CN106950306A - A kind of method of cysteine content in measure Amino Acid Compound Injection - Google Patents
A kind of method of cysteine content in measure Amino Acid Compound Injection Download PDFInfo
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Abstract
The invention provides a kind of method for determining cysteine content in Amino Acid Compound Injection, aqueous cystein solution is prepared, reference substance solution is used as；It regard Amino Acid Compound Injection as need testing solution；Cysteine in the reference substance solution and need testing solution is derived using N (1 pyrene) maleimides；Reference substance solution after obtained derivative and need testing solution are subjected to efficient liquid phase chromatographic analysis, according to the reference substance solution and the liquid chromatogram of need testing solution after derivative, the content of cysteine in Amino Acid Compound Injection is obtained using external standard method.The method provided using the present invention can realize the Accurate Determining of cysteine content in Amino Acid Compound Injection, and specificity is good, precision is good, the rate of recovery is higher.
Description
Technical field
The present invention relates to medical detection technique field, and in particular to one kind determines cysteine in Amino Acid Compound Injection
The method of content.
Background technology
The Amino Acid Compound Injection that current country has been approved by has more than ten of kind, such as 18AA, 18AA-I, 18AA-II,
18AA-IV, 18AA-V etc..In 20 kinds of amino acid of synthetic protein, cysteine is least stablized, and is easily oxidized to and does not dissolve in
The materials such as the cystine of water.Therefore, in Amino Acid Compound Injection, whether the content of cysteine meets regulation directly reflection
Product quality and technological level.
The content using colorimetric method for determining cysteine is had been reported that in the prior art, specifically utilizes developer and half Guang ammonia
Base acid carries out chromogenic reaction under the conditions of certain pH value, and is detected using UV methods；But the specificity of colorimetric method is poor, show
Toner also has different degrees of colour developing, interference measurement result to other amino acid under corresponding pH value condition.In the prior art also
Have been reported that and cysteine is oxidized to cystine using strong oxidizer, then using ninhydrin post-column derivation-amino-acid analyzer
It is measured；But this method oxidation cysteine condition it is violent, whard to control, and amino-acid analyzer sensitivity compared with
It is low, it is desirable to have the cysteine of higher concentration, it is not suitable for the measure of cysteine content in Amino Acid Compound Injection.In recent years
Come, high performance liquid chromatography is widely used in amino acid analysis, but usually require to derive amino acid to improve detection spirit
Sensitivity.Have been reported that iodoacetic acid-OPA, iodoacetic acid-PICOTAG and N- ethyl maleoyl-s in the prior art
Imines derives as derivating agent to cysteine, but there are the problem of derivative step is more, other amino acid are disturbed, it is impossible to
The content of cysteine in Accurate Determining Amino Acid Compound Injection.
The content of the invention
It is an object of the invention to provide a kind of method for determining cysteine content in Amino Acid Compound Injection, the party
Method specificity is good, precision is good, the rate of recovery is higher.
The invention provides a kind of method for determining cysteine content in Amino Acid Compound Injection, including following step
Suddenly：
(1) aqueous cystein solution is prepared, reference substance solution is used as；
It regard Amino Acid Compound Injection as need testing solution；
Cysteine in the reference substance solution and need testing solution is derived using N- (1- pyrenes) maleimides；
(2) reference substance solution and need testing solution after the derivative for obtaining the step (1) carries out high performance liquid chromatography
Analysis, according to the reference substance solution and the liquid chromatogram of need testing solution after derivative, amino acid is obtained using external standard method
The content of cysteine in parenteral solution.
It is preferred that, cysteine and the mol ratio of N- (1- pyrenes) maleimide are 1 when derived from the step (1)：(1
~3).
It is preferred that, N- (1- pyrenes) maleimides are with N- (1- pyrenes) maleimide acetonitrile solution in the step (1)
Form is used.
It is preferred that, the mass concentration of N- (1- pyrenes) the maleimide acetonitrile solution is 0.15~0.25mg/mL.
It is preferred that, the temperature derived from the step (1) is 15~25 DEG C.
It is preferred that, the time derived from the step (1) is 15~25min.
It is preferred that, efficient liquid phase chromatographic analysis uses PDA detectors in the step (2).
It is preferred that, the Detection wavelength of efficient liquid phase chromatographic analysis is 274nm in the step (2).
It is preferred that, efficient liquid phase chromatographic analysis uses octadecylsilane chemically bonded silica chromatographic column in the step (2).
It is preferred that, the column temperature of efficient liquid phase chromatographic analysis is 30~40 DEG C in the step (2).
The invention provides a kind of method for determining cysteine content in Amino Acid Compound Injection, cysteine is prepared
The aqueous solution, is used as reference substance solution；It regard Amino Acid Compound Injection as need testing solution；Using N- (1- pyrenes) maleimide
Cysteine in the reference substance solution and need testing solution is derived；By the reference substance solution after obtained derivative and confession
Test sample solution carries out efficient liquid phase chromatographic analysis, according to the reference substance solution and the liquid chromatogram of need testing solution after derivative,
The content of cysteine in Amino Acid Compound Injection is obtained using external standard method.The method provided using the present invention can be realized multiple
The Accurate Determining of cysteine content in square amino acid injection, and specificity is good, precision is good, the rate of recovery is higher, Neng Goutong
Cysteine content change is crossed, other amino acid degradation situations in reflection Amino Acid Compound Injection, effectively control compound indirectly
The quality of amino acid injection product.
Brief description of the drawings
Fig. 1 is the uv absorption spectra of cysteine derivative；
Fig. 2 is the uv absorption spectra of N- (1- pyrenes) maleimide；
Fig. 3 is the liquid chromatogram of negative control solution in embodiment 1；
Fig. 4 is the liquid chromatogram of need testing solution in embodiment 1；
Fig. 5 is the liquid chromatogram of negative control solution in embodiment 2；
Fig. 6 is the liquid chromatogram of need testing solution in embodiment 2；
Fig. 7 is the liquid chromatogram of negative control solution in embodiment 3；
Fig. 8 is the liquid chromatogram of need testing solution in embodiment 3.
Embodiment
The invention provides a kind of method for determining cysteine content in Amino Acid Compound Injection, including following step
Suddenly：
(1) aqueous cystein solution is prepared, reference substance solution is used as；
It regard Amino Acid Compound Injection as need testing solution；
Cysteine in the reference substance solution and need testing solution is derived using N- (1- pyrenes) maleimides；
(2) reference substance solution and need testing solution after the derivative for obtaining the step (1) carries out high performance liquid chromatography
Analysis, according to the reference substance solution and the liquid chromatogram of need testing solution after derivative, amino acid is obtained using external standard method
The content of cysteine in parenteral solution.
The present invention prepares aqueous cystein solution, is used as reference substance solution.The present invention is for the dense of the reference substance solution
Degree is without special restriction, using the concentration well known to those skilled in the art matched with cysteine in need testing solution i.e.
Can.The present invention does not have special restriction for preparing the method for the reference substance solution solution, ripe using those skilled in the art
The technical scheme for the preparation solution known.
The present invention regard Amino Acid Compound Injection as need testing solution.The present invention is for the Amino Acid Compound Injection
Species there is no special restriction, be using the Amino Acid Compound Injection well known to those skilled in the art containing cysteine
Can, specific kind such as 18AA-V, 18AA- VII or 18AA-IX.
The present invention is entered using N- (1- pyrenes) maleimides to cysteine in the reference substance solution and need testing solution
Row derives.N- (1- pyrenes) maleimide structure is stable, not degradable, and the present invention uses N- (1- pyrenes) maleimides for derivative
Agent, the cysteine derivative obtained after deriving to cysteine has very strong absorption in ultra-violet (UV) band, can shield compound
The interference of other amino acid in amino acid injection, realizes the Accurate Determining of cysteine content in Amino Acid Compound Injection.
In the present invention, the reaction equation of N- (1- pyrenes) maleimides and cysteine is as follows：
In the present invention, the mol ratio of the cysteine and N- (1- pyrenes) maleimide is preferably 1 when derivative：(1~
3), concretely 1：1、1：2 or 1：3.
In the present invention, N- (1- pyrenes) maleimide is preferably with the shape of N- (1- pyrenes) maleimide acetonitrile solution
Formula is used.In the present invention, the mass concentration of N- (1- pyrenes) the maleimide acetonitrile solution is preferably 0.15~0.25mg/
ML, more preferably 0.18~0.22mg/mL, most preferably 0.20mg/mL.
In the present invention, the pH value of reaction system is preferably when N- (1- pyrenes) maleimide derives to cysteine
5.6~6.4.The present invention to the cysteine when deriving, preferably according to the pH value of the Amino Acid Compound Injection
And contained total amino acid concentration is diluted to the Amino Acid Compound Injection.Reactant when so on the one hand meeting derivative
The pH value requirement of system, on the other hand when carrying out follow-up efficient liquid phase chromatographic analysis, will not cause chromatogram column overload or to efficient
Liquid chromatography device produces harmful effect.In an embodiment of the present invention, to Amino Acid Compound Injection 18AA-V, 18AA-
It is specifically that the Amino Acid Compound Injection is diluted 5 times, then when cysteine content is measured in VII or 18AA-IX
Mixed with N- (1- pyrenes) maleimide acetonitrile solution, carry out derivatization reaction.
In the present invention, the derivative temperature is preferably 15~25 DEG C, more preferably 18~22 DEG C, most preferably 20
℃；The derivative time is preferably 15~25min, more preferably 18~22min, most preferably 20min.
Complete after the derivative, the reference substance solution after obtained derivative and need testing solution are carried out efficient liquid by the present invention
Analysis of hplc, according to the reference substance solution and the liquid chromatogram of need testing solution after derivative, compound is obtained using external standard method
The content of cysteine in amino acid injection.
The mobile phase and type of elution used when the present invention is for the efficient liquid phase chromatographic analysis does not have special limit
It is fixed, according to the species of surveyed Amino Acid Compound Injection, using mobile phase well known to those skilled in the art and type of elution
.
Specifically, in the present invention, during using Amino Acid Compound Injection 18AA-V as sample, the high performance liquid chromatography point
Analysis is preferred to use following gradient elution program：
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 18 | 40 | 60 |
| 25 | 10 | 90 |
| 33 | 10 | 90 |
| 34 | 90 | 10 |
| 40 | 90 | 10 |
Wherein, mobile phase A is 0.01mol/L sodium dihydrogen phosphate, and the pH value of the mobile phase A is 2.4~3.4；
Mobile phase B is acetonitrile.
In the present invention, the pH value of the mobile phase A is preferably 2.4~3.4, and more preferably 2.8 ± 0.1.The present invention is excellent
Choosing adjusts the pH value of the mobile phase A using phosphoric acid；The mass percentage concentration of the phosphoric acid is preferably 8~12%, more preferably
10%.
Specifically, in the present invention, during with Amino Acid Compound Injection 18AA- VII for sample, the high performance liquid chromatography
Analysis is preferred to use following gradient elution program：
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 8 | 40 | 60 |
| 18 | 10 | 90 |
| 24 | 10 | 90 |
| 25 | 90 | 10 |
Wherein, mobile phase A is 0.015mol/L sodium acetate solution, and the pH value of the mobile phase A is 2.8 ± 0.1；Flowing
Phase B is acetonitrile.
Present invention preferably employs the pH value that phosphoric acid adjusts the mobile phase A；The mass percentage concentration of the phosphoric acid is preferably 8
~12%, more preferably 10%.
Specifically, in the present invention, during using Amino Acid Compound Injection 18AA-IX as sample, the high performance liquid chromatography
Analysis is preferred to use following gradient elution program：
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 8 | 40 | 60 |
| 18 | 10 | 90 |
| 24 | 10 | 90 |
| 25 | 90 | 10 |
| 30 | 90 | 10 |
Wherein, mobile phase A is 0.01mol/L sodium dihydrogen phosphate, and the pH value of the mobile phase A is 2.8 ± 0.1；
Mobile phase B is acetonitrile.
Present invention preferably employs the pH value that phosphoric acid adjusts the mobile phase A；The mass percentage concentration of the phosphoric acid is preferably 8
~12%, more preferably 10%.
In the present invention, the flow velocity of the mobile phase is preferably 0.8~1.2mL/min, more preferably 1.0mL/min.
In the present invention, the efficient liquid phase chromatographic analysis is preferred to use PDA detectors.
In the present invention, the Detection wavelength of the efficient liquid phase chromatographic analysis is preferably 274nm.The present invention is using PDA inspections
Survey device to detect cysteine derivative and N- (1- pyrenes) maleimide, obtain cysteine derivative and N- (1- pyrenes)
The uv absorption spectra of maleimide, is shown in Fig. 1 and Fig. 2.From Fig. 1 and Fig. 2, cysteine derivative and N- (1- pyrenes)
The ultra-violet absorption spectrum of maleimide is similar, is to have larger absworption peak, and big portion at that wavelength at 274nm in wavelength
Point amino acid not appearance, it is to avoid the interference of other amino acid.
In the present invention, the efficient liquid phase chromatographic analysis is preferred to use octadecylsilane chemically bonded silica chromatographic column.
In the present invention, the column temperature of the efficient liquid phase chromatographic analysis is preferably 30~40 DEG C, more preferably 33~37 DEG C,
Most preferably 35 DEG C.
In the present invention, the sample size of the efficient liquid phase chromatographic analysis is preferably 10 μ L.
The liquid chromatogram of reference substance solution and need testing solution after being derived through the efficient liquid phase chromatographic analysis,
The present invention obtains the content of cysteine in Amino Acid Compound Injection using external standard method.In practical application, can be according to need
Will, the content of cysteine hydrochloride is converted into by the content of cysteine, the Guang of hydrochloric acid half in Amino Acid Compound Injection is realized
The measure of histidine content.
Below in conjunction with the embodiment in the present invention, the technical scheme in the present invention is clearly and completely described.It is aobvious
So, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based on the reality in the present invention
Example is applied, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made all belongs to
In the scope of protection of the invention.
Embodiment 1
Determine the content of cysteine hydrochloride in Amino Acid Compound Injection 18AA-V
(1) using the Hubei amino acid 18AA-V parenteral solutions that in a day or two prepared by pharmaceutical Co. Ltd as sample, determine multiple
The method of CYSTEAMINE HCL acid content, comprises the following steps in square amino acid injection 18AA-V：
(11) precision weighs cysteine, is mixed with water, prepares 0.06mg/mL aqueous cystein solution, is used as control
Product solution；
Amino Acid Compound Injection is prepared according to Amino Acid Compound Injection 18AA-V formula, 5 times are diluted with water, as
Need testing solution；
N- (1- pyrenes) maleimide is mixed with acetonitrile, N- (1- pyrenes) Malaysia acyl that mass concentration is 0.2mg/mL is obtained
Imines acetonitrile solution, is used as derivative agent solution；
(12) it is respectively that 0.5mL reference substance solutions in the step (11) and 0.5mL need testing solutions and 1mL derivating agents is molten
Liquid is mixed, derivative 20min under the conditions of 20 DEG C；
(13) reference substance solution and need testing solution after the derivative for obtaining the step (12) carries out high-efficient liquid phase color
Analysis of spectrum, according to the reference substance solution and the liquid chromatogram of need testing solution after derivative, after being derived using external standard method
The content of cysteine in need testing solution, and then obtain the content of cysteine in Amino Acid Compound Injection 18AA-V；It is logical
The content for crossing cysteine is converted into the content of cysteine hydrochloride, obtains the Guang of hydrochloric acid half in Amino Acid Compound Injection 18AA-V
The content of propylhomoserin；
Wherein, the condition of the efficient liquid phase chromatographic analysis is specific as follows：
Gradient elution program：
Wherein, mobile phase A is 0.01mol/L sodium dihydrogen phosphate, and the pH value of the mobile phase A is 2.8 ± 0.1；
Mobile phase B is acetonitrile；
The flow velocity of mobile phase is 1.0mL/min；
Using PDA detectors, Detection wavelength is 274nm；
Using octadecylsilane chemically bonded silica chromatographic column, column temperature is 35 DEG C；
Sample size is 10 μ L.
(2) method specificity is tested
According to Amino Acid Compound Injection 18AA-V formula, the mixed solution without cysteine is prepared, 5 are diluted with water
Times, it is used as negative control solution；
Amino Acid Compound Injection is prepared according to Amino Acid Compound Injection 18AA-V formula, 5 times are diluted with water, as
Need testing solution；
After the negative control solution and need testing solution are derived according to the method described in step (12), according to step
(13) method described in carries out efficient liquid phase chromatographic analysis, as a result sees Fig. 3 and Fig. 4.Fig. 3 is the liquid chromatogram of negative control solution
A is miscellaneous peak at 12.153min in figure, figure, and 2 be N- (1- pyrenes) maleimide；Fig. 4 is the liquid chromatogram of need testing solution,
1 is cysteine derivative in figure, and 2 be N- (1- pyrenes) maleimide.From Fig. 3 and Fig. 4, negative control solution is in half Guang
It is noiseless to cysteine assay without miscellaneous peak at chromatographic peak, and 12.153min at the retention time of threonine derivative；After derivative not
The measure of N- (1- pyrenes) maleimides also without interference with cysteine of reaction.
The chromatogram peak purity of cysteine derivative in need testing solution is detected by PDA detectors, as a result seen
Table 1.As shown in Table 1, the chromatographic peak of cysteine derivative is single pure color spectral peak, and method specificity is good.
The chromatographic peak purity detecting result of cysteine derivative in the need testing solution of table 1
(3) linear test
According to Amino Acid Compound Injection 18AA-V formula, the mixed solution without cysteine is prepared, 5 are diluted with water
Times, it is used as negative control solution；
Precision weighs cysteine 74.2mg, is mixed with negative control solution described in 500mL, obtains cysteine standard molten
Liquid；Precision measures cysteine standard solution and is diluted with water respectively, and it is respectively 10.2 μ g/mL, 30.7 μ g/ to obtain mass concentration
ML, 61.4 μ g/mL, 71.6 μ g/mL and 102.3 μ g/mL cysteine standard solution；
After the cysteine standard solution of obtained different diluted concentrations is derived according to the method described in step (12), press
Efficient liquid phase chromatographic analysis is carried out according to the method described in step (13), using the peak area of cysteine derivative as ordinate, half
The concentration of cystine is abscissa, and peak area makees linear equation to concentration, obtains the standard curve of cysteine；The half Guang ammonia
Calibration curve equation, coefficient correlation and the range of linearity of acid are specifically shown in Table 2.As shown in Table 2, cysteine is in 10.2~102.3 μ
It is linear good in the range of g/mL, coefficient R2For 0.9995.
Calibration curve equation, coefficient correlation and the range of linearity of the cysteine of table 2
In table 2, X is the mass concentration of cysteine, the liquid chromatogram peak area of Y correspondence cysteine derivatives.
(4) recovery of standard addition is tested
Precision weighs cysteine, is mixed with water, prepares 0.060mg/mL aqueous cystein solution, molten as reference substance
Liquid；
Amino Acid Compound Injection is prepared according to Amino Acid Compound Injection 18AA-V formula, 5 times are diluted with water, as
Need testing solution；
Precision weighs cysteine respectively, is mixed with the need testing solution, prepares cysteine mass concentration and is respectively
(semicystinol concentration is respectively measure concentration to 0.048mg/mL, 0.060mg/mL and 0.072mg/mL mixed solution
80%, the 100% of 0.060mg/mL and 120%) each three parts, it is used as mark-on reclaims testing liquid；
After the reference substance solution and mark-on reclaims testing liquid are derived according to the method described in step (12), according to step
Suddenly the method described in (13) carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, and be converted into by the content of cysteine
The content of cysteine hydrochloride, produces the rate of recovery of cysteine hydrochloride, the results are shown in Table 3.As shown in Table 3, cysteine hydrochloride
The rate of recovery between 97.51%~101.78%, average recovery rate be 100.2%, RSD (%) be 1.4 (n=9), illustrate survey
Determine result accurate.
The recovery of standard addition result of the test of table 3
(5) replica test and Intermediate precision experiment
Replica test
Amino Acid Compound Injection using the Hubei batch number that in a day or two prepared by pharmaceutical Co. Ltd as 20150101
18AA-V is sample, and the Amino Acid Compound Injection 18AA-V is diluted into 5 times with water, need testing solution is used as；
Precision weighs cysteine, is mixed with water, prepares 0.06mg/mL aqueous cystein solution, molten as reference substance
Liquid；
After the need testing solution and reference substance solution are derived according to the method described in step (12), according to step (13)
Described method carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, and is converted into hydrochloric acid half by the content of cysteine
The content of cystine, continuous experiment 6 times, the peak area of each cysteine derivative of comparison and the sign of cysteine hydrochloride contain
Amount, calculates RSD (%), the results are shown in Table 4.
The replica test result of table 4
| Replica test | Cysteine derivative peak area | The sign content (%) of cysteine hydrochloride |
| 1 | 3224110 | 90.16 |
| 2 | 3223125 | 90.14 |
| 3 | 3168773 | 88.62 |
| 4 | 3308234 | 92.52 |
| 5 | 3264078 | 91.28 |
| 6 | 3216250 | 89.95 |
| RSD (%) | 1.46 | 1.46 |
Intermediate precision is tested
Laboratory technician A and laboratory technician B, in different test periods, using different instruments, carries out following Intermediate precision examination
Test：
Amino Acid Compound Injection using the Hubei batch number that in a day or two prepared by pharmaceutical Co. Ltd as 20150101
18AA-V is sample, and the Amino Acid Compound Injection 18AA-V is diluted into 5 times with water, need testing solution is used as；
Precision weighs cysteine, is mixed with water, prepares 0.06mg/mL aqueous cystein solution, molten as reference substance
Liquid；
After the need testing solution and reference substance solution are derived according to the method described in step (12), according to step (13)
Described method carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, and is converted into hydrochloric acid half by the content of cysteine
The content of cystine, continuous experiment 6 times, the peak area of each cysteine derivative of comparison and the sign of cysteine hydrochloride contain
Amount, calculates RSD (%), the results are shown in Table 5.
The Intermediate precision result of the test of table 5
| Intermediate precision is tested | Cysteine derivative peak area | The sign content (%) of cysteine hydrochloride |
| 1 | 3297537 | 90.17 |
| 2 | 3189865 | 87.22 |
| 3 | 3238807 | 88.56 |
| 4 | 3308464 | 90.47 |
| 5 | 3206619 | 87.68 |
| 6 | 3286616 | 89.87 |
| RSD (%) | 1.54 | 1.54 |
Calculate the average value of 12 sign content datas of cysteine hydrochloride in replica test and Intermediate precision experiment
It is 1.7% for 89.72%, RSD (%), illustrates that the method that the present invention is provided has good precision.
(6) stability test
Amino Acid Compound Injection using the Hubei batch number that in a day or two prepared by pharmaceutical Co. Ltd as 20150101
Under the conditions of 18AA-V is sample, room temperature (22 DEG C), open respectively at 0h, 1h, 2h and 4h sampling after plug, respectively will be described with water
Amino acid 18AA-V parenteral solutions dilute 5 times, after deriving according to the method described in step (12), according to described in step (13)
Method carries out efficient liquid phase chromatographic analysis, calculates RSD (%), the results are shown in Table 6, illustrates behind the sample Kaifeng in 4h internal stabilities
Well.
The stability test result of table 6
(7) sample is determined
The Amino Acid Compound Injection 18AA-V prepared using Hubei pharmaceutical Co. Ltd's pilot scale in a day or two as sample, (advise by sample
Lattice are respectively 100mL and 500mL), the Amino Acid Compound Injection 18AA-V is diluted 5 times with water, need testing solution is used as；
Precision weighs cysteine, is mixed with water, prepares 0.06mg/mL aqueous cystein solution, molten as reference substance
Liquid；
After the need testing solution and reference substance solution are derived according to the method described in step (12), according to step (13)
Described method carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, and is converted into hydrochloric acid half by the content of cysteine
The content of cystine, the results are shown in Table 7 and table 8.
Table 7 sample (specification 100mL) measurement result
Table 8 sample (specification 500mL) measurement result
As can be seen that the method provided using the present invention can realize Amino Acid Compound Injection 18AA- from embodiment 1
The Accurate Determining of cysteine content in V, and specificity is good, precision is good, the rate of recovery is higher.The method that the present invention is provided can
The quality of Amino Acid Compound Injection 18AA-V products is effectively controlled, while consider the factors such as big production, storage and transport,
In the case of 70% that CYSTEAMINE HCL acid content is not less than sign content, indirectly in reflection Amino Acid Compound Injection 18AA-V
Other amino acid are without degraded.
Embodiment 2
Determine the content of cysteine in Amino Acid Compound Injection 18AA- VII
(1) using the Hubei parenteral solutions of amino acid 18AA- VII that in a day or two prepared by pharmaceutical Co. Ltd as sample, determine multiple
The method of cysteine content, comprises the following steps in square amino acid injection 18AA- VII：
(11) precision weighs cysteine, is mixed with water, prepares 0.07mg/mL aqueous cystein solution, is used as control
Product solution；
Amino Acid Compound Injection is prepared according to Amino Acid Compound Injection 18AA- VII formula, 5 times, work are diluted with water
For need testing solution；
N- (1- pyrenes) maleimide is mixed with acetonitrile, N- (1- pyrenes) Malaysia acyl that mass concentration is 0.2mg/mL is obtained
Imines acetonitrile solution, is used as derivative agent solution；
(12) it is respectively that 0.5mL reference substance solutions in the step (11) and 0.5mL need testing solutions and 1mL derivating agents is molten
Liquid is mixed, derivative 20min under the conditions of 20 DEG C；
(13) reference substance solution and need testing solution after the derivative for obtaining the step (12) carries out high-efficient liquid phase color
Analysis of spectrum, according to the reference substance solution and the liquid chromatogram of need testing solution after derivative, after being derived using external standard method
The content of cysteine in need testing solution, and then obtain the content of cysteine in Amino Acid Compound Injection 18AA- VII；
Wherein, the condition of the efficient liquid phase chromatographic analysis is specific as follows：
Gradient elution program：
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 8 | 40 | 60 |
| 18 | 10 | 90 |
| 24 | 10 | 90 |
| 25 | 90 | 10 |
Wherein, mobile phase A is 0.015mol/L sodium acetate solution, and the pH value of the mobile phase A is 2.8 ± 0.1；Flowing
Phase B is acetonitrile.
The flow velocity of mobile phase is 1.0mL/min；
Using PDA detectors, Detection wavelength is 274nm；
Using octadecylsilane chemically bonded silica chromatographic column, column temperature is 35 DEG C；
Sample size is 10 μ L.
(2) method specificity is tested
According to Amino Acid Compound Injection 18AA- VII formula, the mixed solution without cysteine is prepared, is diluted with water
5 times, it is used as negative control solution；
Amino Acid Compound Injection is prepared according to Amino Acid Compound Injection 18AA- VII formula, 5 times, work are diluted with water
For need testing solution；
After the negative control solution and need testing solution are derived according to the method described in step (12), according to step
(13) method described in carries out efficient liquid phase chromatographic analysis, as a result sees Fig. 5 and Fig. 6.Fig. 5 is the liquid chromatogram of negative control solution
2 be N- (1- pyrenes) maleimide in figure, figure；Fig. 6 is the liquid chromatogram of need testing solution, and 1 is cysteine derivatives in figure
Thing, 2 be N- (1- pyrenes) maleimide.From Fig. 5 and Fig. 6, negative control solution is in the reservation of cysteine derivative
Between place without chromatographic peak；The unreacted measure of N- (1- pyrenes) maleimides also without interference with cysteine after derivative.
The chromatogram peak purity of cysteine derivative in need testing solution is detected by PDA detectors, as a result seen
Table 9.As shown in Table 9, the chromatographic peak of cysteine derivative is single pure color spectral peak, and method specificity is good.
The chromatographic peak purity detecting result of cysteine derivative in the need testing solution of table 9
(3) linear test
According to Amino Acid Compound Injection 18AA- VII formula, the mixed solution without cysteine is prepared, is diluted with water
5 times, it is used as negative control solution；
Precision weighs cysteine 100.0mg, is mixed with negative control solution described in 100mL, obtains cysteine standard
Solution；Precision measures cysteine standard solution and is diluted with water respectively, and it is respectively 10.0 μ g/mL, 30.0 μ to obtain mass concentration
G/mL, 50.0 μ g/mL, 70.0 μ g/mL and 90.0 μ g/mL cysteine standard solution；
After the cysteine standard solution of obtained different diluted concentrations is derived according to the method described in step (12), press
Efficient liquid phase chromatographic analysis is carried out according to the method described in step (13), using the peak area of cysteine derivative as ordinate, half
The concentration of cystine is abscissa, and peak area makees linear equation to concentration, obtains the standard curve of cysteine；The half Guang ammonia
Calibration curve equation, coefficient correlation and the range of linearity of acid are specifically shown in Table 10.As shown in Table 10, cysteine is 10.0~90.0
It is linear good in the range of μ g/mL, coefficient R2For 0.9997.
Calibration curve equation, coefficient correlation and the range of linearity of the cysteine of table 10
In table 10, X is the mass concentration of cysteine, the liquid chromatogram peak area of Y correspondence cysteine derivatives.
(4) recovery of standard addition is tested
Precision weighs cysteine, is mixed with water, prepares 0.070mg/mL aqueous cystein solution, molten as reference substance
Liquid；
Amino Acid Compound Injection is prepared according to Amino Acid Compound Injection 18AA- VII formula, 5 times, work are diluted with water
For need testing solution；
Precision weighs cysteine respectively, is mixed with the need testing solution, prepares cysteine mass concentration and is respectively
(semicystinol concentration is respectively measure concentration to 0.056mg/mL, 0.070mg/mL and 0.084mg/mL mixed solution
80%, the 100% of 0.070mg/mL and 120%) each three parts, it is used as mark-on reclaims testing liquid；
After the reference substance solution and mark-on reclaims testing liquid are derived according to the method described in step (12), according to step
Suddenly the method described in (13) carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, produces the rate of recovery of cysteine, as a result see
Table 11.As shown in Table 11, the rate of recovery of cysteine is between 99.10%~100.90%, and average recovery rate is 100.08%,
RSD (%) is 0.63 (n=9), illustrates that measurement result is accurate.
The recovery of standard addition result of the test of table 11
(5) replica test and Intermediate precision experiment
Replica test
Amino Acid Compound Injection using the Hubei batch number that in a day or two prepared by pharmaceutical Co. Ltd as 20140601
18AA-VII is sample, and the Amino Acid Compound Injection 18AA-VII is diluted into 5 times with water, need testing solution is used as；
Precision weighs cysteine, is mixed with water, prepares 0.07mg/mL aqueous cystein solution, molten as reference substance
Liquid；
After the need testing solution and reference substance solution are derived according to the method described in step (12), according to step (13)
Described method carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, continuous experiment 6 times, relatively each cysteine derivatives
The peak area of thing and the sign content of cysteine, calculate RSD (%), the results are shown in Table 12.
The replica test result of table 12
| Replica test | Cysteine derivative peak area | The sign content (%) of cysteine |
| 1 | 3547609 | 90.5 |
| 2 | 3578264 | 91.2 |
| 3 | 3631192 | 92.6 |
| 4 | 3591465 | 91.6 |
| 5 | 3541780 | 90.3 |
| 6 | 3551058 | 90.5 |
| RSD (%) | 0.97 | 0.97 |
Intermediate precision is tested
Laboratory technician A and laboratory technician B, in different test periods, using different instruments, carries out following Intermediate precision examination
Test：
Amino Acid Compound Injection using the Hubei batch number that in a day or two prepared by pharmaceutical Co. Ltd as 20140601
18AA-VII is sample, and the Amino Acid Compound Injection 18AA-VII is diluted into 5 times with water, need testing solution is used as；
Precision weighs cysteine, is mixed with water, prepares 0.07mg/mL aqueous cystein solution, molten as reference substance
Liquid；
After the need testing solution and reference substance solution are derived according to the method described in step (12), according to step (13)
Described method carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, continuous experiment 6 times, relatively each cysteine derivatives
The peak area of thing and the sign content of cysteine, calculate RSD (%), the results are shown in Table 13.
The Intermediate precision result of the test of table 13
| Intermediate precision is tested | Cysteine derivative peak area | The sign content (%) of cysteine |
| 1 | 3541534 | 90.3 |
| 2 | 3545456 | 90.4 |
| 3 | 3576832 | 91.2 |
| 4 | 3470939 | 88.5 |
| 5 | 3537612 | 90.2 |
| 6 | 3557222 | 90.7 |
| RSD (%) | 1.01 | 1.01 |
Calculating 12 average values for indicating content datas of cysteine in replica test and Intermediate precision experiment is
90.67%, RSD (%) are 1.08%, illustrate that the method that the present invention is provided has good precision.
(6) stability test
Amino Acid Compound Injection using the Hubei batch number that in a day or two prepared by pharmaceutical Co. Ltd as 20140601
Under the conditions of 18AA-VII is sample, room temperature (22 DEG C), open respectively at 0h, 1h, 2h, 3h, 4h sampling after plug, respectively will with water
The amino acid 18AA-VII parenteral solutions dilute 5 times, after deriving according to the method described in step (12), according to step (13)
Described method carries out efficient liquid phase chromatographic analysis, calculates RSD (%), the results are shown in Table 14, illustrates behind the sample Kaifeng in 4h
Internal stability is good.
The stability test result of table 14
(7) sample is determined
With Hubei each three crowdes of Amino Acid Compound Injection 18AA-VII that in a day or two prepared by pharmaceutical Co. Ltd's lab scale, pilot scale
For sample, the Amino Acid Compound Injection 18AA-VII is diluted 5 times with water, need testing solution is used as；
Precision weighs cysteine, is mixed with water, prepares 0.07mg/mL aqueous cystein solution, molten as reference substance
Liquid；
After the need testing solution and reference substance solution are derived according to the method described in step (12), according to step (13)
Described method carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, obtains the sign content of cysteine, the results are shown in Table
15。
The sample measurement result of table 15
As can be seen that the method provided using the present invention can realize Amino Acid Compound Injection 18AA- from embodiment 2
The Accurate Determining of cysteine content in VII, and specificity is good, precision is good, the rate of recovery is higher.The method energy that the present invention is provided
The quality of enough effectively control Amino Acid Compound Injection 18AA-VII products, while considering big production, storage and transport etc.
Factor, in the case of 80% that cysteine content is not less than sign content, reflects Amino Acid Compound Injection 18AA-V indirectly
In other amino acid without degraded.
Embodiment 3
Determine the content of cysteine in Amino Acid Compound Injection 18AA-IX
(1) using the Hubei amino acid 18AA-IX parenteral solutions that in a day or two prepared by pharmaceutical Co. Ltd as sample, determine multiple
The method of cysteine content, comprises the following steps in square amino acid injection 18AA-IX：
(11) precision weighs cysteine, is mixed with water, prepares 0.05mg/mL aqueous cystein solution, is used as control
Product solution；
Amino Acid Compound Injection is prepared according to Amino Acid Compound Injection 18AA-IX formula, 5 times, work are diluted with water
For need testing solution；
N- (1- pyrenes) maleimide is mixed with acetonitrile, N- (1- pyrenes) Malaysia acyl that mass concentration is 0.2mg/mL is obtained
Imines acetonitrile solution, is used as derivative agent solution；
(12) it is respectively that 0.5mL reference substance solutions in the step (11) and 0.5mL need testing solutions and 1mL derivating agents is molten
Liquid is mixed, derivative 20min under the conditions of 20 DEG C；
(13) reference substance solution and need testing solution after the derivative for obtaining the step (12) carries out high-efficient liquid phase color
Analysis of spectrum, according to the reference substance solution and the liquid chromatogram of need testing solution after derivative, after being derived using external standard method
The content of cysteine in need testing solution, and then obtain the content of cysteine in Amino Acid Compound Injection 18AA-IX；
Wherein, the condition of the efficient liquid phase chromatographic analysis is specific as follows：
Gradient elution program：
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 8 | 40 | 60 |
| 18 | 10 | 90 |
| 24 | 10 | 90 |
| 25 | 90 | 10 |
| 30 | 90 | 10 |
Wherein, mobile phase A is 0.01mol/L sodium dihydrogen phosphate, and the pH value of the mobile phase A is 2.8 ± 0.1；
Mobile phase B is acetonitrile；
The flow velocity of mobile phase is 1.0mL/min；
Using PDA detectors, Detection wavelength is 274nm；
Using octadecylsilane chemically bonded silica chromatographic column, column temperature is 35 DEG C；
Sample size is 10 μ L.
(2) method specificity is tested
According to Amino Acid Compound Injection 18AA-IX formula, the mixed solution without cysteine is prepared, is diluted with water
5 times, it is used as negative control solution；
Amino Acid Compound Injection is prepared according to Amino Acid Compound Injection 18AA-IX formula, 5 times, work are diluted with water
For need testing solution；
After the negative control solution and need testing solution are derived according to the method described in step (12), according to step
(13) method described in carries out efficient liquid phase chromatographic analysis, as a result sees Fig. 7 and Fig. 8.Fig. 7 is the liquid chromatogram of negative control solution
2 be N- (1- pyrenes) maleimide in figure, figure；Fig. 8 is the liquid chromatogram of need testing solution, and 1 is cysteine derivatives in figure
Thing, 2 be N- (1- pyrenes) maleimide.From Fig. 7 and Fig. 8, negative control solution is in the reservation of cysteine derivative
Between place without chromatographic peak；The unreacted measure of N- (1- pyrenes) maleimides also without interference with cysteine after derivative.
The chromatogram peak purity of cysteine derivative in need testing solution is detected by PDA detectors, as a result seen
Table 16.As shown in Table 16, the chromatographic peak of cysteine derivative is single pure color spectral peak, and method specificity is good.
The chromatographic peak purity detecting result of cysteine derivative in the need testing solution of table 16
(3) linear test
According to Amino Acid Compound Injection 18AA-IX formula, the mixed solution without cysteine is prepared, is diluted with water
5 times, it is used as negative control solution；
Precision weighs cysteine 97.6mg, is mixed with negative control solution described in 100mL, obtains cysteine standard molten
Liquid；Precision measures cysteine standard solution and is diluted with water respectively, and it is respectively 9.8 μ g/mL, 19.7 μ g/ to obtain mass concentration
ML, 39.4 μ g/mL, 59.0 μ g/mL and 78.7 μ g/mL cysteine standard solution；
After the cysteine standard solution of obtained different diluted concentrations is derived according to the method described in step (12), press
Efficient liquid phase chromatographic analysis is carried out according to the method described in step (13), using the peak area of cysteine derivative as ordinate, half
The concentration of cystine is abscissa, and peak area makees linear equation to concentration, obtains the standard curve of cysteine；The half Guang ammonia
Calibration curve equation, coefficient correlation and the range of linearity of acid are specifically shown in Table 17.As shown in Table 17, cysteine is in 9.8~78.7 μ
It is linear good in the range of g/mL, coefficient R2For 0.9998.
Calibration curve equation, coefficient correlation and the range of linearity of the cysteine of table 17
In table 2, X is the mass concentration of cysteine, the liquid chromatogram peak area of Y correspondence cysteine derivatives.
(4) recovery of standard addition is tested
Precision weighs cysteine, is mixed with water, prepares 0.05mg/mL aqueous cystein solution, molten as reference substance
Liquid；
Amino Acid Compound Injection is prepared according to Amino Acid Compound Injection 18AA-IX formula, 5 times, work are diluted with water
For need testing solution；
Precision weighs cysteine respectively, is mixed with the need testing solution, prepares cysteine mass concentration and is respectively
(semicystinol concentration is respectively to determine concentration 0.05mg/mL to 0.04mg/mL, 0.05mg/mL and 0.06mg/mL mixed solution
80%, 100% and 120%) each three parts, be used as mark-on reclaims testing liquid；
After the reference substance solution and mark-on reclaims testing liquid are derived according to the method described in step (12), according to step
Suddenly the method described in (13) carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, produces the rate of recovery of cysteine, as a result see
Table 18.As shown in Table 18, the rate of recovery of cysteine is between 97.76%~100.74%, and average recovery rate is 99.28%,
RSD (%) is 1.06 (n=9), illustrates that measurement result is accurate.
The recovery of standard addition result of the test of table 18
(5) replica test and Intermediate precision experiment
Replica test
Amino Acid Compound Injection using the Hubei batch number that in a day or two prepared by pharmaceutical Co. Ltd as 20140601
18AA-IX is sample, and the Amino Acid Compound Injection 18AA-IX is diluted into 5 times with water, need testing solution is used as；
Precision weighs cysteine, is mixed with water, prepares 0.05mg/mL aqueous cystein solution, molten as reference substance
Liquid；
After the need testing solution and reference substance solution are derived according to the method described in step (12), according to step (13)
Described method carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, continuous experiment 6 times, relatively each cysteine derivatives
The peak area of thing and the sign content of cysteine, calculate RSD (%), the results are shown in Table 19.
The replica test result of table 19
Intermediate precision is tested
Laboratory technician A and laboratory technician B, in different test periods, using different instruments, carries out following Intermediate precision examination
Test：
Amino Acid Compound Injection using the Hubei batch number that in a day or two prepared by pharmaceutical Co. Ltd as 20140601
18AA-IX is sample, and the Amino Acid Compound Injection 18AA-IX is diluted into 5 times with water, need testing solution is used as；
Precision weighs cysteine, is mixed with water, prepares 0.05mg/mL aqueous cystein solution, molten as reference substance
Liquid；
After the need testing solution and reference substance solution are derived according to the method described in step (12), according to step (13)
Described method carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, continuous experiment 6 times, relatively each cysteine derivatives
The peak area of thing and the sign content of cysteine, calculate RSD (%), the results are shown in Table 20.
The Intermediate precision result of the test of table 20
| Intermediate precision is tested | Cysteine derivative peak area | The sign content (%) of cysteine |
| 1 | 2012490 | 87.24 |
| 2 | 2025097 | 87.79 |
| 3 | 2145202 | 93.00 |
| 4 | 2111130 | 91.52 |
| 5 | 2111368 | 91.53 |
| 6 | 2113104 | 91.61 |
| RSD (%) | 2.59 | 2.59 |
Calculating 12 average values for indicating content datas of cysteine in replica test and Intermediate precision experiment is
90.32%, RSD (%) are 1.81%, illustrate that the method that the present invention is provided has good precision.
(6) stability test
Amino Acid Compound Injection using the Hubei batch number that in a day or two prepared by pharmaceutical Co. Ltd as 20140601
Under the conditions of 18AA-IX is sample, room temperature (22 DEG C), open after plug respectively at 0min, 80min, 160min, 240min and
320min is sampled, and the amino acid 18AA-IX parenteral solutions is diluted into 5 times with water respectively, according to the side described in step (12)
After method derives, efficient liquid phase chromatographic analysis is carried out according to the method described in step (13), RSD (%) is calculated, the results are shown in Table 21, say
It is good in 5h internal stabilities behind the bright sample Kaifeng.
The stability test result of table 21
(7) sample is determined
With Hubei each three crowdes of Amino Acid Compound Injection 18AA-IX that in a day or two prepared by pharmaceutical Co. Ltd's lab scale, pilot scale
For sample, the Amino Acid Compound Injection 18AA-IX is diluted 5 times with water, need testing solution is used as；
Precision weighs cysteine, is mixed with water, prepares 0.05mg/mL aqueous cystein solution, molten as reference substance
Liquid；
After the need testing solution and reference substance solution are derived according to the method described in step (12), according to step (13)
Described method carries out efficient liquid phase chromatographic analysis, is calculated by external standard method, obtains the sign content of cysteine, the results are shown in Table
22。
The sample measurement result of table 22
As can be seen that the method provided using the present invention can realize Amino Acid Compound Injection 18AA- from embodiment 3
The Accurate Determining of cysteine content in IX, and specificity is good, precision is good, the rate of recovery is higher.The method energy that the present invention is provided
The quality of enough effectively control Amino Acid Compound Injection 18AA-IX products, at the same consider big production, storage and transport etc. because
Element, in the case of 80% that cysteine content is not less than sign content, indirectly in reflection Amino Acid Compound Injection 18AA-IX
Other amino acid are without degraded.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method for determining cysteine content in Amino Acid Compound Injection, comprises the following steps：
(1) aqueous cystein solution is prepared, reference substance solution is used as；
It regard Amino Acid Compound Injection as need testing solution；
Cysteine in the reference substance solution and need testing solution is derived using N- (1- pyrenes) maleimides；
(2) reference substance solution and need testing solution after the derivative for obtaining the step (1) carries out efficient liquid phase chromatographic analysis,
According to the reference substance solution and the liquid chromatogram of need testing solution after derivative, Amino Acid Compound Injection is obtained using external standard method
The content of middle cysteine.
2. according to the method described in claim 1, it is characterised in that cysteine and N- (1- when derived from the step (1)
Pyrene) maleimide mol ratio be 1：(1~3).
3. method according to claim 1 or 2, it is characterised in that in the step (1) N- (1- pyrenes) maleimides with
The form of N- (1- pyrenes) maleimide acetonitrile solution is used.
4. method according to claim 3, it is characterised in that the quality of N- (1- pyrenes) the maleimide acetonitrile solution
Concentration is 0.15~0.25mg/mL.
5. according to the method described in claim 1, it is characterised in that the temperature derived from the step (1) is 15~25 DEG C.
6. method according to claim 1 or 5, it is characterised in that time derived from the step (1) for 15~
25min。
7. according to the method described in claim 1, it is characterised in that efficient liquid phase chromatographic analysis uses PDA in the step (2)
Detector.
8. the method according to claim 1 or 7, it is characterised in that the inspection of efficient liquid phase chromatographic analysis in the step (2)
Survey wavelength is 274nm.
9. according to the method described in claim 1, it is characterised in that efficient liquid phase chromatographic analysis uses ten in the step (2)
Eight alkyl silane bonded silica gel chromatographic columns.
10. the method according to claim 1 or 9, it is characterised in that efficient liquid phase chromatographic analysis in the step (2)
Column temperature is 30~40 DEG C.
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| CN112697727A (en) * | 2020-12-10 | 2021-04-23 | 武汉久安药业有限公司 | Method for determining content of cysteine hydrochloride in compound amino acid injection |
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| CN107907603A (en) * | 2017-10-31 | 2018-04-13 | 华仁药业股份有限公司 | A kind of measure assay method of the amino acids parenteral solution tryptophan in relation to material |
| CN110095535A (en) * | 2019-04-22 | 2019-08-06 | 山东理工职业学院 | The content assaying method of cystine in a kind of Amino Acid Compound Injection (5%) |
| CN110095535B (en) * | 2019-04-22 | 2022-04-15 | 山东理工职业学院 | Method for measuring content of cystine in compound amino acid injection (5 percent) |
| CN111443151A (en) * | 2020-05-27 | 2020-07-24 | 费森尤斯卡比华瑞制药有限公司 | Method for detecting content of trace cysteine in compound amino acid injection |
| CN112697727A (en) * | 2020-12-10 | 2021-04-23 | 武汉久安药业有限公司 | Method for determining content of cysteine hydrochloride in compound amino acid injection |
| CN114280190A (en) * | 2021-12-27 | 2022-04-05 | 无锡市江原实业技贸有限公司 | Kit for detecting related substances of bicysteine |
| CN114280190B (en) * | 2021-12-27 | 2024-05-28 | 无锡市江原实业技贸有限公司 | Kit for detecting related substances of double cysteines |
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