CN106556651B - The method for detecting alanine, lysine, glutamic acid and/or tyrosine - Google Patents
The method for detecting alanine, lysine, glutamic acid and/or tyrosine Download PDFInfo
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Abstract
The present invention relates to Pharmaceutical Analysis technical fields, more particularly to the method for detection alanine, lysine, glutamic acid and/or tyrosine.Method provided by the invention detects product to be tested using LC/MS, by quantified by external standard method, can have good accuracy and sensitivity, and can be suitably used for the quality testing of acetic acid copaxone.It is accurately quantitative that experiment shows that method provided by the invention can accurately carry out alanine, lysine, glutamic acid and/or tyrosine, and recovery of standard addition is 98.1%~101.7%；Repeated good, 5 needle RSD of continuous sample introduction is smaller than 2%；Also, minimum detection limit, less than 1.1 μ g/mL, sensitivity is good.
Description
Technical field
The present invention relates to Pharmaceutical Analysis technical fields, more particularly to detection alanine, lysine, glutamic acid and/or junket ammonia
The method of acid.
Background technique
Currently, the method being often used to the analysis of amino acid is to use derivatization reagent to derive, then by ultraviolet
Detector measurement.This method sensitivity is lower, pretreatment process is complicated, is not suitable for the detection of the free amino acid of low content.
Acetic acid copaxone is called and makees l-alanine-L-glutamic acid-L-lysine-l-tyrosine polypeptide polymer acetic acid
Salt, acetic acid lattice card replace thunder, and this product is a kind of polypeptide compounds of synthesis, rely ammonia by following 4 kinds of amino acids L-alanines, L-
Acid, Pidolidone, l-tyrosine composition, drug are white to linen sterilizing, freeze crystalline flour end, pack another attached water for injection with
It is modulated into injection solution to be used, mainly as single subcutaneous injection to treat multiple sclerosis.Wherein:
L-Alanine, English name: L-Alanine, molecular formula: C3H7NO2.Molecular weight is 89.9, No. CAS: 56-51-7,
To be colourless to white crystalline powder, it is dissolved in water, ethyl alcohol, does not dissolve in ether and acetone.It is mainly used for Biochemical Research, tissue training
It supports, liver functional testing, flavoring agent, the flavor effect that flavouring can be increased, also act as tart flavour corrigent, improve the acid of organic acid
Taste.
L-lysine, English name: L-Lysine, molecular formula: C6H14N2O2.Molecular weight is No. 182.65.CAS: 657-
27-2 is insoluble in ethyl alcohol and ether for white or the crystalline powder of near-white free-flowing, soluble easily in water and formic acid.Mainly
For feed addictive, food additive and pharmacy.
Pidolidone, English name: L-Glutamic, molecular formula: C5H9NO4Molecular weight is No. 147.13.CAS: 56-
86-0 is slightly soluble in cold water, is soluble in hot water, be practically insoluble in ether, ethyl alcohol and acetone for white or colourless flake shape crystal.
Mainly for the production of monosodium glutamate, fragrance, and it is used as salt agent, nutritional supplement and biochemical reagents etc..
L-tyrosine, English name: L-Tyrosine, molecular formula: C9H11NO3.Molecular weight is No. 181.19.CAS: 60-
18-4, be fine needle crystal or crystalline powder, be soluble in buck, do not dissolve in center organic solvent, as dehydrated alcohol, ether,
Acetone etc..Pharmaceutically it is used as treatment hyperthyroidism, also can be used as food additives.
Polypeptide acetic acid copaxone completely by these four L-type amino acid in sequence and mode combines, Dichlorodiphenyl Acetate lattice
It draws and carries out orderly enzymatic hydrolysis for thunder C-terminal, obtained product is necessarily these four free amino acid, by measuring four kinds of free ammonias
The content of base acid can reflect out each amino acid content and ratio of acetic acid copaxone C-terminal, for the qualitative, fixed of drug
Quantity research and control of product quality have important practical value.
But there is no the method that can detect simultaneously l-Alanine, L-lysine, Pidolidone, l-tyrosine at present, and
The free aminoacid content generated after acetic acid copaxone enzymatic hydrolysis is extremely low, and UV detector can not carry out accurately fixed after derivatization
Amount, so discomfort shares general efficient liquid-phase chromatography method and is measured.Therefore, establishing one kind can measure in sample simultaneously
Alanine, lysine, glutamic acid, tyrosine content LC-MS method, have highly important practical value.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that provide detection alanine, lysine, glutamic acid and/or
The method of tyrosine, method accuracy provided by the invention, sensitivity, repeatability are all good.
The method provided by the invention for detecting alanine, lysine, glutamic acid and/or tyrosine, sample to be tested is dissolved
Afterwards, it is analyzed with LC/MS, it is quantitative to alanine, lysine, glutamic acid and/or tyrosine with external standard method；The flowing of LC/MS analysis
Phase A phase is ammonium formate solution；Mobile phase B is mutually acetonitrile
In an embodiment of the present invention, alanine is l-Alanine；Lysine is L-lysine；Glutamic acid is Pidolidone；
Tyrosine is l-tyrosine.
LC/MS method combines the separating capacity of chromatography with mass spectrographic qualitative function, can be realized to COMPLEX MIXED
Object is more accurately qualitatively and quantitatively analyzed, and can simplify the pretreatment process of sample.Substance is detected using LC/MS,
It needs to carry out system suitability analysis to this method, and detects the sensitivity of this method, specifically:
System suitability: after mixed reference substance solution sample detection, alanine, lysine, glutamic acid and/or tyrosine
Four main peaks can all efficiently separate.5 needle of reference substance mother liquor continuous sample introduction, the RSD of 5 detection alanine peak areas are 1.2%；5
The RSD of secondary detection lysine peak area is 0.93%；The RSD of 5 detection glutamic acid peak areas is 1.0%；5 detection tyrosine
The RSD of peak area is 1.5%.Proof system applicability meets the requirements, and method repeatability provided by the invention is good.
Sensitivity: each substance lowest detection is limited to the concentration of ingredient when signal-to-noise ratio is 3.With method provided by the invention progress
Detection, wherein the lowest detection of alanine is limited to 1.028 μ g/mL；The lowest detection of lysine is limited to 0.503 μ g/mL；Paddy ammonia
The lowest detection of acid is limited to 0.3036 μ g/mL；The lowest detection of lysine is limited to 0.2012 μ g/mL；Illustrate provided by the invention
The sensitivity of method is good.
In an embodiment of the present invention, the solvent of dissolution is acetonitrile solution.
In an embodiment of the present invention, the volume fraction of acetonitrile is 40%~90% in acetonitrile solution.
In some embodiments, the volume fraction of acetonitrile is 80% in acetonitrile solution.
In an embodiment of the present invention, the dissolved concentration of sample to be tested is 10mg/mL~20mg/mL.
In some embodiments, the dissolved concentration of sample to be tested is 20mg/mL.
Liquid chromatogram be sample component between column packing and mobile phase mass exchange and achieve the purpose that separate, therefore want
It asks flowing relative sample that there is certain solvability and does not generate chemical reaction with sample, viscosity is small as far as possible, so as to
To good separating effect；And the physico-chemical property of mobile phase will be adapted with the detector used.The method provided by the present invention can protect
Better detection effect is demonstrate,proved, better than the testing result of other mobile phases.
In an embodiment of the present invention, the mobile phase A of LC/MS is mutually ammonium formate solution；Mobile phase B is mutually acetonitrile, wherein
The concentration of ammonium formate is 25mmol/L in ammonium formate solution.
In an embodiment of the present invention, the gradient of LC/MS are as follows:
0min~10min, the volume fraction of mobile phase A phase are 50%；
10min~18min, the volume fraction of mobile phase A phase is by 50%~80%；
18min~25min, the volume fraction of mobile phase A phase are 80%；
25min~25.1min, the volume fraction of mobile phase A phase is by 80%~50%；
25.1min~35min, the volume fraction of mobile phase A phase are 50%.
In an embodiment of the present invention, the flow velocity of the mobile phase of LC/MS is 0.5mL/min.
For method provided by the invention, C18 color is selected according to the polarity of alanine, lysine, glutamic acid, tyrosine
Compose column.The size of chromatographic column can have an impact separating resulting, and internal diameter can have an impact the flow velocity of mobile phase, and length is shorter
Chromatographic column runing time it is short, column pressure it is lower；The longer chromatography column resolution original text of length, but runing time increases.
In an embodiment of the present invention, the chromatographic column of LC/MS is Welch Ultimate SIO2.
In some embodiments, the size of chromatographic column be 4.6 × 150mm, 3 μm.
The characteristic of substance to be separated itself need to be considered the selection of column temperature, and column temperature influences dissolution of the mobile phase to test substance
Degree also will affect column pressure.Under normal circumstances, improve column temperature be conducive to improve separating degree, but temperature it is excessively high will lead to column press through it is low,
It is unfavorable for the detection of substance.
In an embodiment of the present invention, the column temperature of LC/MS is 35 DEG C.
In an embodiment of the present invention, the sample volume of LC/MS is 10 μ L.
In an embodiment of the present invention, the detector of LC/MS is LTQ XL linear ion trap mass spectrograph.
In an embodiment of the present invention, the ion source of LC/MS is ESI；Ion transfer tube temperature is 380 DEG C；Atomizer temperature
180 DEG C of degree；Ion mode is cation；Scanning range is 50Da~500Da；Sweep time is 35min.
In an embodiment of the present invention, the sheath gas of LC/MS is 15arb；Auxiliary gas is 9arb；Blowback air is 3arb；Corona pin
Voltage is 4.0kV.
In an embodiment of the present invention, with mass spectrometry；With quantified by external standard method.
In some embodiments, the method for obtaining alanine content is to be detected according to alanine standard concentration and LC/MS
The peak area of gained alanine draws standard curve, and the content of alanine is obtained according to standard curve.
In some embodiments, the method for drafting of alanine standard curve are as follows: using the concentration of alanine as abscissa, with
The peak area of LC/MS detection gained alanine is that ordinate draws standard curve.
The linear equation of the alanine obtained in method provided by the invention is y=1648x-6564；R2=0.998.
In some embodiments, the method for obtaining lysine content is to be detected according to lysine standard concentration and LC/MS
The peak area of gained lysine draws standard curve, and the content of lysine is obtained according to standard curve.
In some embodiments, the method for drafting of lysine standard curve are as follows: using the concentration of lysine as abscissa, with
The peak area of LC/MS detection gained lysine is that ordinate draws standard curve.
The linear equation of the lysine obtained in method provided by the invention is y=3418x-1543；R2=0.999.
In some embodiments, the method for obtaining content of glutamic acid is to be detected according to glutamic acid standard concentration and LC/MS
The peak area of gained glutamic acid draws standard curve, and the content of glutamic acid is obtained according to standard curve.
In some embodiments, the method for drafting of glutamic acid standard curve are as follows: using the concentration of glutamic acid as abscissa, with
The peak area of LC/MS detection gained glutamic acid is that ordinate draws standard curve.
The linear equation of the glutamic acid obtained in method provided by the invention is y=5537x-1300；R2=0.998.
In some embodiments, the method for obtaining tyrosine content is to be detected according to tyrosine standard concentration and LC/MS
The peak area of gained tyrosine draws standard curve, and the content of tyrosine is obtained according to standard curve.
In some embodiments, the method for drafting of tyrosine standard curve are as follows: using the concentration of tyrosine as abscissa, with
The peak area of LC/MS detection gained tyrosine is that ordinate draws standard curve.
The linear equation of the tyrosine obtained in method provided by the invention is y=5303x-14004；R2=0.998.
Acetic acid copaxone is made of l-Alanine, L-lysine, Pidolidone, l-tyrosine, and therefore, the present invention provides
Method can be applied to the detection of acetic acid copaxone.Acetic acid copaxone of the present invention is acetic acid copaxone injection
Liquid, including acetic acid copaxone and mannitol, the content of acetic acid copaxone is 20mg/ml；The content of mannitol is
40mg/mL。
The present invention also provides a kind of quality determining methods of acetic acid copaxone, comprising: by acetic acid copaxone with carboxylic
It is used as sample to be tested after peptase Y enzymatic hydrolysis, using method provided by the invention to alanine, lysine, glutamic acid and/or tyrosine
It is quantitative.
The quality determining method of acetic acid copaxone provided by the invention using acetic acid copaxone standard items as reference substance, with
The acetic acid copaxone that self-control or market are bought is detection product, under same enzymatic hydrolysis condition, is measured in standard items and detection product
L-Alanine, L-lysine, Pidolidone, l-tyrosine content and/or mass ratio unanimously then detect the up-to-standard of product.
Acetic acid copaxone standard items are acetic acid copaxone injection, are purchased from TEVA.
Content of the present invention unanimously refers to l-Alanine in detection product, L-lysine, Pidolidone, l-tyrosine
Content and l-Alanine in standard items, L-lysine, Pidolidone, l-tyrosine content differ and be no more than ± 2%；The present invention
The mass ratio unanimously refers to l-Alanine in detection product, L-lysine, Pidolidone, the mass ratio of l-tyrosine and standard
The ratio difference of l-Alanine, L-lysine, Pidolidone, l-tyrosine is no more than ± 2% in product.
Carboxypeptidase y is a kind of digestive ferment.It can degrade one by one since the C-terminal of peptide chain in specific manner, release free amine group
A kind of exopeptidase of acid.
In an embodiment of the present invention, the concentration of carboxypeptidase y is 1mg/ml.
In an embodiment of the present invention, the temperature of carboxypeptidase y enzymatic hydrolysis is 25 DEG C, time 5min.
In the embodiment of this law people, the mass ratio of carboxypeptidase y and acetic acid copaxone is 200:1.
Quality testing of the method provided by the invention for acetic acid copaxone injection has good accuracy, mark-on
Recycling the result shows that, after various concentration rate of recovery stock solution is added in the sample, alanine, lysine, glutamic acid, tyrosine
The rate of recovery is 98.1%~101.7%.Meet the requirement of accuracy.
The present invention provides the method for detection alanine, lysine, glutamic acid and/or tyrosine, this method uses LC/MS
Product to be tested is detected, by quantified by external standard method, can have good accuracy and sensitivity, and can be suitably used for acetic acid lattice
Draw the quality testing for replacing thunder.Experiment show method provided by the invention can accurately to alanine, lysine, glutamic acid and/or
Tyrosine carries out accurately quantitatively, and recovery of standard addition is 98.1%~101.7%；Repeated good, 5 needle RSD of continuous sample introduction is all
Less than 2%；Also, minimum detection limit, less than 1.1 μ g/mL, sensitivity is good.
Detailed description of the invention
Fig. 1 shows the mass spectrogram of reference standards；The mass spectrogram of alanine such as Fig. 1-a；The mass spectrogram of lysine such as Fig. 1-b；
The mass spectrogram of glutamic acid such as Fig. 1-c；The mass spectrogram of tyrosine such as Fig. 1-d；
Fig. 2 shows the mass spectrogram of quantitative limit solution；The mass spectrogram of alanine such as Fig. 2-a；The mass spectrogram of lysine such as Fig. 2-b；
The mass spectrogram of glutamic acid such as Fig. 2-c；The mass spectrogram of tyrosine such as Fig. 2-d；
Fig. 3 shows the mass spectrogram of product to be tested；The mass spectrogram of alanine such as Fig. 3-a；The mass spectrogram of lysine such as Fig. 3-b；Paddy ammonia
Mass spectrogram such as Fig. 3-c of acid；The mass spectrogram of tyrosine such as Fig. 3-d.
Specific embodiment
The present invention provides the method for detection alanine, lysine, glutamic acid and/or tyrosine, those skilled in the art
Present disclosure can be used for reference, realization of process parameters is suitably modified.In particular, it should be pointed out that all similar substitutions and modifications pair
It is it will be apparent that they are considered as being included in the present invention for those skilled in the art.Method and application of the invention is
Through being described by preferred embodiment, related personnel obviously can not depart from the content of present invention, in spirit and scope to this
The methods and applications of text are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The instrument or reagent that the present invention uses are all common commercially available product, can all be bought in market.
Reference substance l-Alanine, L-lysine, Pidolidone, l-tyrosine are purchased from Sigma；
Acetic acid copaxone standard items are purchased from TEVA.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1
1, control mother liquor is prepared:
Precision measures l-Alanine 10.28mg, sets in 100ml volumetric flask, is dissolved to scale with water, obtains l-Alanine pair
According to mother liquor；
Precision measures L-lysine 10.60mg, sets in 100ml volumetric flask, is dissolved to scale with water, obtains L-lysine pair
According to mother liquor；
Precision measures Pidolidone 10.12mg, sets in 100ml volumetric flask, is dissolved to scale with water, obtains Pidolidone pair
According to mother liquor；
Precision measures l-tyrosine 10.06mg, sets in 100ml volumetric flask, is dissolved to scale with water, obtains l-tyrosine pair
According to mother liquor.
2,4 parts of control mother liquors respectively take 50 μ L to mix, and add 800 μ l dilution in acetonitrile, sample detection, LC/MS condition are as follows:
Chromatographic column: Welch Ultimate SIO2,4.6 × 150mm, 3 μm；
Flow velocity: 0.5ml/min；
Column temperature: 35 DEG C；
Sample volume: 10 μ L；
Mobile phase A: 25mmol/L ammonium formate solution；
Mobile phase B: acetonitrile；
Condition of gradient elution such as table 1:
1 elution requirement of table
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 10 | 50 | 50 |
| 18 | 80 | 20 |
| 25 | 80 | 20 |
| 25.1 | 50 | 50 |
| 35 | 50 | 50 |
Ion source: ESI；
Sheath gas 15arb, auxiliary gas 9arb；Blowback air: 3arb；
Corona pin voltage: 4.0KV；
Ion transfer tube temperature: 380 DEG C；Atomizer temperature: 180 DEG C；
Ion mode: cation；
Scanning range: 50~500Da；Sweep time: 35min.
Through detecting, the mass spectrogram of alanine such as Fig. 1-a；The mass spectrogram of lysine such as Fig. 1-b；The mass spectrogram of glutamic acid is as schemed
1-c；The mass spectrogram of tyrosine such as Fig. 1-d.
As seen from the figure, four main peaks can all efficiently separate, method provided by the invention can be used for detecting simultaneously l-Alanine,
L-lysine, Pidolidone, l-tyrosine.
Embodiment 2
4 parts of control mother liquors in Example 1, each 50 μ L are mixed.800 μ l dilution in acetonitrile are added and obtain mixing comparison liquid.Even
Continuous 5 needle of sample introduction, LC/MS condition are same as Example 1.The peak area detected every time is recorded, as a result such as table 2:
25 sample introduction peak areas of table
As shown in table 2, each peak area RSD is respectively 1.2%, 0.93%, 1.0%, 1.5%, it is seen that system precision is good
It is good.
Embodiment 3
Each substance lowest detection is limited to the concentration of ingredient when signal-to-noise ratio is 3.
Signal-to-noise ratio computation formula is s/n=2h/hn, in which:
H=peak height corresponding with component
Hn=is being equal to five times of half eminence peak width
Therefore, alanine concentration is 1.028 μ g/mL, lysine concentration is 0.503 μ g/mL, aminoglutaric acid concentration 0.3036
μ g/mL, tyrosine concentration are 0.2012 μ g/mL,
The quantitative limit solution for preparing each amino acid (prepares alanine concentration with 80% acetonitrile solution as 1.028 μ g/
ML, lysine concentration are 0.503 μ g/mL, aminoglutaric acid concentration is 0.3036 μ g/mL, tyrosine concentration is the molten of 0.2012 μ g/mL
Liquid, sample detection.
Through detecting, the mass spectrogram of alanine such as Fig. 2-a；The mass spectrogram of lysine such as Fig. 2-b；The mass spectrogram of glutamic acid is as schemed
2-c；The mass spectrogram of tyrosine such as Fig. 2-d.
As it can be seen that each amino acid can also obtain good detection in minimum detection limit concentration.
Embodiment 4
Four kinds of amino acid are taken, are dissolved using the acetonitrile solution that volume fraction is 80%, the work of concentration shown in table 3 is diluted to
For linear solvent.Sample introduction, LC/MS condition are same as Example 1 respectively.
3 linear solvent concentration of table
| μg/ml | 50% linear solvent | 80% linear solvent | 100% linear solvent | 120% linear solvent | 150% linear solvent |
| Alanine | 25.7 | 41.12 | 51.4 | 61.68 | 77.1 |
| Lysine | 5.3 | 8.48 | 10.6 | 12.72 | 15.9 |
| Glutamic acid | 2.53 | 4.048 | 5.06 | 6.072 | 7.59 |
| Tyrosine | 25.15 | 40.24 | 50.3 | 60.36 | 75.45 |
Using the concentration of each amino acid as abscissa, using peak area obtained by LC/MS detection corresponding concentration amino acid as ordinate
Draw standard curve.
As a result are as follows: the linear equation of alanine is y=1648x-6564；R2=0.998.
The linear equation of lysine is y=3418x-1543；R2=0.999.
The linear equation of glutamic acid is y=5537x-1300；R2=0.998.
The linear equation of tyrosine is y=5303x-14004；R2=0.998.
As it can be seen that method provided by the invention is linearly good.
Embodiment 5
1mg carboxypeptidase y is weighed, adds purified water 1ml to dissolve, is configured to enzymolysis liquid.
By 50 μ L of acetic acid copaxone injection standard items；The mixing of 5 μ L enzymolysis liquids is added, is placed in 25 DEG C of preheated dry types
Insulating box is incubated for 5min.After time arrives, sample introduction after 220 μ L dilution in acetonitrile is added, LC/MS condition is same as Example 1.Gained matter
Spectrogram such as Fig. 3.Wherein, the mass spectrogram of alanine such as Fig. 2-a；The mass spectrogram of lysine such as Fig. 2-b；The mass spectrogram of glutamic acid is as schemed
2-c；The mass spectrogram of tyrosine such as Fig. 2-d.
As it can be seen that using method provided by the invention, it can be by alanine, lysine, paddy in acetic acid copaxone after enzymatic hydrolysis
Propylhomoserin, tyrosine are separated well and are quantified.
Embodiment 6
Accuracy stock solution is prepared: alanine mother liquor 1ml, the lysine mother liquor 0.2ml, glutamic acid of the preparation of Example 1
Mother liquor 0.1ml, tyrosine mother liquor 1ml, set in 5ml volumetric flask, are diluted to scale with water.
By 50 μ L of acetic acid copaxone injection standard items, wherein the concentration of acetic acid copaxone is 20mg/mL, in parallel
9 digestion pipe enzymes are placed in, it is accurate respectively to be added each 3 parts of the rate of recovery stock solution 0.5ml, 1ml, 1.5ml, it mixes, is denoted as respectively
50% accuracy solution, 100% accuracy solution, 150% accuracy solution.Sample after mixing is placed in preheated 25
DEG C dry type insulating box is incubated for 5min.After time arrives, the acetonitrile solution that volume fraction is 80% is added and is diluted to 4ml.Sample introduction inspection
It surveys, four kinds of the recovery of amino acid of 9 parts of samples are obtained by calculation with embodiment 1 in LC/MS condition.As a result 4 be see the table below:
4 recovery of standard addition of table
As it can be seen that the rate of recovery is 98.1%~101.7%, illustrate that accuracy is good.
Embodiment 7
It takes the carboxypeptidase y of 1mg that purified water 1ml is added to dissolve, is configured to enzymolysis liquid.
The acetic acid copaxone solution that 50 μ L are measured using liquid-transfering gun precision, is placed in digestion pipe, and it is mixed that 5 μ L enzymolysis liquids are added
It is even.Sample after mixing is placed in 25 DEG C of preheated dry type insulating boxs and is incubated for 5min.After time arrives, addition adds 220 μ
L dilution in acetonitrile mixes.
6 parts of simultaneously operating, the sample solution after obtaining 6 digestions in parallel, into LC-MS system, LC/MS condition is same
Embodiment 1, is analyzed.It is quantitative with standard curve made from embodiment 4.Calculate the content of four kinds of free amino acids.Obtain four
Amino acid respectively proportion is planted,
The respective percentage of four kinds of amino acid after 56 parts of sample digestions of table
As shown in table 5, each peak area RSD is respectively 1.5%, 1.4%, 0.9%, 0.76%., it is seen that system accuracy is good
It is good.
Embodiment 8
It takes the carboxypeptidase y of 1mg that purified water 1ml is added to dissolve, is configured to enzymolysis liquid.
Acetic acid copaxone standard items and each a batch of homemade acetic acid copaxone product to be detected are taken, according to the following steps in parallel
Processing:
It using the accurate acetic acid copaxone solution for measuring 50 μ L of liquid-transfering gun difference, is placed in digestion pipe, 5 μ L enzymatic hydrolysis is added
Liquid mixes.
Sample after mixing is placed in 25 DEG C of preheated dry type insulating boxs and is incubated for 5min.After time arrives, 220 μ L are added
Dilution in acetonitrile.Sample detection, LC/MS condition are analyzed with embodiment 1.It is quantitative with standard curve made from embodiment 4.
By data processing, the mass fraction of amino acid is obtained.As a result such as table 6:
The mass fraction of each amino acid of table 6
| Alanine | Lysine | Glutamic acid | Tyrosine | |
| Original is ground | 26.89% | 7.26% | 4.82% | 61.05% |
| Self-control | 25.54% | 7.89% | 4.39% | 62.18% |
It is found that each free amino acid proportion that acetic acid copaxone obtains after carboxypeptidase y digestion, and original is ground
Sample is not different substantially with self-control sample, and product is qualified.
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (6)
1. the method for detecting alanine, lysine, glutamic acid and/or tyrosine in the carboxypeptidase y zymolyte of acetic acid copaxone,
It is characterized in that, being analyzed after sample to be tested is dissolved with LC/MS, with external standard method to alanine, lysine, glutamic acid and/or junket
Propylhomoserin is quantitative；
The chromatographic column of the LC/MS is Welch Ultimate SIO2；
The mobile phase A of the LC/MS analysis is mutually 25mmol/L ammonium formate solution；Mobile phase B is mutually acetonitrile；
The gradient of the LC/MS are as follows: 0min~10min, the volume fraction of mobile phase A phase are 50%；
10min~18min, the volume fraction of mobile phase A phase is by 50%~80%；
18min~25min, the volume fraction of mobile phase A phase are 80%；
25min~25.1min, the volume fraction of mobile phase A phase is by 80%~50%；
25.1min~35min, the volume fraction of mobile phase A phase are 50%；
The ion source of the LC/MS is ESI；Ion transfer tube temperature is 380 DEG C；180 DEG C of atomizer temperature；Ion mode is positive
Ion；Scanning range is 50Da~500Da；Sweep time is 35min.
2. the method according to claim 1, wherein the solvent of the dissolution is acetonitrile solution.
3. according to the method described in claim 2, it is characterized in that, the volume fraction of acetonitrile is 40% in the acetonitrile solution
~90%.
4. the method according to claim 1, wherein the flow velocity of the mobile phase of the LC/MS is 0.5mL/min.
5. a kind of quality determining method of acetic acid copaxone, which is characterized in that after digesting acetic acid copaxone with carboxypeptidase y
As sample to be tested, using the described in any item methods of Claims 1 to 4 to alanine, lysine, glutamic acid and/or junket ammonia
Acid cut amount.
6. quality determining method according to claim 5, which is characterized in that the temperature of the carboxypeptidase y enzymatic hydrolysis is 25 DEG C,
Time is 5min.
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