is 4.6mm × 250mm, and flowing includes that A liquid and B liquid, A liquid are methanol mutually, and B liquid is 0.025mol/L Acetic acid-sodium acetate buffering
Liquid, the pH 3.6 of B liquid, in elution process, the volume ratio of A liquid and B liquid is 68:32, and flow velocity is 1ml/min, and column temperature is 35 DEG C, enters
Sample amount is 10 μ l, and high performance liquid chromatography detector is fluorescence detector, and excitation wavelength 337nm launches wavelength 454nm.
Embodiment 2
(1) accurately weigh lactalbumin glucosamine glycation product 60mg, be added to hydrolyze in bottle, add 6mol/L's
Hydrochloric acid 5ml seals, and hydrolyzes 3h, takes hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water at 100 DEG C.
(2) taking raw milk albumin 60mg, be added to hydrolyze in bottle, the hydrochloric acid 5ml adding 6mol/L seals, water at 100 DEG C
Solve 3h, take hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water.
(3) sample obtained by step (1) and step (2) and space management liquid, add 0.7ml borate buffer solution and
0.3ml derivative reagent mixes, and derivative reagent is that 50mg o-phthalaldehyde(OPA) is dissolved in 1ml methanol, 10ml borate buffer and
0.1mL3-mercaptopropionic acid, 25 DEG C of dark places derive 12min.
(4) the sample liquid after deriving carries out rp-hplc analysis, and chromatographic column is Elite C18, and chromatographic column is joined
Number is 4.6mm × 250mm, and flowing includes that A liquid and B liquid, A liquid are methanol mutually, and B liquid is 0.025mol/L Acetic acid-sodium acetate buffering
Liquid, the pH 3.6 of B liquid, in elution process, the volume ratio of A liquid and B liquid is 60:40, and flow velocity is 1ml/min, and column temperature is 35 DEG C, enters
Sample amount is 10 μ l, and high performance liquid chromatography detector is fluorescence detector, and excitation wavelength 337nm launches wavelength 454nm.
Embodiment 3
(1) accurately weigh lactalbumin glucosamine glycation product 60mg, be added to hydrolyze in bottle, add 6mol/L's
Hydrochloric acid 5ml seals, and hydrolyzes 5h, takes hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water at 100 DEG C.
(2) taking raw milk albumin 60mg, be added to hydrolyze in bottle, the hydrochloric acid 5ml adding 6mol/L seals, water at 100 DEG C
Solve 5h, take hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water.
(3) sample obtained by step (1) and step (2) and space management liquid, add 0.7ml borate buffer solution and
0.3ml derivative reagent mixes, and derivative reagent is that 50mg o-phthalaldehyde(OPA) is dissolved in 1ml methanol, 10ml borate buffer and
0.1mL3-mercaptopropionic acid, 25 DEG C of dark places derive 18min.
(4) the sample liquid after deriving carries out rp-hplc analysis, and chromatographic column is Elite C18, and chromatographic column is joined
Number is 4.6mm × 250mm, and flowing includes that A liquid and B liquid, A liquid are methanol mutually, and B liquid is 0.025mol/L Acetic acid-sodium acetate buffering
Liquid, the pH 3.6 of B liquid, in elution process, the volume ratio of A liquid and B liquid is 60:40, and flow velocity is 1ml/min, and column temperature is 35 DEG C, enters
Sample amount is 10 μ l, and high performance liquid chromatography detector is fluorescence detector, and excitation wavelength 337nm launches wavelength 454nm.
Embodiment 4
(1) accurately weigh lactalbumin glucosamine glycation product 60mg, be added to hydrolyze in bottle, add 6mol/L's
Hydrochloric acid 5ml seals, and hydrolyzes 4h, takes hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water at 100 DEG C.
(2) taking raw milk albumin 60mg, be added to hydrolyze in bottle, the hydrochloric acid 5ml adding 6mol/L seals, water at 100 DEG C
Solve 4h, take hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water.
(3) sample obtained by step (1) and step (2) and space management liquid, add 0.7ml borate buffer solution and
0.3ml derivative reagent mixes, and derivative reagent is that 50mg o-phthalaldehyde(OPA) is dissolved in 1ml methanol, 10ml borate buffer and
0.1mL3-mercaptopropionic acid, 25 DEG C of dark places derive 18min.
(4) the sample liquid after deriving carries out rp-hplc analysis, and chromatographic column is Elite C18, and chromatographic column is joined
Number is 4.6mm × 250mm, and flowing includes that A liquid and B liquid, A liquid are methanol mutually, and B liquid is 0.025mol/L Acetic acid-sodium acetate buffering
Liquid, the pH 3.6 of B liquid, in elution process, the volume ratio of A liquid and B liquid is 76:24, and flow velocity is 1ml/min, and column temperature is 35 DEG C, enters
Sample amount is 10 μ l, and high performance liquid chromatography detector is fluorescence detector, and excitation wavelength 337nm launches wavelength 454nm.
Embodiment 5
(1) accurately weigh lactalbumin glucosamine glycation product 60mg, be added to hydrolyze in bottle, add 6mol/L's
Hydrochloric acid 5ml seals, and hydrolyzes 3h, takes hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water at 100 DEG C.
(2) taking raw milk albumin 60mg, be added to hydrolyze in bottle, the hydrochloric acid 5ml adding 6mol/L seals, water at 100 DEG C
Solve 3h, take hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water.
(3) sample obtained by step (1) and step (2) and space management liquid, add 0.7ml borate buffer solution and
0.3ml derivative reagent mixes, and derivative reagent is that 50mg o-phthalaldehyde(OPA) is dissolved in 1ml methanol, 10ml borate buffer and
0.1mL3-mercaptopropionic acid, 25 DEG C of dark places derive 15min.
(4) the sample liquid after deriving carries out rp-hplc analysis, and chromatographic column is Elite C18, and chromatographic column is joined
Number is 4.6mm × 250mm, and flowing includes that A liquid and B liquid, A liquid are methanol mutually, and B liquid is 0.025mol/L Acetic acid-sodium acetate buffering
Liquid, the pH 3.6 of B liquid, in elution process, the volume ratio of A liquid and B liquid is 76:24, and flow velocity is 1ml/min, and column temperature is 35 DEG C, enters
Sample amount is 10 μ l, and high performance liquid chromatography detector is fluorescence detector, and excitation wavelength 337nm launches wavelength 454nm.
Embodiment 6
(1) accurately weigh lactalbumin glucosamine glycation product 60mg, be added to hydrolyze in bottle, add 6mol/L's
Hydrochloric acid 5ml seals, and hydrolyzes 3h, takes hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water at 100 DEG C.
(2) taking raw milk albumin 60mg, be added to hydrolyze in bottle, the hydrochloric acid 5ml adding 6mol/L seals, water at 100 DEG C
Solve 3h, take hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water.
(3) sample obtained by step (1) and step (2) and space management liquid, add 0.7ml borate buffer solution and
0.3ml derivative reagent mixes, and derivative reagent is that 50mg o-phthalaldehyde(OPA) is dissolved in 1ml methanol, 10ml borate buffer and
0.1mL3-mercaptopropionic acid, 25 DEG C of dark places derive 12min.
(4) the sample liquid after deriving carries out rp-hplc analysis, and chromatographic column is Elite C18, and chromatographic column is joined
Number is 4.6mm × 250mm, and flowing includes that A liquid and B liquid, A liquid are methanol mutually, and B liquid is 0.025mol/L Acetic acid-sodium acetate buffering
Liquid, the pH 3.6 of B liquid, in elution process, the volume ratio of A liquid and B liquid is 76:24, and flow velocity is 1ml/min, and column temperature is 35 DEG C, enters
Sample amount is 10 μ l, and high performance liquid chromatography detector is fluorescence detector, and excitation wavelength 337nm launches wavelength 454nm.
Claims (4)
1. utilize a method for glucosamine content in rp-hplc analysis lactalbumin glycation product, its
Being characterised by, the method comprises the following steps: (1) accurately weighs lactalbumin glucosamine glycation product 60mg, is added to
In hydrolysis bottle, add the hydrochloric acid 5ml of 6mol/L, seal, hydrolyze 3-5h at 100 DEG C, take hydrolyzed solution 1ml, regulate pH to 7.0, use
Ultra-pure water is settled to 25ml；(2) take raw milk albumin 60mg, be added to hydrolyze in bottle, add the hydrochloric acid 5ml of 6mol/L, seal,
Hydrolyze 3-5h at 100 DEG C, take hydrolyzed solution 1ml, regulate pH to 7.0, be settled to 25ml with ultra-pure water；(3) by step (1) and step
(2) sample obtained and space management liquid, add 0.7ml borate buffer solution and the mixing of 0.2-0.4ml derivative reagent, and 25 DEG C dark
The derivative 12-18min in place, derivative reagent is that 50mg o-phthalaldehyde(OPA) is dissolved in 1ml methanol, 10ml borate buffer and 0.1mL3-
Mercaptopropionic acid；(4) the sample liquid after deriving carries out rp-hplc analysis, and chromatographic column is Elite C18, and chromatographic column is joined
Number is 4.6mm × 250mm, and flowing includes that A liquid and B liquid, A liquid are methanol mutually, and B liquid is 0.025mol/L Acetic acid-sodium acetate buffering
Liquid, the pH 3.4-3.8 of B liquid, in elution process, the volume ratio of A liquid and B liquid is 60:40-76:24, and flow velocity is 1ml/min, column temperature
Being 35 DEG C, sample size is 10 μ l, and high performance liquid chromatography detector is fluorescence detector, excitation wavelength 337nm, launches wavelength
454nm。
One the most according to claim 1 utilizes amino in rp-hplc analysis lactalbumin glycation product
The method of glucose content, it is characterised in that described hydrolysising condition is: hydrolyze 4h at 100 DEG C.
One the most according to claim 1 utilizes amino in rp-hplc analysis lactalbumin glycation product
The method of glucose content, it is characterised in that the liquid-derived condition of described sample is: take 0.3ml respectively by step (1) and step
(2) sample obtained and space management liquid, add 0.7ml borate buffer solution and the mixing of 0.3ml derivative reagent, spreads out in 25 DEG C of dark places
Raw 15min.
One the most according to claim 1 utilizes amino in rp-hplc analysis lactalbumin glycation product
The method of glucose content, it is characterised in that described analysis chromatographic condition is: chromatographic column is Elite C18, chromatographic column parameter
For 4.6mm × 250mm, described flowing includes A liquid and B liquid mutually, and A liquid is methanol, and B liquid is 0.025mol/L Acetic acid-sodium acetate
Buffer, the pH 3.6 of B liquid, in elution process, the volume ratio of A liquid and B liquid is 68:32, and sample size is 10 μ l.
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| CN109164197A (en) * | 2018-09-20 | 2019-01-08 | 中国食品发酵工业研究院有限公司 | The liquid chromatography detecting method of sodium cyclohexylsulfamate in a kind of white wine |
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