CN105319282B - Content measurement method of compound amino acid (15) dipeptide (2) injection - Google Patents
Content measurement method of compound amino acid (15) dipeptide (2) injection Download PDFInfo
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Abstract
The invention relates to the field of medicine production and particularly relates to a content measurement method of a compound amino acid (15) dipeptide (2) injection. In the method, an ion exchange resin column is employed with following conditions: column temperature is 52-62 DEG C, flow quantity is 0.4 ml/min, detection wavelengths are 570 nm and 440 nm, a mobile phase is composed of buffer liquids B1, B2, B3, B4 and B5 and elution time is 60 min, wherein the elution in the 0-2.5 min is carried out with the B1 being 100%, the elution in the 2.6-5 min is carried out with the B2 being 100%, the elution in the 6.1-13.8 min is carried out with the B3 being 100%, the elution in the 13.9-29.0 min is carried out with the B4 being 100%, the elution in the 29.1-33 min is carried out with the B5 being 100%, the elution in the 33.1-34.0 min is carried out with the B2 being 100% and the elution in the 34.1-53 min is carried out with the B1 being 100%. The content measurement method achieves excellent separation between glycyl glutamine and alanine in the compound amino acid (15) dipeptide (2) injection, satisfies requirement of separation degree on different components in the sample and achieves accurate detection of the contents of fourteen amino acids and one dipeptide in the sample.
Description
Technical field
The present invention relates to pharmaceutical production field, and in particular to a kind of double peptide (2) the injection liquid hold-ups of amino acid (15) are surveyed
Determine method.
Background technology
Amino acid is the important substance in the fundamental structural unit and bio-metabolic process of protein, Analytical Technology of Amino Acid
Protein chemistry, biochemistry and whole life science and product development, quality control box production management etc. are had
Significance, is widely used in the analysis of food processing, the medicine of medical and health industry, food, health products etc..Its analysis is surveyed
Determine method, according to detection method chemical analysis, electrochemical methods, AAS etc. can be divided into；According to derivatization reaction
Successively, column front derivation and post-column derivation can be divided into.
In above analysis method, the specificity of chemical analysis, electrochemical methods and AAS is poor, it is impossible to real
Existing several amino acids are while separation determination；Post-column derivation method before and after post, although can be using automatic sampler and chromatograph joint used reality
The separation determination of existing several amino acids, but deriving technology is complex, and the cycle is longer, and derivatization conditions are wayward, repeatability
It is poor, it is difficult to promote.
Amino-acid analyzer can be separated and assay to several amino acids simultaneously, and without carrying out post before and
Post-column derivation, can be determined with direct injection analysis, and sensitive, quick, the data precision is high；High resolution, simple to operate, reappearance
Good, peak shape is good, stable system, not there is a problem of that liquid phase process generates two grades of derivatives, so far qualitative and quantitative point of amino acid
One of most widely used method in analysis.
Conventional Contents of Amino Acids, what is generally adopted is all the separation that amino-acid analyzer instrument producer itself is recommended
Condition, including pH of cushioning fluid, buffer exchange time and column temperature etc..
But, 14 kinds of amino acid and 1 in using amino-acid analyzer to double peptide (2) parenteral solutions of amino acid (15)
When double peptides (Gly-Glu) carry out assay, when the separation condition recommended using instrument itself is tested, wherein
The separating effect of two components of Gly-Glu and alanine in sample is bad, does not reach test to Component seperation degree
Requirement, cause assay result precision not good.
The content of the invention
The present invention is directed to deficiency of the prior art, there is provided a kind of double peptide (2) the injection liquid hold-ups of amino acid (15) are surveyed
Determine method.
The technical scheme is that：
Double peptide (2) the parenteral solution content assaying methods of a kind of amino acid (15), using ion exchange resin column, column temperature
52-62 DEG C, flow is 0.4ml/min, and Detection wavelength is 570nm and 440nm, and mobile phase is by buffer B 1, B2, B3, B4, B5
Composition, elution time is 60 minutes, and 0-2.5 minutes are eluted using 100% B1, and 2.6-5 minutes are entered using 100% B2
Row wash-out, 6.1-13.8 minutes are eluted using 100% B3, and 13.9-29.0 minutes are eluted using 100% B4,
29.1-33 minutes are eluted using 100% B5, and 33.1-34.0 minutes are eluted using 100% B2,34.1-53 point
Clock is eluted using 100% B1；
The buffer B 1 consists of 6.19g sodium citrate 2H2O, 1M NaOH6-7mL, 5.66g sodium chloride, 19.80g
Citric acid H2O, 135.0ml ethanol, 1-3g citric acids, with distilled water 1000mL solution is configured to；
The buffer B 2 consists of 7.74g sodium citrate 2H2O, 1M NaOH20-22mL, 7.07g sodium chloride,
22.00g citric acid H2O, 25.0ml ethanol, with distilled water 1000mL solution is configured to；
The buffer B 3 consists of 13.31g sodium citrate 2H2O, 3.74g sodium chloride, 12.80g citric acid H2O、
9.00ml ethanol, with distilled water 1000mL solution is configured to；
The buffer B 4 consists of 26.67g sodium citrate 2H2O, 54.35g sodium chloride, 6.10g citric acid H2O,
1000mL solution is configured to distilled water；
The buffer B 5 consists of 8.00g NaOH, 100.0ml ethanol, is configured to 1000mL with distilled water molten
Liquid.
Preferably, described column temperature is 57 DEG C.
Preferably, described buffer B 1 consists of 6.19g sodium citrate 2H2O, 1M NaOH6-7mL, 5.66g chlorination
Sodium, 19.80g citric acid H2O, 135.0ml ethanol, 2g citric acids, with distilled water 1000mL solution is configured to.
The invention has the beneficial effects as follows：
Double peptide (2) the parenteral solution content assaying methods of amino acid (15) of the present invention, realize amino acid (15) double
Glycylglutamine and the good separation of alanine, meet separation of the test to sample different component in peptide (2) parenteral solution
The requirement of degree, realizes the Accurate Determining to amino acid in sample 14 and a kind of double peptide content.
Description of the drawings
Accompanying drawing 1 is the separating spectrum obtained by the embodiment of the present invention.
Specific embodiment
The specific embodiment of the present invention is as follows, and separating spectrum is as shown in Figure 1：
Using Hitachi's L-8900 amino-acid analyzers
Test specimen：Double peptide (2) parenteral solutions of amino acid (15) (Huaren Pharmaceutical Co., Ltd.'s production)
Reference substance：Institute, lot number 140624-200805, purity 100% are examined in source；
Post pressure：The post pressure of pump 1 is 11Mpa or so, and the post of pump 2 pressure is 2.0Mpa；
Applied sample amount：10μL
Using ion exchange resin column (being purchased from Hitachi), 57 DEG C of column temperature, flow is 0.4ml/min, and Detection wavelength is 570nm
And 440nm, mobile phase is made up of buffer B 1, B2, B3, B4, B5, and elution time is 60 minutes, and 0-2.5 minutes adopt
100% B1 is eluted, and 2.6-6 minutes are eluted using 100% B2, and 6.1-13.8 minutes are entered using 100% B3
Row wash-out, 13.9-29.0 minutes are eluted using 100% B4, and 29.1-33 minutes are eluted using 100% B5,
33.1-34.0 minutes are eluted using 100% B2, and 34.1-53 minutes are eluted using 100% B1；
Table 1：Eluent system
| Time(min) | %B1 | %B2 | %B3 | %B4 | %B5 |
| 0.0 | 100 | 0 | 0 | 0 | 0 |
| 2.5 | 100 | 0 | 0 | 0 | 0 |
| 2.6 | 0 | 100 | 0 | 0 | 0 |
| 6.0 | 0 | 100 | 0 | 0 | 0 |
| 6.1 | 0 | 0 | 100 | 0 | 0 |
| 13.8 | 0 | 0 | 100 | 0 | 0 |
| 13.9 | 0 | 0 | 0 | 100 | 0 |
| 29.0 | 0 | 0 | 0 | 100 | 0 |
| 29.1 | 0 | 0 | 0 | 0 | 100 |
| 33.0 | 0 | 0 | 0 | 0 | 100 |
| 33.1 | 0 | 100 | 0 | 0 | 0 |
| 34.0 | 0 | 100 | 0 | 0 | 0 |
| 34.1 | 100 | 0 | 0 | 0 | 0 |
| 53.0 | 100 | 0 | 0 | 0 | 0 |
Note：% in table refers to percent by volume.
The buffer B 1 consists of 6.19g sodium citrate 2H2O, 1M NaOH6-7mL, 5.66g sodium chloride, 19.80g
Citric acid H2O, 135.0ml ethanol, 2g citric acids, with distilled water 1000mL solution is configured to；
The buffer B 2 consists of 7.74g sodium citrate 2H2O, 1M NaOH20-22mL, 7.07g sodium chloride,
22.00g citric acid H2O, 25.0ml ethanol, with distilled water 1000mL solution is configured to；
The buffer B 3 consists of 13.31g sodium citrate 2H2O, 3.74g sodium chloride, 12.80g citric acid H2O、
9.00ml ethanol, with distilled water 1000mL solution is configured to；
The buffer B 4 consists of 26.67g sodium citrate 2H2O, 54.35g sodium chloride, 6.10g citric acid H2O,
1000mL solution is configured to distilled water；
The buffer B 5 consists of 8.00g NaOH, 100.0ml ethanol, is configured to 1000mL with distilled water molten
Liquid.
Table 2：Buffer solution with tabulation
As a result as shown in figure 1, glycylglutamine is good with alanine in double peptide (2) parenteral solutions of amino acid (15)
It is good to separate, the requirement for testing the separating degree to sample different component is met, realize double to amino acid in sample 14 and a kind
The Accurate Determining of peptide content.
Test example：
Test has carried out one for the condition such as the column temperature of pH value, the conversion time of buffer solution and splitter of buffer solution
The test of row, as a result finds splitter column temperature at 57 DEG C, and separating effect is optimal, such as table 3.
Table 3：Column temperature affects on separating degree
On the premise of the such as buffer system of table 1, in the buffer B 1 in 1000mL citric acid, Malaysia are separately added into
The buffer salts such as acid, fumaric acid and vitamin C, in the case of the citric acid of 2g amounts is added in the buffer B 1 of 1000mL, sample
In component separation situation preferably, other buffer salts are as a result undesirable.
In process of the test, add maleic acid appropriate in the buffer B 1 of 1000mL, ultrasonic 5min is mixed, 0.45 μm of Jing
Membrane filtration process, it is stand-by.After adding different amounts of maleic acid, the separation situation of each material is as shown in the table in sample component,
The separating resulting of constituent part is undesirable, such as table 4.
Table 4：Impact of the maleic acid to separating degree is added in buffer B 1
Add fumaric acid appropriate in the buffer B 1 of 1000mL, ultrasonic 5min is mixed, at 0.45 μm of membrane filtration of Jing
Reason, it is stand-by.After adding different amounts of fumaric acid, the separation situation of each material is as shown in the table in sample component, constituent part
Separating resulting is still undesirable, while during using the condition, the column pressure of splitter has rising trend (such as table 5).Table 5：Buffer solution
Impact of the fumaric acid to separating degree is added in B1
Add vitamin C appropriate in the buffer B 1 of 1000mL, ultrasonic 5min is mixed, at 0.45 μm of membrane filtration of Jing
Reason, it is stand-by.After adding different amounts of maleic acid, the separation situation of each material is as shown in the table in sample component, constituent part
Separating resulting is still undesirable, and the reappearance of sample result poor (such as table 6).
Table 6：Impact of the vitamin C to separating degree is added in buffer B 1
The citric acid of 2g amounts, ultrasonic 5min is added to mix, at 0.45 μm of membrane filtration of Jing in the buffer B 1 of 1000mL
Reason, it is stand-by.The amount of citric acid in by changing buffer B 1, so that the pH value of buffer B 1 is changed, sample component
In each material separation situation it is as shown in the table, should under the conditions of Gly-Glu in sample component and alanine point
It is good from degree, while the separation situation between other components also very well, meets requirement of the test to sample separating degree (such as table 7).
Table 7：Impact of the citric acid to separating degree is added in buffer B 1
。
Claims (3)
1. double peptide (2) parenteral solution content assaying methods of a kind of amino acid (15), it is characterised in that：Using ion exchange resin
Post, 52-62 DEG C of column temperature, flow is 0.4ml/min, and Detection wavelength is 570nm and 440nm, mobile phase be by buffer B 1, B2,
B3, B4, B5 are constituted, and elution time is 53 minutes, and 0-2.5 minutes are eluted using 100% B1, and 2.6-6 minutes adopt
100% B2 is eluted, and 6.1-13.8 minutes are eluted using 100% B3, and 13.9-29.0 minutes are using 100%
B4 is eluted, and 29.1-33 minutes are eluted using 100% B5, and 33.1-34.0 minutes are washed using 100% B2
De-, 34.1-53 minutes are eluted using 100% B1；
The buffer B 1 consists of 6.19g sodium citrate 2H2O, 1M NaOH 6-7mL, 5.66g sodium chloride, 19.80g lemons
Sour H2O, 135.0ml ethanol, 1-3g citric acids, with distilled water 1000mL solution is configured to；
The buffer B 2 consists of 7.74g sodium citrate 2H2O, 1M NaOH 20-22mL, 7.07g sodium chloride, 22.00g lemons
Lemon acid H2O, 25.0ml ethanol, with distilled water 1000mL solution is configured to；
The buffer B 3 consists of 13.31g sodium citrate 2H2O, 3.74g sodium chloride, 12.80g citric acid H2O、
9.00ml ethanol, with distilled water 1000mL solution is configured to；
The buffer B 4 consists of 26.67g sodium citrate 2H2O, 54.35g sodium chloride, 6.10g citric acid H2O, with steaming
Distilled water is configured to 1000mL solution；
The buffer B 5 consists of 8.00g NaOH, 100.0ml ethanol, and with distilled water 1000mL solution is configured to.
2. double peptide (2) parenteral solution content assaying methods of amino acid (15) according to claim 1, it is characterised in that：
Described column temperature is 57 DEG C.
3. double peptide (2) parenteral solution content assaying methods of amino acid (15) according to claim 1, it is characterised in that institute
The buffer B 1 stated consists of 6.19g sodium citrate 2H2O, 1M NaOH 6-7mL, 5.66g sodium chloride, 19.80g lemons
Sour H2O, 135.0ml ethanol, 2g citric acids, with distilled water 1000mL solution is configured to.
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| CN110907584B (en) * | 2019-12-06 | 2020-08-11 | 鹤山市东古调味食品有限公司 | Method for detecting alcohol degree of non-alcoholic liquid |
| CN113384523B (en) * | 2021-06-29 | 2022-06-03 | 四川科伦药业股份有限公司 | Production and preparation method of compound amino acid (15) dipeptide (2) injection |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102749397A (en) * | 2012-07-19 | 2012-10-24 | 天津金耀集团有限公司 | Analysis method of compound amino acid and dipeptide injection |
| CN103134892A (en) * | 2011-11-30 | 2013-06-05 | 天津金耀集团有限公司 | Analytical method of compound amino acid (15) double peptide (2) injection |
Family Cites Families (1)
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-
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- 2014-06-20 CN CN201410279676.9A patent/CN105319282B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103134892A (en) * | 2011-11-30 | 2013-06-05 | 天津金耀集团有限公司 | Analytical method of compound amino acid (15) double peptide (2) injection |
| CN102749397A (en) * | 2012-07-19 | 2012-10-24 | 天津金耀集团有限公司 | Analysis method of compound amino acid and dipeptide injection |
Non-Patent Citations (8)
| Title |
|---|
| llets dried by far-infrared assisted heat pump drying.《Food Control》.2014,第36卷(第1期),第102-110页. * |
| Method Validation of 16 Types of Structural Amino Acids using an Automated Amino Acid Analyzer;You-Shin Shim 等;《Food Sci. Biotechnol.》;20131231;第22卷(第6期);第1567-1571页 * |
| Yun Deng 等.Thermal behavior, microstructure and protein quality of squid fi * |
| 复方氨基酸（15）双肽（2）注射液中两组分的含量测定;叶晓林 等;《药物鉴定》;20081231;第17卷(第7期);第18-19页 * |
| 日立L-8900型氨基酸分析仪用缓冲液的研究;高翠红 等;《氨基酸和生物资源》;20101231;第32卷(第4期);第81-84页 * |
| 炒焦对山楂中氨基酸含量的影响分析;张韵 等;《氨基酸和生物资源》;20131231;第35卷(第3期);第28-31页 * |
| 纪银福 等.饲料中氨基酸的测定-酸水解法测试研究.《草食家畜》.2007,(第2期),第39-41页. * |
| 高效液相色谱法测定复方氨基酸（15）双肽（2）注射液中2种杂质的含量测定;陆明 等;《药物分析杂志》;20110531;第31卷(第5期);第867-870页 * |
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