CN102269737B - HPLC (high performance liquid chromatography) detection method of arginine ketoglutarate - Google Patents
HPLC (high performance liquid chromatography) detection method of arginine ketoglutarate Download PDFInfo
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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
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- G—PHYSICS
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
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Abstract
The invention aims at providing a method for determining the content of L-arginine alpha ketoglutarate and a preparation thereof as well as related substances through high performance liquid chromatography. The chromatographic conditions are as follows: the chromatographic column is an amino column; the mobile phase is a methanol-acetonitrile-phosphate buffer solution according to a ratio of (0-20): (30-70): (20-50) in parts by volume; and the detection wavelength is 200-220nm.
Description
Technical field
The present invention relates to a kind of method of measuring α-ketoglutaric acid-L-arginine salt of biomedicine field, particularly a kind of content of high effective liquid chromatography for measuring α-ketoglutaric acid-L-arginine salt and preparation thereof and method of related substance of adopting.
Background technology
α-ketoglutaric acid-L-arginine salt (1:2) and preparation thereof are the liver-protecting medicines of French auspicious flange pharmaceutical factory development.It is lacked of proper care for liver function, can be directly and Quick for nutrition, provide energy to liver cell, safeguard rapidly liver normal function and health thereof.
In the new drug research process, the content of medicine and the Accurate Determining of impurity thereof and analysis are the essential condition that guarantees its quality.α-ketoglutaric acid-L-arginine salt legibility from, therefore, when its content, purity, related substance etc. are analyzed, run into larger difficulty.Prior art only has pair α-ketoglutaric acid or L-arginine monomer to carry out the report of content or determination of related substances, Jing Hongwu etc. disclose employing spectrophotometry α-ketoglutaric acid (SIMULTANEOUS DETERMINATION of α-ketoglutaric acid and pyruvic acid. Tongji University's journal, 1986,14 (4): 531-536), but spectrophotometric method low precision, accuracy is not high, and only can measure the content of α-ketoglutaric acid.Mao Ying etc. disclose the content that adopts the high effective liquid chromatography for measuring ketoglutaric acid, and it adopts Agilent ODS C
18Post is with 0.5% (NH
4)
2HPO
4-H
3PO
4Damping fluid is mobile phase, flow velocity is 110ml/min, it is the content that the 233nm(HPLC method is measured ketoglutaric acid that ultraviolet detects wavelength. Chinese Pharmaceutical Affairs, 2008,22 (10): 598-599), but the method also only can be used for measuring the content of ketoglutaric acid, can't carry out the inspection of content detection and related substance to α-ketoglutaric acid-L-arginine salt.From a few days ago on, in the prior art not to the report of the quality control of α-ketoglutaric acid-L-arginine salt.
Dimension, prior art need that a kind of that α-ketoglutaric acid-L-arginine salt and preparation thereof are carried out the accuracy of quality testing and purity test is high, the method for favorable reproducibility.
Summary of the invention
The object of the present invention is to provide a kind of content of high effective liquid chromatography for measuring α-ketoglutaric acid-L-arginine salt and preparation thereof and method of related substance of adopting, thereby guarantee the purity of α-ketoglutaric acid-L-arginine salt, realize the quality control of its raw material and preparation.Adopt method of the present invention, can detect by a high performance liquid chromatography, simultaneously Accurate Determining goes out content and the related substance of α-ketoglutaric acid and L-arginine in α-ketoglutaric acid-L-arginine salt or its preparation, main ingredient all can reach good separating with impurity, can be advantageously used in the α-ketoglutaric acid-content of L-arginine salt and the detection of related substance thereof.
In the method for the present invention, the α-ketoglutaric acid-α-ketoglutaric acid of L-arginine salt and the ratio of L-arginine are 1:1 or 1:2, preferred 1:2, i.e. α-ketoglutaric acid-L-arginine salt (1:2).
The chromatographic condition of high performance liquid chromatography of the present invention is:
Chromatographic column is nh 2 column;
Mobile phase is methyl alcohol-acetonitrile-phosphate buffer=(0-20): (30-70): (20-50), preferred (0-10): (50-60): 40,0:60:40 most preferably is in volume parts;
The detection wavelength is 200-220nm, preferred 205nm.
In the method for the present invention, column temperature is 5-50 ℃, is preferably 10-40 ℃, most preferably is room temperature (15-30 ℃).
In the method for the present invention, the damping fluid in the mobile phase is phosphate buffer, and its pH value is 2.5-7.0, preferred 4.0-7.0, most preferably 5.2.Wherein, the acid or the alkali that are used for the pH value adjusting comprise hydrochloric acid, sulfuric acid, hydrosulfate, citric acid, tartrate, methane-sulforic acid, xylene monosulfonic acid, ethane diacid, formic acid, oxalic acid, phosphoric acid,diluted, acetic acid, phosphate dihydrogen salt solution, phosphoric acid hydrogen two salt solusions, preferably phosphoric acid disodium hydrogen or potassium, most preferably disodium phosphate soln.Randomly, can also add triethylamine or tetrahydrofuran in the phosphate solution, wherein, phosphate solution, tetrahydrofuran, triethylamine ratio are: 980-1000:0-20:(0-5); Preferred 1000:0:0-3.
Particularly, detection method of the present invention may further comprise the steps:
1, with α-ketoglutaric acid-L-arginine salt test sample dissolving;
2, the need testing solution after the dissolving injects high performance liquid chromatograph;
3, record chromatogram.
Wherein, chromatographic condition is:
Chromatographic column is nh 2 column;
Mobile phase is methyl alcohol-acetonitrile-phosphate buffer=(0-20): (30-70): (20-50), preferred (0-10): (50-60): 40,0:60:40 most preferably is in volume parts;
Flow velocity: 0.8-2.0ml/min, preferred 0.8-1.5ml/min, most preferably 0.8-1.2ml/min.
The detection wavelength is 200-220nm, preferred 205nm.
In the above method, column temperature is preferably room temperature.
In the above method, the pH value of the phosphate buffer in the mobile phase is 2.5-7.0, preferred 4.0-7.0, most preferably 5.2.Wherein, the acid or the alkali that are used for the pH value adjusting comprise hydrochloric acid, sulfuric acid, hydrosulfate, citric acid, tartrate, methane-sulforic acid, xylene monosulfonic acid, ethane diacid, formic acid, oxalic acid, phosphoric acid,diluted, acetic acid, phosphate dihydrogen salt solution, phosphoric acid hydrogen two salt solusions, preferably phosphoric acid disodium hydrogen or potassium, most preferably disodium phosphate soln.Can also add triethylamine or tetrahydrofuran in the phosphate solution, wherein, phosphate solution, tetrahydrofuran, triethylamine ratio are: 980-1000:0-20:(0-5); Preferred 1000:0:0-3.
In the method for the present invention, be below 100 minutes the writing time of chromatogram, preferred 80 minutes, and more preferably 60 minutes.
In the assay method of the present invention, the mensuration concentration of α-ketoglutaric acid-L-arginine salt solusion is 0.1-3.0mg/mL, preferred 0.5-1.5mg/mL, more preferably 1.0mg/mL.
Adopt method of the present invention, the appearance time of major component is: the retention time of two main peaks is: arginine (3-50min, preferred 5-20min, most preferably 9-14min), ketoglutaric acid (5-50min, preferred 15-30min, most preferably 20-25min).
The present invention adopts amino chromatographic column, can effectively separate α-ketoglutaric acid-L-arginine salt and related substance thereof, the purity of Accurate Determining α-ketoglutaric acid-L-arginine salt; The invention solves the separation problem of α-ketoglutaric acid-L-arginine salt and related substance thereof, thereby guarantee the quality controllable of α-ketoglutaric acid-L-arginine salt and preparation thereof.
Description of drawings
HPLC when Fig. 1 mobile phase is acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is regulated pH value to 5.2 with the disodium phosphate soln of 0.02mol/L)=60:40 schemes
HPLC figure when Fig. 2 mobile phase is acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate adds 0.2% triethylamine, with phosphoric acid,diluted adjust pH to 5.2)=60:40
HPLC figure when Fig. 3 mobile phase is acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate adds 0.2% triethylamine, with ethane diacid adjust pH to 5.2)=60:40
HPLC when Fig. 4 mobile phase is acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is regulated pH value to 6.5 with the disodium phosphate soln of 0.02mol/L)=60:40 schemes
HPLC when Fig. 5 mobile phase is acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is regulated pH value to 5.5 with the disodium phosphate soln of 0.02mol/L)=50:50 schemes
HPLC figure when Fig. 6 mobile phase is acetonitrile-phosphate buffered saline(PBS) (0.02mol/L sodium dihydrogen phosphate)=70:30
HPLC figure when Fig. 7 mobile phase is methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.01mol/L sodium dihydrogen phosphate adds 0.2% triethylamine, with phosphoric acid,diluted adjust pH to 7.0)=15:45:40
HPLC figure when Fig. 8 mobile phase is methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.03mol/L sodium dihydrogen phosphate adds 0.2% triethylamine and 2% tetrahydrofuran, with phosphoric acid,diluted adjust pH to 4.0)=15:50:35
HPLC figure when Fig. 9 mobile phase is methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L potassium dihydrogen phosphate adds 0.375% triethylamine and 1% tetrahydrofuran, with phosphoric acid,diluted adjust pH to 6.0)=20:30:50
HPLC figure when Figure 10 mobile phase is methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (0.02mol/L sodium dihydrogen phosphate, with phosphoric acid,diluted adjust pH to 6.0)=10:50:40
HPLC when Figure 11 mobile phase is methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L potassium dihydrogen phosphate is regulated pH value to 6.0 with the dipotassium hydrogen phosphate solution of 0.02mol/L)=10:60:30 schemes
HPLC figure when Figure 12 mobile phase is methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (0.02mol/L disodium phosphate soln, with phosphoric acid,diluted adjust pH to 6.0)=10:60:30
The HPLC figure of Figure 13 methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is with citric acid adjust pH to 5.0)=when the 5:55:40 mobile phase is
HPLC figure when Figure 14 mobile phase is methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is with the salt acid for adjusting pH value to 3.5 of 0.005mol/L)=10:50:40
HPLC when Figure 15 mobile phase is methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is regulated pH value to 4.5 with the sodium hydrogen phosphate of 0.02mol/L)=10:50:40 schemes
Embodiment
Following examples of the present invention only are used for illustrating realization technical scheme of the present invention, and these embodiments do not consist of further restriction to the present invention.Those skilled in the art are equal to replacement or corresponding logic improvement according to existing knowledge to the present invention, all belong to scope of the present invention.In following examples, the high performance liquid chromatograph of employing is Japanese Shimadzu: LC-10ATvp, SPD-10Avp; Chromatographic column is Agilent NH
25 μ m, 4.6 * 250mm; Sampling volume is 20 μ l.Certainly, also can adopt amino chromatographic column and other sampling volumes of high performance liquid chromatograph, other particle diameters and the length of other producers and model, can both reach purpose of the present invention.
Chromatographic condition
Mobile phase: acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is regulated pH value to 5.2 with the disodium phosphate soln of 0.02mol/L)=60:40;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 1.As shown in Figure 1, peak 1-3 is impurity peaks, and peak 4 is the L-arginine peak, and peak 5 is the α-ketoglutaric acid peak.Peak 1-3 and main peak degree of separation are all greater than 2.5.The mensuration wavelength that α-ketoglutaric acid-L-arginine salt is selected is in terminal the absorption, is subject to the impact of mobile phase.Adopt mobile phase pH value 6.8(aqueous pH values 5.2 in the present embodiment), baseline more tends towards stability, and flow phase system is simple and easy to prepare, and the mensuration process is not subject to external environmental interference; And two main peak appearance times are suitable, and are good with the impurity degree of separation.
Chromatographic condition
Mobile phase: acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate adds 0.2% triethylamine, with phosphoric acid,diluted adjust pH to 5.2)=60:40;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 2.As shown in Figure 2, main peak separates well with each impurity peaks in the sample.Peak 1-2, peak 4-5 are impurity peaks, and peak 3 is the L-arginine peak, and peak 6 is the α-ketoglutaric acid peak.Peak 1-2, peak 4-5 and main peak degree of separation are all greater than 2.5.
Chromatographic condition
Mobile phase: acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate adds 0.2% triethylamine, with ethane diacid adjust pH to 5.2)=60:40;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 3.As shown in Figure 3, main peak separates well with each impurity peaks in the sample.Peak 1-3 is impurity peaks, and peak 4 is L arginine peak, and peak 5 is the α-ketoglutaric acid peak.Peak 1-3 and main peak degree of separation are all greater than 2.5.
Chromatographic condition
Mobile phase: acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is regulated pH value to 6.5 with the disodium phosphate soln of 0.02mol/L)=60:40;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 4.As shown in Figure 4, main peak separates well with each impurity peaks in the sample.Peak 1-3, peak 5 are impurity peaks, and peak 4 is the L-arginine peak, and peak 6 is the α-ketoglutaric acid peak.Peak 1-3 and main peak degree of separation are all greater than 2.5, and peak 5 is 2.4 with the main peak degree of separation.
Chromatographic condition
Mobile phase: acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is regulated pH value to 5.5 with the disodium phosphate soln of 0.02mol/L)=50:50;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 5.As shown in Figure 5, main peak separates well with each impurity peaks in the sample.Peak 1-5 is impurity peaks, and peak 6 is L arginine peak, and peak 7 is the α-ketoglutaric acid peak.Peak 1-5 and main peak degree of separation are all greater than 2.5.
Chromatographic condition
Mobile phase: acetonitrile-phosphate buffered saline(PBS) (0.02mol/L sodium dihydrogen phosphate)=70:30;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 6.As shown in Figure 6, main peak separates well with each impurity peaks in the sample.Peak 2-4 is impurity peaks, and peak 1 is the L-arginine peak, and peak 5 is the α-ketoglutaric acid peak.Peak 2-4 and main peak degree of separation are all greater than 2.5.
Chromatographic condition
Mobile phase: methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.01mol/L sodium dihydrogen phosphate adds 0.2% triethylamine, with phosphoric acid,diluted adjust pH to 7.0)=15:45:40;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 7.As shown in Figure 7, main peak separates well with each impurity peaks in the sample.Peak 1-3 is impurity peaks, and peak 4 is the α-ketoglutaric acid peak, and peak 5 is the L-arginine peak.Peak 2-4 and main peak degree of separation are all greater than 2.5.
Chromatographic condition
Mobile phase: methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.03mol/L sodium dihydrogen phosphate adds 0.2% triethylamine and 2% tetrahydrofuran, with phosphoric acid,diluted adjust pH to 4.0)=15:50:35;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 8.As shown in Figure 8, main peak separates well with each impurity peaks in the sample.Peak 1, peak 3-5 are impurity peaks, and peak 2 is the α-ketoglutaric acid peak, and peak 6 is the L-arginine peak.Peak 3 is 2.2 with the main peak degree of separation, and all the other each impurity peaks and main peak degree of separation are all greater than 2.5.
Embodiment 9
Chromatographic condition
Mobile phase: methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L potassium dihydrogen phosphate adds 0.375% triethylamine and 1% tetrahydrofuran, with phosphoric acid,diluted adjust pH to 6.0)=20:30:50;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 9.As shown in Figure 9, main peak separates well with each impurity peaks in the sample.Peak 1-5 is impurity peaks, and peak 6 is the α-ketoglutaric acid peak, and peak 7 is the L-arginine peak.Peak 1-5 and main peak degree of separation are all greater than 2.5.
Chromatographic condition
Mobile phase: methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is with phosphoric acid,diluted adjust pH to 6.0)=10:50:40;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: 20 ℃
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 25ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 10.As shown in Figure 10, main peak separates well with each impurity peaks in the sample.Peak 1-2, peak 4-5 are impurity peaks, and peak 3 is the α-ketoglutaric acid peak, and peak 6 is the L-arginine peak.Peak 1-2, peak 4-5 and main peak degree of separation are all greater than 2.5.
Embodiment 11
Chromatographic condition
Mobile phase: methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L potassium dihydrogen phosphate is regulated pH value to 6.0 with the dipotassium hydrogen phosphate solution of 0.02mol/L)=10:60:30;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: 40 ℃
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 11.As shown in Figure 11, main peak separates well with each impurity peaks in the sample.Peak 1,2,4,6,7 is impurity peaks, and peak 3 is the α-ketoglutaric acid peak, and peak 5 is the L- arginine peak.Peak 1,2,4,6,7 and the main peak degree of separation all greater than 2.5.
Embodiment 12
Chromatographic condition
Mobile phase: methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L disodium phosphate soln is with phosphoric acid,diluted adjust pH to 6.0)=10:60:30;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 20mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 12.As shown in Figure 12, main peak separates well with each impurity peaks in the sample.Peak 1-4, peak 6 are impurity peaks, and peak 5 is the α-ketoglutaric acid peak, and peak 7 is the L-arginine peak.Peak 1-4, peak 6 and main peak degree of separation are all greater than 2.5.
Embodiment 13
Chromatographic condition
Mobile phase: methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L disodium phosphate soln,, with citric acid adjust pH to 5.0)=5:55:40;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 13.As shown in Figure 13, main peak separates well with each impurity peaks in the sample.Peak 1-4 is impurity peaks, and peak 5 is the L-arginine peak, and peak 6 is the α-ketoglutaric acid peak.Peak 1-4 and main peak degree of separation are all greater than 2.5.
Embodiment 14
Chromatographic condition
Mobile phase: methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is with the salt acid for adjusting pH value to 3.5 of 0.005mol/L)=10:50:40;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 14.As shown in Figure 14, main peak separates well with each impurity peaks in the sample.Peak 1, peak 3-4 are impurity peaks, and peak 2 is the L-arginine peak, and peak 5 is the α-ketoglutaric acid peak.Peak 1 is 2.1 with the main peak degree of separation, and all the other each impurity peaks and main peak degree of separation are all greater than 2.5.
Embodiment 15
Chromatographic condition
Mobile phase: methyl alcohol-acetonitrile-phosphate buffered saline(PBS) (the 0.02mol/L sodium dihydrogen phosphate is regulated pH value to 4.5 with the sodium hydrogen phosphate of 0.02mol/L)=10:50:40;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm
Column temperature: room temperature
Experimental procedure:
Need testing solution: take by weighing α-ketoglutaric acid-L-arginine salt (1:2) 10mg and place the 10ml measuring bottle, add the mobile phase dissolving and be diluted to scale, shake up, and get final product.
The accurate need testing solution 20 μ l that draw inject high performance liquid chromatograph, and the record chromatogram the results are shown in accompanying drawing 15.As shown in Figure 15, main peak separates well with each impurity peaks in the sample.Peak 1-7 is impurity peaks, and peak 8 is the L-arginine peak, and peak 9 is the α-ketoglutaric acid peak.Peak 1-7 and main peak degree of separation are all greater than 2.5.
Claims (18)
1. one kind is adopted the content of high effective liquid chromatography for measuring α-ketoglutaric acid-L-arginine salt and preparation thereof and the method for related substance, and its chromatographic condition is:
Chromatographic column: nh 2 column;
Mobile phase is methyl alcohol: acetonitrile: phosphate buffer=(0-20): (30-60): (20-50), in volume parts; Detect wavelength: 200-220nm;
PH:4.0-7.0。
2. according to claim 1 method, the α-ketoglutaric acid wherein-α-ketoglutaric acid of L-arginine salt and the ratio of L-arginine are 1:1 or 1:2.
3. according to claim 2 method, the α-ketoglutaric acid wherein-α-ketoglutaric acid of L-arginine salt and the ratio of L-arginine are 1:2.
4. either method according to claim 1～3, mobile phase wherein are methyl alcohol: acetonitrile: phosphate buffer=(0-10): (50-60): 40, in volume parts.
5. according to claim 4 method, mobile phase wherein is methyl alcohol: acetonitrile: phosphate buffer=10:50:40, in volume parts.
6. according to claim 5 method, detection wavelength wherein is 205nm.
7. according to claim 6 method, the column temperature that wherein adopts is room temperature.
8. according to claim 1 method, wherein the pH value of phosphate buffer is 6.0.
9. either method according to claim 1～3 wherein also adds triethylamine and/or tetrahydrofuran in the phosphate buffer, phosphate solution, tetrahydrofuran, triethylamine ratio are: (980-1000): (0-20): (0-5).
10. according to claim 9 method, wherein phosphate solution, tetrahydrofuran, triethylamine ratio are 1000:0:0-3.
11. method according to claim 1 may further comprise the steps:
(1) with α-ketoglutaric acid-L-arginine salt test sample dissolving;
(2) need testing solution after the dissolving injects high performance liquid chromatograph;
(3) record chromatogram.
12. method according to claim 11, the mensuration concentration of α-ketoglutaric acid-L-arginine salt solusion is 0.1-3.0mg/mL.
13. method according to claim 12, the mensuration concentration of α-ketoglutaric acid-L-arginine salt solusion is 0.5-1.5mg/mL.
14. method according to claim 13, the mensuration concentration of α-ketoglutaric acid-L-arginine salt solusion is 1.0mg/mL.
15. one kind is adopted the content of high effective liquid chromatography for measuring α-ketoglutaric acid-L-arginine salt and preparation thereof and the method for related substance, its chromatographic condition is:
Chromatographic column: nh 2 column;
Mobile phase: acetonitrile-phosphate buffered saline(PBS)=60:40; Phosphate buffered saline(PBS) wherein is the 0.02mol/L sodium dihydrogen phosphate, and regulates pH value to 5.2 with the disodium phosphate soln of 0.02mol/L;
Flow velocity: 1.0ml/min;
Detect wavelength: 205nm;
The ratio that takes by weighing α-ketoglutaric acid and L-arginine is that the α-ketoglutaric acid of 1:2-L-arginine salt places measuring bottle, adds the mobile phase dissolving and is diluted to 1mg/ml, shakes up, and gets need testing solution:
Accurate absorption need testing solution is an amount of, injects high performance liquid chromatograph, the record chromatogram.
16. method according to claim 15, nh 2 column wherein are Agilent NH
2, 5 μ m, the nh 2 column of 4.6 * 250mm.
17. according to claim 15 or 16 method, column temperature wherein is room temperature.
18. according to claim 15 or 16 method, sampling volume is 20 μ l.
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| CN101250563A (en) * | 2008-03-20 | 2008-08-27 | 江南大学 | Method for realizing excessive accumulation of alpha-ketoglutarate acid by adding alpha-ketoglutarate acid dehydrogenase inhibitor |
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