CN104483406B - The high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid - Google Patents
The high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid Download PDFInfo
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- 229940009533 alpha-ketoglutaric acid Drugs 0.000 title claims abstract description 107
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 16
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 claims abstract description 108
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims abstract description 52
- 229930064664 L-arginine Natural products 0.000 claims abstract description 52
- 235000014852 L-arginine Nutrition 0.000 claims abstract description 52
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 claims abstract description 52
- 239000012071 phase Substances 0.000 claims abstract description 46
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 12
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 8
- 239000007791 liquid phase Substances 0.000 claims abstract description 8
- 238000012360 testing method Methods 0.000 claims description 46
- 239000013558 reference substance Substances 0.000 claims description 38
- 239000007788 liquid Substances 0.000 claims description 10
- 239000012467 final product Substances 0.000 claims description 8
- 238000005070 sampling Methods 0.000 claims description 5
- 238000010812 external standard method Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 3
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical group [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 6
- 239000000203 mixture Substances 0.000 abstract description 5
- 238000000926 separation method Methods 0.000 abstract description 5
- 238000011067 equilibration Methods 0.000 abstract description 3
- 238000004440 column chromatography Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 39
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 12
- 238000011084 recovery Methods 0.000 description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 235000009697 arginine Nutrition 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- -1 ketoglutaric acid arginine salt Chemical class 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- PAVHERCAUPDRGZ-UHFFFAOYSA-N OC(O)CC[SiH3] Chemical group OC(O)CC[SiH3] PAVHERCAUPDRGZ-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- FGDQGIKMWOAFIK-UHFFFAOYSA-N acetonitrile;phosphoric acid Chemical compound CC#N.OP(O)(O)=O FGDQGIKMWOAFIK-UHFFFAOYSA-N 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 229960000484 ceftazidime Drugs 0.000 description 1
- NMVPEQXCMGEDNH-TZVUEUGBSA-N ceftazidime pentahydrate Chemical compound O.O.O.O.O.S([C@@H]1[C@@H](C(N1C=1C([O-])=O)=O)NC(=O)\C(=N/OC(C)(C)C(O)=O)C=2N=C(N)SC=2)CC=1C[N+]1=CC=CC=C1 NMVPEQXCMGEDNH-TZVUEUGBSA-N 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000019837 monoammonium phosphate Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
Abstract
The invention discloses a kind of high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid, relate to column chromatography methods field.Its chromatographic condition is: chromatographic column: C
18chromatographic column; Mobile phase: 0.02mol/L ~ 0.08mol/L phosphate buffer, pH3.0-4.0; Flow rate of mobile phase: 0.5-1.3ml/min; Determined wavelength: 200-210nm; Chromatographic column column temperature: 15 DEG C ~ 30 DEG C, carry out efficient liquid phase chromatographic analysis, measures the content of L-arginine and α-ketoglutaric acid in L-arginine-α-ketoglutaric acid or its preparation.The present invention can be detected by a high performance liquid chromatography, and Accurate Determining goes out L-arginine and α-ketoglutaric acid content in L-arginine-α-ketoglutaric acid or its preparation simultaneously, and two kinds of compositions can reach good separation, and C
18chromatographic column equilibration time is short, about need 30-40 minute just can, improve work efficiency.
Description
Technical field
The present invention relates to column chromatography methods field.
Background technology
L-arginine-α-ketoglutaric acid is a kind of amidates health products that on current international market, sales volume increases day by day, is mainly used in the physique etc. improving human body protein and strengthen people.Have 1: 1 and 2: 1(mol ratio of arginine and ketoglutaric acid) two kinds of specifications, L-arginine-α-ketoglutaric acid (2: 1) and preparation thereof are the liver-protecting medicines of French auspicious flange pharmaceutical factory development, for liver disorder can directly and Quick for nutrition, there is provided energy to liver cell, safeguard rapidly liver normal function and health.
In prior art, the grand force of scape etc. disclose and adopt the 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 (HPLC method measures the content of ketoglutaric acid, Chinese Pharmaceutical Affairs, 2008,22(10) adopting high effective liquid chromatography for measuring ketoglutaric acid: 598-599), it adopts AgilentODSC
18post, with 0.5%(NH
4)
2hPO
4-H
3pO
4damping fluid is mobile phase, and flow velocity is 1.0ml/min, and UV detect wavelength is 233nm, but the method also only can be used for the content measuring ketoglutaric acid, cannot carry out content detection to L-arginine-α-ketoglutaric acid.
Under 2010 editions Chinese Pharmacopoeia ceftazidime for inj kind items, in assay, arginine content measures, be filling agent with dihydroxypropyl silane group silica gel, (ammonium dihydrogen phosphate (ADP) 1.15g is taken with acetonitrile-pH2.0 phosphate buffer, after the 800ml that adds water makes dissolving, by phosphoric acid adjust ph to 2.0, then be diluted with water to 1000ml, mixing) (750: 250) be mobile phase, determined wavelength is 206nm, but the method only can measure the content of L-arginine.
In the HPLC detection method (Beijing JiaShiLianBo Pharmaceutical Technology Co., Ltd) of a kind of ketoglutaric acid arginine salt of patent of application number 201010192571.1, chromatographic condition is: mobile phase acetonitrile-phosphate buffer=60: 40, phosphate buffer is wherein 0.02mol/L sodium dihydrogen phosphate, and by the disodium phosphate soln adjust ph to 5.2 of 0.02mol/L; Flow velocity 1.0ml/L, determined wavelength 205nm, chromatographic column is AgilentNH
2, 5 μm, the nh 2 column of 4.6 × 250mm, but nh 2 column equilibration time is long, approximately needs 1-2 hour, and even longer, baseline is not easily stablized.And the bonded functional group aminopropyl of nh 2 column is than C
18, C
8the bonded functional group C of post
18, C
8easy hydrolysis, so serviceable life and C
18, C
8post is compared slightly inferior.When using nh 2 column to carry out the detection of acidic materials, the existence of acidic materials means the existence of proton, the amido functional group of slightly negative charge can be made protonated, thus after causing using a period of time, the analysis thing retention properties of some class changed to some extent or show that post effect declines, affect assay accuracy.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid, adopts C
18chromatographic column, carry out efficient liquid phase chromatographic analysis, can be detected by a high performance liquid chromatography, Accurate Determining goes out the content of L-arginine and α-ketoglutaric acid in L-arginine-α-ketoglutaric acid or its preparation simultaneously, two kinds of compositions can reach good separation, the detection of L-arginine-α-ketoglutaric acid content can be advantageously used in, guarantee that it is quality controllable, and C
18chromatographic column equilibration time is short, about need 30-40 minute just can, improve work efficiency.
For solving the problems of the technologies described above, the technical solution used in the present invention is: a kind of high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid, and its chromatographic condition is:
Chromatographic column: C
18chromatographic column;
Mobile phase: 0.02mol/L ~ 0.08mol/L phosphate buffer, pH3.0-4.0;
Flow rate of mobile phase: 0.5-1.3ml/min;
Determined wavelength: 200-210nm;
Chromatographic column column temperature: 15 DEG C ~ 30 DEG C;
Carry out efficient liquid phase chromatographic analysis, measure the content of L-arginine and α-ketoglutaric acid in L-arginine-α-ketoglutaric acid or its preparation.
Preferably, the high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid, comprises the following steps:
(1) prepare mobile phase, adjust ph, filter, degassed;
(2) high performance liquid chromatograph condition is regulated, start balance chromatographic column;
(3) configuration of reference substance and need testing solution: take L-arginine and α-ketoglutaric acid reference substance and L-arginine-α-ketoglutaric acid test sample, dissolve constant volume with mobile phase respectively, to obtain final product;
(4) reference substance solution, need testing solution are injected high performance liquid chromatograph after filtering, record chromatogram and peak area, adopt external standard method to calculate content.
Adopt the inventive method can make L-arginine, separation that α-ketoglutaric acid two kinds of compositions are good, be applicable to the mensuration of all size L-arginine-α-ketoglutaric acid product (mol ratio of L-arginine and α-ketoglutaric acid can be arbitrary value) content.
Preferably, in L-arginine-α-ketoglutaric acid, the mol ratio of L-arginine and α-ketoglutaric acid is 1: 1 or 2: 1.
Preferably, mobile phase is 0.05mol/L phosphate buffer.
Preferred further, phosphate buffer is sodium dihydrogen phosphate or potassium phosphate buffer.
Further preferred, the pH value of phosphate buffer is 3.4.
Preferably, flow rate of mobile phase is 1.0ml/min; Determined wavelength is 205nm.
Preferably, the mol ratio of L-arginine and α-ketoglutaric acid is the concentration of the L-arginine-α-ketoglutaric acid need testing solution of 1: 1 is 0.3-1.0mg/mL; The mol ratio of L-arginine and α-ketoglutaric acid is the concentration of the L-arginine-α-ketoglutaric acid need testing solution of 2: 1 is 0.5-1.5mg/mL.
Further preferably, the mol ratio of L-arginine and α-ketoglutaric acid is the concentration of the L-arginine-α-ketoglutaric acid need testing solution of 1: 1 is 0.64mg/mL; The mol ratio of L-arginine and α-ketoglutaric acid is the concentration of the L-arginine-α-ketoglutaric acid need testing solution of 2: 1 is 0.96mg/mL.
Preferred further, sampling volume is 20 μ l.
The selection of mobile phase of the present invention: L-arginine-α-ketoglutaric acid is soluble in water, insoluble in organic solvent, adopt pure water phase mobile phase, sample dissolution is more abundant, and ODSC
18chromatographic column is water wettability chromatographic column, and be applicable to pure water phase mobile phase, both combinations make testing result more accurate.Mobile phase likely causes sample to separate out containing organic phase, and serious meeting blocking chromatographic column, causes damage.
The beneficial effect adopting technique scheme to produce is: this method can be detected by a high performance liquid chromatography, Accurate Determining goes out the content of L-arginine and α-ketoglutaric acid in L-arginine-α-ketoglutaric acid or its preparation simultaneously, two kinds of compositions can reach good separation, the detection of L-arginine-α-ketoglutaric acid content can be advantageously used in, guarantee that it is quality controllable.And simple and efficient, save artificial, time and solvent etc., cost is low.Empirical tests, the method has good preci-sion and accuracy.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation;
Fig. 1 is the chromatogram of L-arginine in the embodiment of the present invention 1-α-ketoglutaric acid test sample;
Fig. 2 is the chromatogram of L-arginine in the embodiment of the present invention 2-α-ketoglutaric acid test sample;
Fig. 3 is the canonical plotting of L-arginine linear relationship;
Fig. 4 is the canonical plotting of α-ketoglutaric acid linear relationship.
Embodiment
Embodiments of the invention are only used for explanation and realize technical scheme of the present invention, and these embodiments do not form further restriction to the present invention.In following examples, the high performance liquid chromatograph of employing is Japanese Shimadzu: LC-15C, SPD-15C; Chromatographic column is LunaC
185 μm of 4.6 × 250mm; Sampling volume is 20 μ l(quantitative loop).Also the C of the high performance liquid chromatograph of other producers and model, other particle diameters and length can be adopted
18chromatographic column and other sampling volumes, can reach object of the present invention.
Embodiment 1
Chromatographic condition
Mobile phase: 0.05mol/L phosphate sodium dihydrogen buffer solution (by 0.05mol/L phosphoric acid adjust ph to 3.4)
Flow rate of mobile phase: 1.0ml/min
Determined wavelength: 205nm
Chromatographic column column temperature: 25 DEG C
Experimental procedure:
(1) prepare mobile phase, adjust ph, filter, degassed;
(2) high performance liquid chromatograph condition is regulated, start balance chromatographic column;
(3) configuration of reference substance and need testing solution:
Reference substance solution: take L-arginine reference substance 34.0mg and α-ketoglutaric acid reference substance 29.0mg respectively in 100ml measuring bottle, add mobile phase and be diluted to scale, shake up, obtain reference substance solution.
Need testing solution: take L-arginine-α-ketoglutaric acid (mol ratio of L-arginine and α-ketoglutaric acid is 1: 1) 64mg and be placed in 100ml measuring bottle, add mobile phase and dissolve and be diluted to scale, shake up, to obtain final product.
(4) reference substance solution, need testing solution are injected high performance liquid chromatograph after filtering, record chromatogram and peak area, adopt external standard method to calculate content.
Need testing solution chromatogram is as shown in Figure 1: 2.829min is the peak of L-arginine, and 3.487min is the peak of α-ketoglutaric acid, and all the other are impurity peaks, and two main peak appearance times are suitable, theoretical cam curve is greater than 5000, and good with magazins' layout degree.
Shown in concrete numerical value sees the following form:
Embodiment 2
Chromatographic condition
Mobile phase: 0.05mol/L phosphate sodium dihydrogen buffer solution (by 0.05mol/L phosphoric acid adjust ph to 3.4)
Flow rate of mobile phase: 1.0ml/min
Determined wavelength: 205nm
Chromatographic column column temperature: 25 DEG C
Experimental procedure:
(1) prepare mobile phase, adjust ph, filter, degassed;
(2) high performance liquid chromatograph condition is regulated, start balance chromatographic column;
(3) configuration of reference substance and need testing solution:
Reference substance solution: take L-arginine reference substance 68.0mg and α-ketoglutaric acid reference substance 29.0mg respectively in 100ml measuring bottle, add mobile phase and be diluted to scale, shake up, obtain reference substance solution.
Need testing solution: take L-arginine-α-ketoglutaric acid (mol ratio of L-arginine and α-ketoglutaric acid is 2: 1) 96mg and be placed in 100ml measuring bottle, add mobile phase and dissolve and be diluted to scale, shake up, to obtain final product.
(4) reference substance solution, need testing solution are injected high performance liquid chromatograph after filtering, record chromatogram and peak area, adopt external standard method to calculate content.
Need testing solution chromatogram is as shown in Figure 2: 2.879min is the peak of L-arginine, and 3.710min is the peak of α-ketoglutaric acid, and all the other are impurity peaks, and two main peak appearance times are suitable, theoretical cam curve is greater than 5000, and good with magazins' layout degree.
Shown in concrete numerical value sees the following form:
1, precision test:
Precision means that, under the test condition of regulation, same even test sample, through the degree of closeness repeatedly between sampling and measuring acquired results.The sample of preparation 3 concentration, each concentration respectively prepares 3 parts of need testing solutions respectively, and measure, carry out reproducibility by 9 measurement results, RSD is not more than 2.0%, and Precision test result sees the following form:
Result shows that L-arginine and α-ketoglutaric acid replica test RSD are all less than 2.0%, proves that the method measures L-arginine and α-ketoglutaric acid has good precision.
2, linearly test:
Linearly mean in the scope of design, the degree of the direct proportional relation of measured object concentration in test result and sample.Linearly whether an available stock solution dilutes through precision, and the method preparing a series of test sample measures, and maps with the peak area recorded and measured object concentration, to observe, carry out linear regression.
L-arginine linear relationship sees the following form with shown in Fig. 3:
α-ketoglutaric acid linear relationship sees the following form with shown in Fig. 4:
Linear test result shows the linear equation y=3.3E+5x+4.4E+5 of L-arginine, R
2be 0.9966, α-ketoglutaric acid y=5.5E+5x+40260, R
2be 0.9997, prove that the method mensuration L-arginine and α-ketoglutaric acid are all in good linear relationship.
3, accuracy test:
Accuracy means the result and the close degree of theoretical value that measure by the method, and generally represent by the recovery (%), the recovery can accept in 98.0% ~ 102.0% scope.
Shown in the L-arginine recovery sees the following form:
Shown in the α-ketoglutaric acid recovery sees the following form:
Result shows that the recovery of L-arginine is between 99.41% ~ 100.43%, average recovery rate 99.96%; The recovery of α-ketoglutaric acid between 99.21% ~ 100.28%, average recovery rate 99.82%.Prove that the method measures L-arginine and α-ketoglutaric acid has good accuracy.
Embodiment 3
Be with the difference of embodiment 1, chromatographic condition:
Mobile phase: 0.05mol/L potassium phosphate buffer (by 0.05mol/L phosphoric acid adjust ph to 3.4);
Flow rate of mobile phase: 0.5ml/min;
Determined wavelength: 200nm;
Chromatographic column column temperature: 15 DEG C.
The configuration of reference substance and need testing solution:
Reference substance solution: take L-arginine reference substance 16mg and α-ketoglutaric acid reference substance 14mg respectively in 100ml measuring bottle, add mobile phase and be diluted to scale, shake up, obtain reference substance solution.
Need testing solution, takes L-arginine-α-ketoglutaric acid (mol ratio of L-arginine and α-ketoglutaric acid is 1: 1) 30mg and is placed in 100ml measuring bottle, add mobile phase and dissolve and be diluted to scale, shake up, to obtain final product.
Carry out efficient liquid phase chromatographic analysis, measure the content of L-arginine and α-ketoglutaric acid in L-arginine-α-ketoglutaric acid or its preparation.Its effect also can meet the demands.
Embodiment 4
Be with the difference of embodiment 1, chromatographic condition:
Mobile phase: 0.02mol/L potassium phosphate buffer (by 0.05mol/L phosphoric acid adjust ph to 3.0);
Flow rate of mobile phase: 1.3ml/min;
Determined wavelength: 210nm;
Chromatographic column column temperature: 30 DEG C.
The configuration of reference substance and need testing solution:
Reference substance solution: take L-arginine reference substance 35mg and α-ketoglutaric acid reference substance 15mg respectively in 100ml measuring bottle, add mobile phase and be diluted to scale, shake up, obtain reference substance solution.
Need testing solution, takes L-arginine-α-ketoglutaric acid (mol ratio of L-arginine and α-ketoglutaric acid is 2: 1) 50mg and is placed in 100ml measuring bottle, add mobile phase and dissolve and be diluted to scale, shake up, to obtain final product.
Carry out efficient liquid phase chromatographic analysis, measure the content of L-arginine and α-ketoglutaric acid in L-arginine-α-ketoglutaric acid or its preparation.Its effect also can meet the demands.
Embodiment 5
Be with the difference of embodiment 1, chromatographic condition:
Mobile phase: 0.08mol/L phosphate sodium dihydrogen buffer solution (by 0.05mol/L phosphoric acid adjust ph to 4.0);
The configuration of reference substance and need testing solution:
Reference substance solution: take L-arginine reference substance 106mg and α-ketoglutaric acid reference substance 44mg respectively in 100ml measuring bottle, add mobile phase and be diluted to scale, shake up, obtain reference substance solution.
Need testing solution, takes L-arginine-α-ketoglutaric acid (mol ratio of L-arginine and α-ketoglutaric acid is 2: 1) 150mg and is placed in 100ml measuring bottle, add mobile phase and dissolve and be diluted to scale, shake up, to obtain final product.
Carry out efficient liquid phase chromatographic analysis, measure the content of L-arginine and α-ketoglutaric acid in L-arginine-α-ketoglutaric acid or its preparation.Its effect also can meet the demands.
Embodiment 6
Be with the difference of embodiment 1, the configuration of reference substance and need testing solution:
Reference substance solution: take L-arginine reference substance 55mg and α-ketoglutaric acid reference substance 45mg respectively in 100ml measuring bottle, add mobile phase and be diluted to scale, shake up, obtain reference substance solution.
Need testing solution, takes L-arginine-α-ketoglutaric acid (mol ratio of L-arginine and α-ketoglutaric acid is 1: 1) 100mg and is placed in 100ml measuring bottle, add mobile phase and dissolve and be diluted to scale, shake up, to obtain final product.
Carry out efficient liquid phase chromatographic analysis, measure the content of L-arginine and α-ketoglutaric acid in L-arginine-α-ketoglutaric acid or its preparation.Its effect also can meet the demands.
This method can be detected by a high performance liquid chromatography, Accurate Determining goes out the content of L-arginine and α-ketoglutaric acid in L-arginine-α-ketoglutaric acid or its preparation simultaneously, two kinds of compositions can reach good separation, the detection of L-arginine-α-ketoglutaric acid content can be advantageously used in, guarantee that it is quality controllable.And simple and efficient, save artificial, time and solvent etc., cost is low.Empirical tests, the method has good preci-sion and accuracy.
Claims (9)
1. a high-efficiency liquid chromatography method for detecting for L-arginine-α-ketoglutaric acid, is characterized in that, its chromatographic condition is:
Chromatographic column: C
18chromatographic column;
Mobile phase: 0.02mol/L ~ 0.08mol/L phosphate buffer, pH3.0-4.0;
Flow rate of mobile phase: 0.5-1.3ml/min;
Determined wavelength: 200-210nm;
Chromatographic column column temperature: 15 DEG C ~ 30 DEG C;
Carry out efficient liquid phase chromatographic analysis, measure the content of L-arginine and α-ketoglutaric acid in L-arginine-α-ketoglutaric acid or its preparation;
Comprise the following steps:
(1) prepare mobile phase, adjust ph, filter, degassed;
(2) high performance liquid chromatograph condition is regulated, start balance chromatographic column;
(3) configuration of reference substance and need testing solution: take L-arginine and α-ketoglutaric acid reference substance and L-arginine-α-ketoglutaric acid test sample, dissolve constant volume with mobile phase respectively, to obtain final product;
(4) reference substance solution, need testing solution are injected high performance liquid chromatograph after filtering, record chromatogram and peak area, adopt external standard method to calculate content.
2. the high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid according to claim 1, is characterized in that the mol ratio of L-arginine and α-ketoglutaric acid in described L-arginine-α-ketoglutaric acid is 1: 1 or 2: 1.
3. the high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid according to claim 1, is characterized in that described mobile phase is 0.05mol/L phosphate buffer.
4. the high-efficiency liquid chromatography method for detecting of the L-arginine-α-ketoglutaric acid according to claim 1 or 3, is characterized in that described phosphate buffer is sodium dihydrogen phosphate or potassium phosphate buffer.
5. the high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid according to claim 4, is characterized in that the pH value of described phosphate buffer is 3.4.
6. the high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid according to claim 1, is characterized in that described flow rate of mobile phase is 1.0ml/min; Determined wavelength is 205nm.
7. the high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid according to claim 1, it is characterized in that, in described L-arginine-α-ketoglutaric acid, the mol ratio of L-arginine and α-ketoglutaric acid is 1: 1 or 2: 1, wherein, the mol ratio of L-arginine and α-ketoglutaric acid is the concentration of the L-arginine-α-ketoglutaric acid need testing solution of 1: 1 is 0.3-1.0mg/mL; The mol ratio of L-arginine and α-ketoglutaric acid is the concentration of the L-arginine-α-ketoglutaric acid need testing solution of 2: 1 is 0.5-1.5mg/mL.
8. the high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid according to claim 7, it is characterized in that, the mol ratio of L-arginine and α-ketoglutaric acid is the concentration of the L-arginine-α-ketoglutaric acid need testing solution of 1: 1 is 0.64mg/mL; The mol ratio of L-arginine and α-ketoglutaric acid is the concentration of the L-arginine-α-ketoglutaric acid need testing solution of 2: 1 is 0.96mg/mL.
9. the high-efficiency liquid chromatography method for detecting of L-arginine-α-ketoglutaric acid according to claim 7, is characterized in that, sampling volume is 20 μ l.
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