CN1053128A - The high-efficient liquid phase chromatogram process measuring method of 18 compound amino-acids injection liq - Google Patents
The high-efficient liquid phase chromatogram process measuring method of 18 compound amino-acids injection liq Download PDFInfo
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- CN1053128A CN1053128A CN 90100069 CN90100069A CN1053128A CN 1053128 A CN1053128 A CN 1053128A CN 90100069 CN90100069 CN 90100069 CN 90100069 A CN90100069 A CN 90100069A CN 1053128 A CN1053128 A CN 1053128A
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Abstract
The present invention relates to the drug inspection specialty 18 compound amino-acids injection liq efficiently roll the phase chromatography assay method, it adopts the high-efficient phase chromatogram instrument, with 2, the 4-dinitrofluorobenzene is made derivative reagent, with pre-column derivatization method 18 compound amino-acids injection liq is carried out quantitative measurement.This method adopts quantitative dilution technology preparation to detect liquid, has adopted the mobile phase A of finite concentration and pH value and Mobile phase B to do the gradient elution agent in testing process.This method is only once finished the detection by quantitative process of 18 compound amino-acids injection liq on high performance liquid chromatograph commonly used, have the saving testing cost, and analytical cycle is short, and easy to operate, analyst coverage is wide, accuracy height, the characteristics of favorable reproducibility.
Description
The present invention relates to a kind of high-efficient liquid phase chromatogram process measuring method of 18 compound amino-acids injection liq of drug inspection specialty.
Automatic amino acid analyzer is mainly adopted in the quantitative measurement of moriamin-s of the prior art, perhaps adopt complete high performance liquid chromatography post after, the column front derivation device.The shortcoming of this quasi-instrument and device is: the equipment price costliness, can not separate 18 kinds of compound amino acids simultaneously, and analysis time is long.
The objective of the invention is to provide a kind of employing high performance liquid chromatograph, make the high-efficient liquid phase chromatogram process measuring method of the 18 compound amino-acids injection liq of column front derivation reagent with the 2.4-dinitrofluorobenzene.It can be effectively in 30 minutes first separation go out 18 kinds of compound amino acids, carry out the quantitative test and the mensuration of each component amino acid content.
The detecting instrument that the present invention adopts is: the common high performance liquid chromatograph with gradient elution function.It includes gradient pump, instruments such as sampling valve and carbon-18 anti-phase analytical columns; The ultraviolet-visible spectrophotometric detector; The computer integraph.
The testing conditions that the present invention adopts is: ultraviolet one visible spectrophotometric detector detects at 360nm wavelength place, and the column temperature of analytical column is 30 ℃, and mobile phase A and Mobile phase B are adopted in the gradient elution agent.Mobile phase A is 1: 1 a acetonitrile solution; Mobile phase B is sodium acetate and acetate buffer solution, and its concentration is 0.05mol/L, and pH value is 6.4, and contains 1% N, the dinethylformamide ion pair.Adopt fixing moving phase program, that is: mobile phase A and the Mobile phase B concentration ratio in 0-30 minute gradually changed between 16: 84 to 98: 2, and flow velocity is 1ml/min.The derivatization temperature adopts 60 ℃ ± 2 ℃ water-baths, 60 minutes time.The derivatization damping fluid is that pH value is 7.0 potassium dihydrogen phosphate.The detection sample size is 10uL.The recording chart chart drive speed of computer integraph is 0.5cm/mln.
Detection principle of the present invention is: to most of amino acid that do not produce fluorescence and no uv absorption itself, adopt the 2.4-dinitrofluorobenzene to make the direct derivatization of derivative reagent, the derivant that acquisition can be detected.Under alkali condition, amino acid whose amino can generate yellow derivant dinitro benzene amino acid with the reaction of derivative reagent 2.4-dinitrofluorobenzene quantitatively.Adopt ion pair chromatography, utilize substituting group, aliphatic hydrocarbon, aromatic hydrocarbon, heterocycle, hydroxyl, the difference of sulfydryl and isoelectric point, the derivant dinitro benzene amino acid that generates is separated on the anti-phase analytical column of carbon-18, and with N, dinethylformamide adds in the moving phase, make electronegative carboxyl and N, the positive ion of dinethylformamide forms ion pair, thereby separated amino acid peak face is increased, and retention time prolongs.Post is imitated and is improved, and formed non-polar group and reversed-phase column filler interact, and makes each seed amino acid obtain separating.Each seed amino acid after the chromatographic resolution is made chromatogram through the detection and the processing of ultraviolet one visible spectrophotometric detector and computer integraph, finishes each component amino acid percentage composition according to chromatogram then and analyzes and measure.
Accepted standard reference substance of the present invention is: L-asparatate, L-glutamic acid, L-serine, L-glycocoll, the L-threonine, L-proline, L-alanine, the L-arginine, L-valine, L-methionine, the L-cystine, L-isoleucine, L-leucine, the L-tryptophane, L-phenylalanine, L-histidine, the L-halfcystine, L-lysine, L-tyrosine.Its amino acid content is all more than 99%.
The present invention adopts the detectable of the pure quality of following analysis: acetonitrile, and sodium acetate, acetic acid, sodium bicarbonate and potassium dihydrogen phosphate, 2.4-dinitrofluorobenzene are derivative reagent, through the decompression distillation purifying.
The object of the present invention is achieved like this: adopt above-mentioned detecting instrument, testing conditions detects principle, and standard reference material and detectable are finished the high effective liquid chromatography for measuring process of 18 compound amino-acids injection liq.Accurately getting test sample and reference substance adds water respectively and makes certain density solution, each is accurately measured 1-2ml and places the 10ml volumetric flask, add 0.5mol/l concentration, the sodium bicarbonate solution 1ml of PH9 adds 2.4-dinitrofluorobenzene 10ul, acetonitrile 1ml, jolting is even, put heating taking-up after 60 minutes in dark place in 60 ℃ of water-baths, the potassium phosphate buffer that adds PH7.0 is to scale, and jolting is even.Get the derivative products dinitro benzene amino acid after 10ul quantitatively dilutes, directly inject the anti-phase analytical column of carbon-18 of high performance liquid chromatograph through sampling valve.Simultaneously, gradient elution agent mobile phase A and Mobile phase B are pressed the described moving phase program of testing conditions, send into sampling valve by gradient pump with the flow velocity of 1ml/min.The anti-phase analytical column of carbon-18 through high performance liquid chromatograph carries out chromatographic resolution.Each amino acid derivativges and reversed-phase column filler interact in this course, make each seed amino acid obtain separating.Finish chromatographic resolution through carbon-18 reversed-phase column, each seed amino acid after the chromatographic resolution is made detection by quantitative through the ultraviolet-visible spectrophotometric detector, the detection electric signal is sent into the computer integraph and is carried out data processing, and each seed amino acid chromatogram that draws successively on recording paper strip.The isolated various amino acid sequences of this chromatogram are: L-asparatate, L-glutamic acid, L-serine, L-glycocoll, the L-threonine, L-proline, L-alanine, the L-arginine, L-valine, L-methionine, the L-cystine, L-isoleucine, L-leucine, the L-tryptophane, L-phenylalanine, L-histidine, the L-halfcystine, L-lysine, L-tyrosine.According to each seed amino acid chromatogram of survey test sample and reference substance, calculate the amino acid whose percentage composition of each component in the 18 compound amino-acids injection liq respectively with external standard method.
The present invention only need adopt high-efficient phase chromatogram instrument commonly used and other general utensil, make derivative reagent with the 2.4-dinitrofluorobenzene and can finish detection, with the expensive amino-acid analyzer of employing, device is compared behind the perhaps complete high performance liquid chromatography post, before the post, and the present invention can save testing cost.The present invention can finish the detection by quantitative to 18 compound amino-acids injection liq in 30 minutes, and it is wide to have analyst coverage, and analytical cycle is short, convenience operation.In addition, because the maximum error that detects of the present invention is no more than 5%, measuring accuracy is less than 2%, therefore, originally returns and has the accuracy in detection height, the advantage of favorable reproducibility.
Concrete detection method of the present invention is provided by following embodiment.
Accurately get test sample and reference substance and add water respectively and make certain density solution, detect test and carry out respectively, each solution of accurately measuring 1-2ml places the 10ml volumetric flask, add 0.5mol/l concentration, the sodium bicarbonate solution 1ml of PH9 adds 2.4-dinitrofluorobenzene 10ul, acetonitrile 1ml, jolting is even.Put heating taking-up after 60 minutes in dark place in 60 ℃ of water-baths, the potassium phosphate buffer that adds PH7.0 is to scale, and jolting is even.Get the derivative products dinitro benzene amino acid after 10ul quantitatively dilutes, directly inject the anti-phase analytical column of carbon-18 of high performance liquid chromatograph through sampling valve.Simultaneously, gradient elution agent mobile phase A and Mobile phase B are sent into sampling valve by following moving phase program by gradient pump with flow velocity 1ml/min, promptly mobile phase A and the Mobile phase B concentration ratio in 0-30 minute gradually changed between 16: 84 to 98: 2.The anti-phase analytical column of carbon-18 through high performance liquid chromatograph carries out chromatographic resolution.Each amino acid derivativges and reversed-phase column filler interact, and make each seed amino acid obtain separating.Finish chromatographic resolution through the anti-phase analytical column of carbon-18, each seed amino acid after the chromatographic resolution is made detection by quantitative through the ultraviolet-visible spectrophotometric detector, the detection electric signal is sent into the computer integraph and is carried out data processing, and each seed amino acid chromatogram that draws successively on recording paper strip.Isolated each amino acid sequence of this chromatogram is: L-asparatate, L-glutamic acid, L-serine, L-glycocoll, the L-threonine, L-proline, L-alanine, the L-arginine, L-valine, L-methionine, the L-cystine, L-isoleucine, L-leucine, the L-tryptophane, L-phenylalanine, L-histidine, the L-halfcystine, L-lysine, L-tyrosine.Each seed amino acid chromatogram according to survey test sample and reference substance are represented respectively calculates the amino acid whose percentage composition of each component in the 18 compound amino-acids injection liq respectively with external standard method.
Claims (3)
1, the high-efficient liquid phase chromatogram process measuring method of 18 compound amino-acids injection liq, it is characterized in that the 1 milliliter of acetonitrile of in the pre-column derivatization process, will annotating, jolting is even, place 60 ℃ of water-bath dark place heating to take out after 60 minutes, the potassium phosphate buffer that adds PH7.0 is diluted to scale, jolting is even again, in gradient elution agent process, be the gradient elution agent with mobile phase A and Mobile phase B, and the moving phase program that adopts mobile phase A and Mobile phase B concentration ratio in 0-30 minute between 16: 84 to 98: 2, to gradually change.
2, method according to claim 1 is characterized in that mobile phase A is an acetonitrile solution, and concentration ratio is 1: 1.
3, method according to claim 1 is characterized in that Mobile phase B is sodium acetate and acetate buffer solution, and concentration is 0.05mol/l, and pH value is 6.4, and contains 1% N, dinethylformamide.
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| CN 90100069 CN1053128A (en) | 1990-01-04 | 1990-01-04 | The high-efficient liquid phase chromatogram process measuring method of 18 compound amino-acids injection liq |
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| CN 90100069 CN1053128A (en) | 1990-01-04 | 1990-01-04 | The high-efficient liquid phase chromatogram process measuring method of 18 compound amino-acids injection liq |
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Cited By (12)
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|---|---|---|---|---|
| CN100362346C (en) * | 2006-06-21 | 2008-01-16 | 广州星群（药业）股份有限公司 | Method for detecting amino acid component in Xiasangju preparation |
| CN100394177C (en) * | 2004-06-02 | 2008-06-11 | 昆明贵金属研究所 | High-efficient liquid-phase chromatographic testing method for copper in aspirin |
| CN101963603A (en) * | 2010-09-30 | 2011-02-02 | 湖北新生源生物工程股份有限公司 | Method for analyzing arginine and arginine hydrochloride raw materials and preparations by using HPLC method |
| CN101968472A (en) * | 2010-11-01 | 2011-02-09 | 湖北新生源生物工程股份有限公司 | Method for analyzing raw materials of histidine and histidine monohydrochloride and preparation by HPLC method |
| CN101294938B (en) * | 2008-06-12 | 2011-07-06 | 山东省科学院海洋仪器仪表研究所 | Method for analyzing and distinguishing sea surface excessive oil by ozone oxidization deriving liquid chromatogram coupling technique |
| CN101581703B (en) * | 2008-05-16 | 2012-05-09 | 上海中医药大学 | Method for single-time measuring ten amino bioactivators in organism |
| CN101365555B (en) * | 2006-01-11 | 2013-07-03 | 阿勒里斯铝业科布伦茨有限公司 | Method of manufacturing a brazed assembly |
| CN105021740A (en) * | 2015-08-15 | 2015-11-04 | 杭州新博思生物医药有限公司 | High-performance liquid chromatography analytical method for N1,N1-diisopropyl ethylenediamine |
| CN107817312A (en) * | 2017-12-15 | 2018-03-20 | 湖南千金协力药业有限公司 | The detection method of amino acid content in a kind of Zinc calcium gluconate oral solution |
| 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 |
| CN111829851A (en) * | 2020-07-25 | 2020-10-27 | 重庆医科大学 | Pretreatment method for determination of 18 amino acids in brain tissue and plasma of C57 mouse |
| CN114441681A (en) * | 2022-01-28 | 2022-05-06 | 山东省食品药品检验研究院 | Method for measuring 20 free amino acids and L-hydroxyproline in special medical formula food |
-
1990
- 1990-01-04 CN CN 90100069 patent/CN1053128A/en active Pending
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100394177C (en) * | 2004-06-02 | 2008-06-11 | 昆明贵金属研究所 | High-efficient liquid-phase chromatographic testing method for copper in aspirin |
| CN101365555B (en) * | 2006-01-11 | 2013-07-03 | 阿勒里斯铝业科布伦茨有限公司 | Method of manufacturing a brazed assembly |
| CN100362346C (en) * | 2006-06-21 | 2008-01-16 | 广州星群（药业）股份有限公司 | Method for detecting amino acid component in Xiasangju preparation |
| CN101581703B (en) * | 2008-05-16 | 2012-05-09 | 上海中医药大学 | Method for single-time measuring ten amino bioactivators in organism |
| CN101294938B (en) * | 2008-06-12 | 2011-07-06 | 山东省科学院海洋仪器仪表研究所 | Method for analyzing and distinguishing sea surface excessive oil by ozone oxidization deriving liquid chromatogram coupling technique |
| CN101963603B (en) * | 2010-09-30 | 2013-04-24 | 湖北新生源生物工程股份有限公司 | Method for analyzing arginine and arginine hydrochloride raw materials and preparations by using HPLC method |
| CN101963603A (en) * | 2010-09-30 | 2011-02-02 | 湖北新生源生物工程股份有限公司 | Method for analyzing arginine and arginine hydrochloride raw materials and preparations by using HPLC method |
| CN101968472A (en) * | 2010-11-01 | 2011-02-09 | 湖北新生源生物工程股份有限公司 | Method for analyzing raw materials of histidine and histidine monohydrochloride and preparation by HPLC method |
| CN101968472B (en) * | 2010-11-01 | 2013-04-24 | 湖北新生源生物工程股份有限公司 | Method for analyzing raw materials of histidine and histidine monohydrochloride and preparation by HPLC method |
| CN105021740A (en) * | 2015-08-15 | 2015-11-04 | 杭州新博思生物医药有限公司 | High-performance liquid chromatography analytical method for N1,N1-diisopropyl ethylenediamine |
| 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 |
| CN107817312A (en) * | 2017-12-15 | 2018-03-20 | 湖南千金协力药业有限公司 | The detection method of amino acid content in a kind of Zinc calcium gluconate oral solution |
| CN107817312B (en) * | 2017-12-15 | 2019-11-26 | 湖南千金协力药业有限公司 | The detection method of amino acid content in a kind of Zinc calcium gluconate oral solution |
| CN111829851A (en) * | 2020-07-25 | 2020-10-27 | 重庆医科大学 | Pretreatment method for determination of 18 amino acids in brain tissue and plasma of C57 mouse |
| CN114441681A (en) * | 2022-01-28 | 2022-05-06 | 山东省食品药品检验研究院 | Method for measuring 20 free amino acids and L-hydroxyproline in special medical formula food |
| CN114441681B (en) * | 2022-01-28 | 2024-06-04 | 山东省食品药品检验研究院 | Method for determining 20 free amino acids and L-hydroxyproline in special medical formula food |
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