CN105588900A - Compounded amino acid injection 18AA-II content measurement method - Google Patents
Compounded amino acid injection 18AA-II content measurement method Download PDFInfo
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Abstract
The invention relates to the field of medicine preparation and especially relates to a compounded amino acid injection 18AA-II content measurement method. According to the method, when the time of conversion of a buffer solution from B2 to B3 is pushed back by 0.5 min, the time of conversion of a buffer solution from B3 to B4 is pushed back by 0.8 min, and the temperature of a separation column reaches 57 DEG C, a better separation effect between the components is achieved and further separation degree between cystine and valine satisfies the demand on content measurement, and meanwhile, interference on the cystine due to conversion of a mobile phase is reduced, so that accurate quantitative measurement is achieved even under a low content, and finally, accurate measurement of contents of sixteen amino acids in the compounded amino acid injection 18AA-II with an amino acid analyzer can be achieved.
Description
Technical field
The present invention relates to medicine preparation field, particularly a kind of Amino Acid Compound Injection 18AA-II content assaying method.
Background technology
Amino acid is the important substance in fundamental structural unit and the bio-metabolic process of protein, Analytical Technology of Amino Acid is significant to protein chemistry, biochemistry and whole life science and product development, the production management of quality control box etc., is widely used in the analysis of medicine, food, health products of food processing, medical and health industry etc. Its analysis determining method, can be divided into chemical analysis, electrochemical methods, AAS etc. according to detection method; According to the priority of derivatization reaction, can be divided into column front derivation and post-column derivation.
In above analytical method, the specificity of chemical analysis, electrochemical methods and AAS is poor, can not realize several amino acids separation determination simultaneously; Pre-column derivatization, although can use automatic sampler and the chromatograph joint used separation determination of realizing several amino acids, deriving technology is comparatively complicated, and the cycle is longer, and derivatization conditions is wayward, and repeatability is poor, is difficult to promote.
Amino acid whose method for separating and analyzing is a lot, in recent years it is generally acknowledged that ion-exchange chromatography is quantitative approach more accurately, and amino-acid analyzer and HPLC method are used comparatively general. But HPLC method deriving technology is comparatively complicated, and the cycle is longer, and derivatization conditions is wayward, and repeatability is poor, be difficult to promote. With respect to HPLC method, the sensitive Quick of amino-acid analyzer supplies data accurately and reliably: the high analytical cycle simple to operate of resolution ratio is short; Retention time, peak area, favorable reproducibility detection limit can reach 3pmol, does not have the problem of liquid level derivative, and peak type shows Gaussian distribution, and stable system is fast.
Conventional Contents of Amino Acids, what conventionally adopt is all the separation condition that amino-acid analyzer instrument producer itself is recommended, and comprises pH of cushioning fluid, buffer solution conversion time and column temperature etc.
But, in the time using amino-acid analyzer to carry out assay to Amino Acid Compound Injection (18AA-II), because cystine is less, and cystine appearance time just in time, near mobile phase transformation time, uses the separation condition of instrument itself to test, and causes cystine and valine analytical effect not good, do not reach the requirement of test to component separating degree, cystine fluctuation is simultaneously larger, causes assay result precision not good, poor reproducibility.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of Amino Acid Compound Injection 18AA-II content assaying method.
For achieving the above object, the technical solution used in the present invention is as follows:
A kind of Amino Acid Compound Injection 18AA-II content assaying method, adopt ion exchange resin column, 57 DEG C of column temperatures, flow is 0.4ml/min, detecting wavelength is 570nm and 440nm, mobile phase is by buffer B 1, B2, B3, B4, B5 composition, elution time is 60 minutes, within 0-2.5 minute, adopt 100% B1 to carry out wash-out, within 2.6-5 minute, adopt 100% B2 to carry out wash-out, within 5.1-13.6 minute, adopt 100% B3 to carry out wash-out, 13.7-27.8 the B4 of minute employing 100% carries out wash-out, 27.9-33.8 the B5 of minute employing 100% carries out wash-out, 33.9-34.8 the B2 of minute employing 100% carries out wash-out, 34.9-53.8 the B1 of minute employing 100% carries out wash-out,
Described buffer B 1 consists of 6.19g natrium citricum 2H2O, 1MNaOH6-7mL, 5.66g sodium chloride, 19.80g citric acid H2O, 135.0ml ethanol, be mixed with 1000mL solution with distilled water;
Described buffer B 2 consists of 7.74g natrium citricum 2H2O, 1MNaOH20-22mL, 7.07g sodium chloride, 22.00g citric acid H2O, 25.0ml ethanol, be mixed with 1000mL solution with distilled water;
Described buffer B 3 consists of 13.31g natrium citricum 2H2O, 3.74g sodium chloride, 12.80g citric acid H2O, 9.00ml ethanol, be mixed with 1000mL solution with distilled water;
Described buffer B 4 consists of 26.67g natrium citricum 2H2O, 54.35g sodium chloride, 6.10g citric acid H2O, is mixed with 1000mL solution with distilled water;
Described buffer B 5 consists of 8.00gNaOH, 100.0ml ethanol, is mixed with 1000mL solution with distilled water.
Beneficial effect of the present invention:
The time that the present invention changes from B2 to B3 at buffer solution postpones 0.5min, the time of changing from B3 to B4 at buffer solution postpones 0.8min, splitter temperature reaches 57 while spending, separating effect between each component is better, make the two separating degree of cystine and valine meet the requirement of assay, make cystine reduced by the interference of mobile phase conversion simultaneously, make it also can accurate quantitative analysis in the situation that content is lower, finally realized the Accurate Determining of amino-acid analyzer to Amino Acid Compound Injection (18AA-II) 16 seed amino acid content.
Brief description of the drawings
Accompanying drawing 1 is the separating spectrum of the separation condition of amino-acid analyzer recommendation;
The separating spectrum that accompanying drawing 2 is separation condition of the present invention.
Detailed description of the invention
Further set forth the specific embodiment of the present invention below in conjunction with embodiment as follows, separating spectrum as shown in Figure 2:
Adopt the L-8900 of Hitachi amino-acid analyzer
Test specimen: Amino Acid Compound Injection (18AA-II) (Huaren Pharmaceutical Co., Ltd.'s production)
Reference substance: examine institute in source, lot number 140624-200805, purity 100%;
Post is pressed: the post of pump 1 is pressed the left and right for 11Mpa, and pump 2 posts are pressed as 2.0Mpa;
Applied sample amount: 10 μ L
Adopt ion exchange resin column (purchased from Hitachi), 57 DEG C of column temperatures, flow is 0.4ml/min, detecting wavelength is 570nm and 440nm, mobile phase is by buffer B 1, B2, B3, B4, B5 composition, elution time is 60 minutes, within 0-2.5 minute, adopt 100% B1 to carry out wash-out, within 2.6-5 minute, adopt 100% B2 to carry out wash-out, within 5.1-13.6 minute, adopt 100% B3 to carry out wash-out, 13.7-27.8 the B4 of minute employing 100% carries out wash-out, 27.9-33.8 the B5 of minute employing 100% carries out wash-out, 33.9-34.8 the B2 of minute employing 100% carries out wash-out, 34.9-53.8 the B1 of minute employing 100% carries out wash-out,
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 |
| 5.0 | 0 | 100 | 0 | 0 | 0 |
| 5.1 | 0 | 0 | 100 | 0 | 0 |
| 13.6 | 0 | 0 | 100 | 0 | 0 |
| 13.7 | 0 | 0 | 0 | 100 | 0 |
| 27.8 | 0 | 0 | 0 | 100 | 0 |
| 27.9 | 0 | 0 | 0 | 0 | 100 |
| 33.8 | 0 | 0 | 0 | 0 | 100 |
| 33.9 | 0 | 100 | 0 | 0 | 0 |
| 34.8 | 0 | 100 | 0 | 0 | 0 |
| 34.9 | 100 | 0 | 0 | 0 | 0 |
| 53.8 | 100 | 0 | 0 | 0 | 0 |
Note: the % in table refers to percent by volume.
Described buffer B 1 consists of 6.19g natrium citricum 2H2O, 1MNaOH6-7mL, 5.66g sodium chloride, 19.80g citric acid H2O, 135.0ml ethanol, be mixed with 1000mL solution with distilled water;
Described buffer B 2 consists of 7.74g natrium citricum 2H2O, 1MNaOH20-22mL, 7.07g sodium chloride, 22.00g citric acid H2O, 25.0ml ethanol, be mixed with 1000mL solution with distilled water;
Described buffer B 3 consists of 13.31g natrium citricum 2H2O, 3.74g sodium chloride, 12.80g citric acid H2O, 9.00ml ethanol, be mixed with 1000mL solution with distilled water;
Described buffer B 4 consists of 26.67g natrium citricum 2H2O, 54.35g sodium chloride, 6.10g citric acid H2O, is mixed with 1000mL solution with distilled water;
Described buffer B 5 consists of 8.00gNaOH, 100.0ml ethanol, is mixed with 1000mL solution with distilled water.
Table 2: the preparation table of buffer solution
The condition such as the conversion time of buffer solution and the column temperature of splitter that the present invention is directed to has been carried out a series of conditional filtering. The time of first buffer solution being changed from B2 to B3 under the condition of 4.5min respectively in advance and postpone 0.5min, find in the situation that postponing 0.5 minute namely conversion time at 5.0min, the time of simultaneously B3 being changed to B4 postpones 0.8min, namely putting off until separating effect under 13.7min condition by original 12.9min makes moderate progress, cystine is subject to the content fluctuation of mobile phase conversion less, and it is good that reappearance becomes.
Fig. 1 is that the L-8900 of Hitachi amino-acid analyzer recommendation condition separates, and the separating effect that can find out cystine and front and back two seed amino acids is not very good, and self peak shape neither very goodly be seen, cannot carry out accurate quantitative analysis analysis.
Fig. 2 is the collection of illustrative plates of separation condition of the present invention, can obviously find out that the present invention makes cystine reach effective with former and later two amino acid and separates, optimize peak shape, reduced because of the mobile phase impact quantitative on cystine conversion time, thereby improved accuracy and the reappearance of its assay.
Claims (1)
1. an Amino Acid Compound Injection 18AA-II content assaying method, it is characterized in that, adopt ion exchange resin column, 57 DEG C of column temperatures, flow is 0.4ml/min, detecting wavelength is 570nm and 440nm, mobile phase is by buffer B 1, B2, B3, B4, B5 composition, elution time is 60 minutes, within 0-2.5 minute, adopt 100% B1 to carry out wash-out, within 2.6-5 minute, adopt 100% B2 to carry out wash-out, within 5.1-13.6 minute, adopt 100% B3 to carry out wash-out, 13.7-27.8 the B4 of minute employing 100% carries out wash-out, 27.9-33.8 the B5 of minute employing 100% carries out wash-out, 33.9-34.8 the B2 of minute employing 100% carries out wash-out, 34.9-53.8 the B1 of minute employing 100% carries out wash-out,
Described buffer B 1 consists of 6.19g natrium citricum 2H2O, 1MNaOH6-7mL, 5.66g sodium chloride, 19.80g citric acid H2O, 135.0ml ethanol, be mixed with 1000mL solution with distilled water;
Described buffer B 2 consists of 7.74g natrium citricum 2H2O, 1MNaOH20-22mL, 7.07g sodium chloride, 22.00g citric acid H2O, 25.0ml ethanol, be mixed with 1000mL solution with distilled water;
Described buffer B 3 consists of 13.31g natrium citricum 2H2O, 3.74g sodium chloride, 12.80g citric acid H2O, 9.00ml ethanol, be mixed with 1000mL solution with distilled water;
Described buffer B 4 consists of 26.67g natrium citricum 2H2O, 54.35g sodium chloride, 6.10g citric acid H2O, is mixed with 1000mL solution with distilled water;
Described buffer B 5 consists of 8.00gNaOH, 100.0ml ethanol, is mixed with 1000mL solution with distilled water.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106468695A (en) * | 2016-09-27 | 2017-03-01 | 上海海洋大学 | A kind of method simultaneously measuring bioactive peptide and free amino acid |
| CN108008060A (en) * | 2017-09-21 | 2018-05-08 | 中国农业科学院农业质量标准与检测技术研究所 | The assay method and reagent of hydroxyproline in a kind of feed |
| 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%) |
| CN111323503A (en) * | 2020-03-12 | 2020-06-23 | 河北科星药业有限公司 | Method for measuring content of compound amino acid injection |
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2014
- 2014-10-24 CN CN201410579008.8A patent/CN105588900B/en active Active
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| I.BETNER 等: "New Automated Amino Acid Analysis by HPLC Precolumns Derivatization with Fluorenylmethyloxycarbonylchloride", 《CHROMATOGRAPHIA》 * |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106468695A (en) * | 2016-09-27 | 2017-03-01 | 上海海洋大学 | A kind of method simultaneously measuring bioactive peptide and free amino acid |
| CN106468695B (en) * | 2016-09-27 | 2018-05-15 | 上海海洋大学 | Method that is a kind of while measuring active peptide and free amino acid |
| CN108008060A (en) * | 2017-09-21 | 2018-05-08 | 中国农业科学院农业质量标准与检测技术研究所 | The assay method and reagent of hydroxyproline in a kind of feed |
| CN108008060B (en) * | 2017-09-21 | 2020-07-28 | 中国农业科学院农业质量标准与检测技术研究所 | Method and reagent for determining hydroxyproline in feed |
| 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) |
| CN111323503A (en) * | 2020-03-12 | 2020-06-23 | 河北科星药业有限公司 | Method for measuring content of compound amino acid injection |
| WO2021179535A1 (en) * | 2020-03-12 | 2021-09-16 | 河北科星药业有限公司 | Method for determining content of compound amino acid injection |
| CN111323503B (en) * | 2020-03-12 | 2022-10-11 | 河北科星药业有限公司 | Method for measuring content of compound amino acid injection |
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