CN105699502A - Method for detecting related substances of amino acid calcium components in compound alpha-ketoacid tablets - Google Patents
Method for detecting related substances of amino acid calcium components in compound alpha-ketoacid tablets Download PDFInfo
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- CN105699502A CN105699502A CN201510975446.0A CN201510975446A CN105699502A CN 105699502 A CN105699502 A CN 105699502A CN 201510975446 A CN201510975446 A CN 201510975446A CN 105699502 A CN105699502 A CN 105699502A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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Abstract
The invention discloses a method for detecting related substances of amino acid calcium components in compound alpha-ketoacid tablets. The method specifically includes the steps that by means of an ultraviolet detector and a chromatographic column Hi-Plex H (300*7.7 mm, 8 microns), with a sulfuric acid solution as the flowing phase, an appropriate amount of compound alpha-ketoacid tablet powder (ground) is taken, and the flowing phase is added to prepare a solution to serve as a test article solution, wherein each 1 ml of solution contains 6 mg of compound alpha-ketoacid tablets; 1 ml of test article solution is sucked to be diluted by the flowing phase to prepare a solution to serve as a contrast solution, wherein each 1 ml of solution contains about 60 micrograms of compound alpha-ketoacid tablet powder; the test article solution and the contrast solution are fed respectively, wherein the feeding time is 95 minutes, and the sum of the areas of impurity peaks in a chromatogram of the test article solution shall not be larger than the area of the main peak of the contrast solution. By means of the method, the situations of impurities and degrading products of compound alpha-ketoacid tablets can be rapidly and accurately detected. The method is easy and rapid to implement, high in sensitivity and capable of well controlling product quality.
Description
Technical field
The present invention relates to pharmaceutical analysis detection field, particularly to the relevant substance detecting method of amino acid calcium constituents in a kind of compound alpha-ketoacid tablet preparation。
Background technology
Chronic kidney disease (chronickidneydisease, CKD) refers to the chronic Renal Structure and dysfunction that a variety of causes causes。Epidemiological study shows, since 21 century, chronic kidney disease has become as a principal disease threatening whole world publilc health。In developed country, the people of 6.5%～10.1% suffers from nephropathy in various degree。Ending 2011, the total prevalence rate of China's chronic kidney disease is 9.3%, and the prevalence of its mid-aged population CKD reaches 18.7%。Because the features such as CKD high incidence, high admission rate, high mortality and great number medical expense become the major disease that harm public health worldwide is healthy。
For a long time, keto acid is only applied to delay progression of disease, and supplements the deficiency adopting Very low protein diet treatment uremic patient essential amino acids。And compound alpha-keto acid tablets contains 4 kinds of ketone for amino acid calcium, a kind of hydroxyl for amino acid calcium and 5 seed amino acids, it is possible to correct the disorder of patient's aminoacid composition。
Zoopery simultaneously shows, compound alpha-keto acid tablets can suppress the muscle of Mus to degrade, and reduces blood urea nitrogen and produces, and leucine can promote that albumen synthesizes, and there are some researches show, hypoalbuminemia is closely related with inflammatory reaction。Compared with taking in treatment with strengthening albumen, keto acid treatment can significantly reduce hs-CRP level。
In a word, compound alpha-keto acid tablets can improve insulin resistant, improves skeletal diseases, increases albumin level and appetite, improves lipid and constitutes, can be applicable to chronic kidney disease (CKD) 1-4 phase patient
Owing in α keto acid compound, amino acid calcium crude drug likely introduces retained material and other is about material in building-up process, it is also possible to produce catabolite in storage。The detection method having related substance after making compound alpha-ketoacid tablet preparation without effective amino acid calcium crude drug at present。
Summary of the invention
It is an object of the invention to provide and a kind of adopt UV-detector to the relevant substance detecting method of amino acid calcium constituents in compound recipe α keto acid sheet。
In order to realize foregoing invention purpose, the technical solution adopted in the present invention is as follows:
The relevant substance detecting method of amino acid calcium constituents in a kind of compound alpha-ketoacid tablet preparation, comprises the following steps:
(1) chromatographic condition setting procedure:
In this step, detector adopts UV-detector, column temperature 40 DEG C, flow velocity 0.65ml/min；The mobile phase of chromatographic condition is concentration is the concentrated sulfuric acid solution of 0.0100-0.0180mol/L, detects wavelength 190-205nm, and chromatogram column temperature is 40-60 DEG C, flow velocity 0.65-1.0ml/min；
(2) preparation process of need testing solution:
Taking compound alpha-ketoacid tablet preparation appropriate, the last mobile phase adding step (1) of pulverizing is configured to the solution of 6mg/ml, as need testing solution；
(3) preparation of contrast solution:
Measuring need testing solution prepared by step (2) appropriate, the mobile phase adding step (1) is diluted to the solution that concentration is 1.58～15.81 μ g/ml, as contrast solution；
(4) determination step:
Take contrast solution 10 μ l injection chromatograph of liquid prepared by step (3), regulate detection sensitivity, main constituent chromatograph peak height is made to be about the 20% of full scale, take need testing solution 10 μ l injection chromatograph of liquid prepared by step (2) again, run 95min, record each impurity peak area, adopt main constituent counter point, calculate each impurity content。
In a preferred embodiment of the invention, described mobile phase is concentration is the concentrated sulfuric acid solution of 0.0145mol/L。
In a preferred embodiment of the invention, described flow velocity is 0.65ml/min。
In a preferred embodiment of the invention, in compound alpha-ketoacid tablet preparation in amino acid calcium constituents, the detectability of tung-oil coated urea be 7.90ng, phenylacetic acid detectability be 3.78ng (S/N ≈ 3)。
In a preferred embodiment of the invention, the linearly dependent coefficient before the concentration of peak area and contrast solution is 0.9999。
The relevant substance detecting method of amino acid calcium constituents in compound alpha-ketoacid tablet preparation of the present invention, it is adaptable to various needs carries out the related preparations that amino acid calcium constituents has related substance to detect, such as α keto acid compound。
The present invention having the active effect that compared with the prior art
1. the relevant substance detecting method of amino acid calcium constituents in compound alpha-ketoacid tablet preparation of the present invention, the big compound preparation of evaluating establishing a kind of quicklook has the effective ways of related substance: impurity various in big compound preparation can be carried out accurate quantitative analysis, the problems such as compound preparation dopant species is many, separating degree is poor, detection difficult, ownership are loaded down with trivial details that solve such, well have the evaluation of related substance to provide objective and accurate method in such preparation。
2. the relevant substance detecting method of amino acid calcium constituents in compound alpha-ketoacid tablet preparation of the present invention, can detect amino acid calcium constituents impurity and catabolite situation in compound alpha-ketoacid tablet preparation fast and accurately。Product quality can be controlled preferably。
3. the present invention is applicable to the various related preparations containing such amino acid calcium composition, such as α keto acid compound。
4. detection method is accurate, simple to operation, and favorable reproducibility is highly sensitive, it is possible to fully meet the requirement that Related substances separation measures with catabolite, controls the special impurities in sample preferably, it is ensured that product quality, practical at work。
This test adopts common hydrogen form cation exchange chromatography post Hi-PlexH (300 × 7.7mm, 8 μm) achieve amino acid calcium constituents in α keto acid compound fast and accurately and have the mensuration of related substance, ensure that the quality controllable of α keto acid compound, have important practical significance in the quality control of production process。
Accompanying drawing illustrates:
Fig. 1 is the HPLC collection of illustrative plates of contrast solution。
Fig. 2 is the HPLC collection of illustrative plates of need testing solution。
Fig. 3 is specificity test HPLC collection of illustrative plates。
Detailed description of the invention
Embodiment 1
1. chromatographic condition and system suitability:
1.1. the selection of chromatographic condition:
Instrument: high performance liquid chromatograph Agilent1200 type, its optimum column temperature 40 DEG C, flow velocity 0.65ml/min。Liquid-phase chromatographic column Hydrogen strong cation chromatographic column Hi-PlexH (300 × 7.7mm, 8 μm) with reference to pertinent literature binding tests concrete condition, selection sulfuric acid solution is mobile phase, finally determined by tests such as flow rate of mobile phase screening, concentration screening, the screening of detection wavelength, column temperature screenings, mobile phase optium concentration is 0.0145mol/L, optimum flow rate 0.65ml/min, best detection wavelength 205nm, optimum column temperature is 40 DEG C, sample size 10 μ l。
Under this chromatographic condition, in compound alpha-ketoacid tablet preparation, amino acid calcium constituents has related substance retention time moderate, and peak shape is better。
1.2. to weigh tung-oil coated urea, phenylacetic acid reference substance appropriate for precision, dissolve with mobile phase and make certain concentration solution, in proportion after stepwise dilution, detect according to about the chromatographic condition under substance-measuring item, using signal to noise ratio (S/N) ≈ 3 as detection limit, signal to noise ratio (S/N) ≈ 10 is as quantitative limit。Result proves, the method is highly sensitive, it is possible to fully meet the requirement that Related substances separation measures。Measurement result is in Table 1。
Table 1 detection limit and quantitative limit measurement result
1.3. stability of solution newly prepares a need testing solution, is separately employed in instrument and does not close, when mobile phase is changed without, respectively at 0h, 2h, 4h, 6h, 8h, 10h, 12h sample introduction is once, by to wherein impurity content (observing by normalization method) to investigate the stability of solution, investigate 4 DEG C preserve need testing solution stabilities of solution in 12h, the content of four impurity is basically unchanged, RSD is less than 10%
Illustrate that in 12h, solution-stabilized row is good。Therefore, use mobile phase sample dissolution, and the mode depositing in 4 DEG C carries out follow-up test, or solution selects now with the current。Measurement result is in Table 2。
Table 2: stability of solution measurement result
Above result of the test shows, the method simplicity is sensitive, favorable reproducibility, can preferably the quality of dabigatran in sample be detected preferably。
2. the preparation of need testing solution:
Take the appropriate grind into powder of compound alpha-ketoacid tablet preparation, add mobile phase and be configured to the solution of 6mg/m, as need testing solution。Need testing solution chromatogram is shown in Fig. 1。
3. the preparation of contrast solution:
Measure above-mentioned steps (2) need testing solution appropriate, add the solution that mobile phase is diluted in every 1ml containing about 60 μ g, as contrast solution。Reference substance solution chromatogram is shown in Fig. 2。
4. in compound alpha-ketoacid tablet preparation, amino acid calcium constituents has the detection of related substance:
Take contrast solution 10 μ l and inject chromatograph of liquid, regulate detection sensitivity, main constituent chromatograph peak height is made to be about the 20% of full scale, take need testing solution 10 μ l again and inject chromatograph of liquid, record chromatogram runs 95min to instrument, record each impurity peaks, with main constituent Self-control method, calculate each impurity content。
Embodiment 2
The checking of testing conditions
2.1. specificity
Take need testing solution 10ml injecting chromatograph, as system suitability solution, analyze by above-mentioned chromatographic condition sample introduction。Result shows (see Fig. 3), each main constituent and each impurity, and the equal R of separating degree between impurity and impurity, more than 1.5, meets the requirements。Measurement result is in Table 3。
Table 3 system suitability result
2.2. test sample precision test
Taking the need testing solution of new preparation, repeat sample introduction six times, measure the peak area of impurity in sample, the RSD calculating impurity peak area is respectively less than 10%, it was shown that instrument precision is good。Measurement result is in Table 4。
Table 4 test sample precision measurement result
2.3. comparison liquid precision test
Taking the contrast solution of new preparation, repeat sample introduction six times, measure the peak area of main peak, the RSD calculating main peak peak area is respectively less than 10%, it was shown that instrument precision is good。Measurement result is in Table 5。
Table 5 compares liquid precision measurement result
2.4. repeatability test
Taking the need testing solution 6 parts of same batch, analyze by 1.1 lower chromatographic condition sample introductions, before each sample introduction, sample solution is put in 4 DEG C of preservations, test result indicate that, the RSD of impurity peaks is respectively less than 10%, it was shown that the method has good repeatability。Measurement result is in Table 6。
Table 6 repeatability experimental data
In embodiment 3, compound alpha-ketoacid tablet preparation, amino acid calcium constituents has the mensuration of related substance
Take 20130601 batches of compound alpha-ketoacid tablet preparations appropriate, finely ground, weigh 300mg sample in 50ml volumetric flask, add mobile phase appropriate, ultrasonic 30min makes dissolving, lets cool to room temperature, adds mobile phase and is diluted to scale, shakes up, and filters, takes subsequent filtrate as need testing solution。Precision measures 1ml need testing solution and (dilutes 100 times) in 100ml volumetric flask, adds mobile phase and is diluted to scale, shake up, obtains reference substance solution。Under following selected chromatographic condition: UV-detector (Agilent 1200 type), strong ion-exchange chromatography (the AgilentHi-PlexH of Hydrogen, 300 × 7.7mm) mobile phase: 0.0145mol/L sulfuric acid solution, flow velocity: 0.65ml/min, detection wavelength: 205nm, column temperature: 40 DEG C, sampling volume:
10ul, runs time: 95min
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia two annex V D of version in 2010)。Precision measures reference substance solution 10 μ l and injects chromatograph of liquid, regulates detection sensitivity, makes the peak height of main constituent chromatographic peak be about the 20% of full scale。The accurate blank solution measuring 10 μ l and need testing solution inject chromatograph of liquid again, record chromatogram。Chromatographic peak in deduction system and blank solution, as impurity chromatographic peak occurs in need testing solution chromatogram, record the peak area of each impurity, calculate impurity content by main constituent Self-control method, any negligible less than the peak of main peak area 0.05% in need testing solution chromatogram。
In 20130601 batches of compound alpha-ketoacid tablet preparations, the relevant substance-measuring result of amino acid calcium constituents is in Table 7。
Table 7:20130601 criticizes the relevant substance-measuring result of amino acid calcium constituents in compound alpha-ketoacid tablet preparation
Claims (5)
1. the relevant substance detecting method of amino acid calcium constituents in a compound alpha-ketoacid tablet preparation, it is characterised in that comprise the following steps:
(1) chromatographic condition setting procedure:
In this step, detector adopts UV-detector, column temperature 40 DEG C, flow velocity 0.65ml/min；The mobile phase of chromatographic condition is concentration is the concentrated sulfuric acid solution of 0.0100-0.0180mol/L, detects wavelength 190-205nm, and chromatogram column temperature is 40-60 DEG C, flow velocity 0.65-1.0ml/min；
(2) preparation process of need testing solution:
Taking compound alpha-ketoacid tablet preparation appropriate, the last mobile phase adding step (1) of pulverizing is configured to the solution of 6mg/ml, as need testing solution；
(3) preparation of contrast solution:
Measuring need testing solution prepared by step (2) appropriate, the mobile phase adding step (1) is diluted to the solution that concentration is 1.58～15.81 μ g/ml, as contrast solution；
(4) determination step:
Take contrast solution 10 μ l injection chromatograph of liquid prepared by step (3), regulate detection sensitivity, main constituent chromatograph peak height is made to be about the 20% of full scale, take need testing solution 10 μ l injection chromatograph of liquid prepared by step (2) again, run 95min, record each impurity peak area, adopt main constituent counter point, calculate each impurity content。
2. the relevant substance detecting method of amino acid calcium constituents in a kind of compound alpha-ketoacid tablet preparation as claimed in claim 1, it is characterised in that described mobile phase is concentration is the concentrated sulfuric acid solution of 0.0145mol/L。
3. the relevant substance detecting method of amino acid calcium constituents in a kind of compound alpha-ketoacid tablet preparation as claimed in claim 1, it is characterised in that described flow velocity is 0.65ml/min。
4. the relevant substance detecting method of amino acid calcium constituents in a kind of compound alpha-ketoacid tablet preparation as claimed in claim 1, it is characterized in that, in compound alpha-ketoacid tablet preparation in amino acid calcium constituents, the detectability of tung-oil coated urea be 7.90ng, phenylacetic acid detectability be 3.78ng。
5. the relevant substance detecting method of amino acid calcium constituents in a kind of compound alpha-ketoacid tablet preparation as claimed in claim 1, it is characterised in that the linearly dependent coefficient before the concentration of peak area and contrast solution is 0.9999。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112697934A (en) * | 2020-12-10 | 2021-04-23 | 武汉久安药业有限公司 | Method for detecting content of pyroglutamic acid in compound amino acid injection |
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| CN104101678A (en) * | 2013-04-03 | 2014-10-15 | 天津金耀集团有限公司 | Method for determining content of five calcium salts in compound alpha-ketoacid tablet |
| CN104316641A (en) * | 2014-10-20 | 2015-01-28 | 华东理工大学 | Method for detecting impurity content in ketophenylalanine calcium |
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2016
- 2016-05-09 CN CN201510975446.0A patent/CN105699502A/en active Pending
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| CN102961378A (en) * | 2012-11-27 | 2013-03-13 | 贵州信邦制药股份有限公司 | Compound alpha-keto acid tablets and preparation method and detection method thereof |
| CN104101678A (en) * | 2013-04-03 | 2014-10-15 | 天津金耀集团有限公司 | Method for determining content of five calcium salts in compound alpha-ketoacid tablet |
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| CN112697934A (en) * | 2020-12-10 | 2021-04-23 | 武汉久安药业有限公司 | Method for detecting content of pyroglutamic acid in compound amino acid injection |
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