CN107976508A - A kind of method for analyzing hydrochloric acid ammonia ketone valeric acid content and purity - Google Patents
A kind of method for analyzing hydrochloric acid ammonia ketone valeric acid content and purity Download PDFInfo
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- CN107976508A CN107976508A CN201711201499.2A CN201711201499A CN107976508A CN 107976508 A CN107976508 A CN 107976508A CN 201711201499 A CN201711201499 A CN 201711201499A CN 107976508 A CN107976508 A CN 107976508A
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- hydrochloric acid
- ketone valeric
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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
- G01N30/89—Inverse 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
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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Abstract
The invention discloses a kind of method for analyzing hydrochloric acid ammonia ketone valeric acid content and purity, carried out using high performance liquid chromatography, be specially：1st, mobile phase is prepared：By volume 90:10 mix concentration for 0.005 ~ 0.04mol/L perfluoroetane sulfonic acid sodium water solutions with acetonitrile, and perfluoroetane sulfonic acid sodium water solution adjusts pH value to 1.2 1.6 with perchloric acid before mixing；2nd, with high performance liquid chromatography UV detector to sample detection, ultraviolet detection 200 ~ 220nm of wavelength, 0.5 2ml/min of column flow rate, 25 ~ 40 DEG C of column temperature, 5 30 μ L of sample size；3rd, the preparation of test sample and reference substance solution：Hydrochloric acid ammonia ketone valeric acid reference substance and test sample are weighed respectively, and it is 20 40mg/ml solution to be configured to concentration with water, obtains assay test sample and content reference substance；Take reference substance appropriate, be diluted with water into 0.02 ~ 0.04mg/ml and obtain purity reference substance.
Description
Technical field
The present invention relates to a kind of method for analyzing hydrochloric acid ammonia ketone valeric acid content and purity, belong to the analysis field of medical material.
Background technology
Hydrochloric acid ammonia ketone valeric acid is a kind of photosensitizer, is clinically used for the diseases such as treatment skin neoplasin, but its sample all the time
Analysis method but there are no document report.Although being included in American Pharmacopeia to the raw material, sulfuric acid is used in American Pharmacopeia
As mobile phase, the reservation of hydrochloric acid ammonia ketone valeric acid is very weak, seriously affects the detection of hydrochloric acid ammonia ketone valeric acid content and purity.
The content of the invention
The technical problems to be solved by the invention be to provide a kind of preci-sion and accuracy is good, analysis result accurately and reliably
Hydrochloric acid ammonia ketone valeric acid content and purity analysis determining method.
Its technical problem to be solved can be implemented by the following technical programs.
A kind of method for analyzing hydrochloric acid ammonia ketone valeric acid content and purity, using high performance liquid chromatography to hydrochloric acid ammonia ketone valeric acid
Content and purity are analyzed, and are concretely comprised the following steps：
1）, mobile phase prepare：
By percent by volume 85:15~95:Perfluoroetane sulfonic acid sodium water solution of 5 proportionings by concentration for 0.005mol/L ~ 0.04mol/L
Mixed with acetonitrile, wherein perfluoroetane sulfonic acid sodium water solution adjusts pH value to 1.2-1.6 with perchloric acid before mixing；
2）, using high performance liquid chromatography UV detector sample is detected, ultraviolet detection wavelength is 200 ~ 220nm, color
Spectrum column flow rate is 0.5-2ml/min, and chromatographic column column temperature is 25 DEG C ~ 40 DEG C, and sample size is 5-30 μ L；
3）, test sample and reference substance solution preparation：
Hydrochloric acid ammonia ketone valeric acid reference substance and appropriate test sample are weighed respectively, and it is 20-40mg/ml's to make solvent to be configured to concentration with water
Solution, up to assay test solution and content reference substance solution；Take reference substance solution appropriate, it is diluted with water into 0.02 ~
0.04mg/ml is up to purity reference substance solution.
As one of the preferred embodiment of the present invention, the concentration of the perfluoroetane sulfonic acid sodium water solution is 0.02mol/L, is adjusted
PH value afterwards is 1.3.
Further as the technical program is improved, and the ultraviolet detection wavelength is 205 ~ 210nm, and column flow rate is
1ml/min；Chromatographic column column temperature is 35 DEG C；Sample size is 10-20 μ L.
The further improvement of the technical program is also served as, the chromatographic column used is 5 μm, the anti-phase C18 of 4.6 × 150 ~ 250mm
Chromatographic column.
Further as the technical program is improved, and the chromatographic column used is 5 μm, the anti-phase C18 colors of 4.6 × 150mm
Compose column.
The further improvement of the technical program is also served as, the chromatographic column used is 5 μm, the anti-phase C18 of 4.6 × 250mm
Chromatographic column.
As one of preferred embodiment of the present invention, the chromatographic column used is model Waters Xbridge C18
The anti-phase C18 chromatographic columns of 5um, 4.6 × 150mm.
As another preferred embodiment of the present invention, the chromatographic column used is model Waters Xbridge C18
The anti-phase C18 chromatographic columns of 5um, 4.6 × 250mm.
As one of the preferred embodiment of the present invention, step 1）When middle mobile phase is prepared, perfluoroetane sulfonic acid sodium water solution and second
Nitrile mixed volume mixing ratio is 90:10.
Using the hydrochloric acid ammonia ketone valeric acid content of above-mentioned technical proposal and the analysis determining method of purity, compared to American Pharmacopeia side
Method, this method use high effective liquid chromatography for measuring hydrochloric acid ammonia ketone valeric acid content and purity, belong to retain it is stronger, reappearance is more preferable
A kind of detection method.
The invention discloses a kind of method for analyzing hydrochloric acid ammonia ketone valeric acid content and purity, using high performance liquid chromatography pair
Hydrochloric acid ammonia ketone valeric acid content and purity are analyzed, and the preci-sion and accuracy of method is good, and analysis result is accurately and reliably.
Advantages of the present invention is：Chromatographic column and detector use common anti-phase C18 columns and UV detector, operation letter
Single, main peak peak shape is good, retains and is adapted to, and accuracy and precision through the bright analysis method of method proof list are high, method specificity
It is good with reappearance, easy to normalizing operation.
Brief description of the drawings
Fig. 1 is hydrochloric acid ammonia ketone valeric acid content and purity test sample collection of illustrative plates, and chromatographic column is Waters Xbridge C18 5um,
4.6×150mm；
Fig. 2 is hydrochloric acid ammonia ketone valeric acid content reference substance collection of illustrative plates, and chromatographic column is Waters Xbridge C18 5um, 4.6 ×
250mm；
Embodiment
The embodiment of the present invention is further described below in conjunction with the accompanying drawings.
The present invention provides a kind of method for analyzing hydrochloric acid ammonia ketone valeric acid content and purity, specially with high performance liquid chromatography
Method external standard method is analyzed（Measure）The method of hydrochloric acid ammonia ketone valeric acid content and purity.
HPLC operating conditions are as follows：
Chromatographic column：Waters Xbridge C18 5um, 4.6 × 150mm；
Flow velocity：1ml/min；
UV detector Detection wavelengths：205nm；
Sample size：10μL；
Chromatographic column column temperature：35℃；
Mobile phase：（The perfluoroetane sulfonic acid sodium water solution of 0.02mol/L, pH value is adjusted to 1.3 with perchloric acid）- acetonitrile=90:10（V:
V）；
High performance liquid chromatograph：Waters e2695 high performance liquid chromatographs, configure 2489 UV detector of Waters；
Solution is prepared：Hydrochloric acid ammonia ketone valeric acid reference substance and appropriate test sample are weighed respectively, are dissolved with water and are quantified dilution and are made often
Solution of the 1ml containing about 40mg, up to assay test solution and content reference substance solution；Take reference substance solution appropriate, add
Water is diluted to 0.04mg/ml up to purity reference substance solution.
Method run time：20 minutes
Loading sequence：First 2 pin water of sample introduction, then respectively 5 pin content reference substance solution of sample introduction, 1 pin content and purity test solution,
1 pin water and 1 pin purity reference substance solution
System suitability requirement：The RSD% of 5 pin content reference substance solution main peak areas must not cross 0.73%, the theoretical tray of main peak
Number is not less than 8000, and tailing factor is not more than 2.0.
Computational methods：The content of calculated by peak area hydrochloric acid ammonia ketone valeric acid test sample is pressed with external standard method, salt is calculated with external standard method
The purity of sour ammonia ketone valeric acid test sample.
Precision test：
Table 1 below is hydrochloric acid ammonia ketone valeric acid assay Precision test result, and with reference to the data of table 1, precision test shows：This
Method favorable reproducibility, the precision of method are good.
Table 1
Recovery test：
Table 2 below be hydrochloric acid ammonia ketone valeric acid assay recovery test as a result, with reference to table 2 data, recovery test shows：This
For the method rate of recovery between 99.37% ~ 100.44%, method accuracy is high.
Table 2
Fig. 1 is hydrochloric acid ammonia ketone valeric acid content and purity test sample collection of illustrative plates, and chromatographic column is Waters Xbridge C18 5um, 4.6
×150mm；Abscissa is the time in figure（Unit：min）, ordinate is peak area, and the system suitability that table 3 below is Fig. 1 is reported
As a result, as can be seen that this method main peak peak shape is good from figure, and chlorion peak and major impurity peak can be kept completely separate.
Table 3：
| Retention time min | Peak area | Number of theoretical plate | Tailing factor | Separating degree | |
| 1 | 3.960 | 4722 | 12812 | 0.98 | |
| 2 | 4.362 | 4695596 | 11252 | 1.11 | 2.65 |
Fig. 2 is hydrochloric acid ammonia ketone valeric acid content reference substance collection of illustrative plates, and chromatographic column is Waters Xbridge C18 5um, 4.6 ×
250mm；Abscissa is the time in figure（Unit：min）, ordinate is peak area, the system suitability report knot that table 4 below is Fig. 2
Fruit, as can be seen that this method main peak peak shape is good from figure, and chlorion peak and major impurity peak can be kept completely separate
Table 4：
| Retention time min | Peak area | Number of theoretical plate | Tailing factor | Separating degree | |
| 1 | 5.575 | 5627 | 8529 | 1.06 | |
| 2 | 6.506 | 5895853 | 12225 | 0.98 | 2.95 |
Embodiment described above is merely a preferred embodiment of the present invention, and simultaneously the feasible implementation of non-invention is exhaustive, and foundation
Any equivalent transformation that technical scheme is made, is within the scope of protection of the invention.
Claims (9)
1. a kind of method for analyzing hydrochloric acid ammonia ketone valeric acid content and purity, contains hydrochloric acid ammonia ketone valeric acid using high performance liquid chromatography
Amount and purity are analyzed, and are concretely comprised the following steps：
1）, mobile phase prepare：
By percent by volume 85:15~95:5 proportioning is water-soluble by the perfluorooctane sulfonate that concentration is 0.005mol/L ~ 0.04mol/L
Liquid is mixed with acetonitrile, wherein perfluoroetane sulfonic acid sodium water solution adjusts pH value to 1.2-1.6 with perchloric acid before mixing；
2）, using high performance liquid chromatography UV detector sample is detected, ultraviolet detection wavelength is 200 ~ 220nm, color
Spectrum column flow rate is 0.5-2ml/min, and chromatographic column column temperature is 25 DEG C ~ 40 DEG C, and sample size is 5-30 μ L；
3）, test sample and reference substance solution preparation：
Hydrochloric acid ammonia ketone valeric acid reference substance and appropriate test sample are weighed respectively, and it is 20-40mg/ml's to make solvent to be configured to concentration with water
Solution, up to assay test solution and content reference substance solution；Take reference substance solution appropriate, it is diluted with water into 0.02 ~
0.04mg/ml is up to purity reference substance solution.
2. the method for hydrochloric acid ammonia ketone valeric acid content and purity is analyzed according to claim 1, it is characterised in that the octane sulphur
The concentration of acid sodium aqueous solution is 0.02mol/L, and the pH value after adjusting is 1.3.
3. the method for hydrochloric acid ammonia ketone valeric acid content and purity is analyzed according to claim 1, it is characterised in that the ultraviolet inspection
Survey wavelength is 205 ~ 210nm, column flow rate 1ml/min；Chromatographic column column temperature is 35 DEG C；Sample size is 10-20 μ L.
4. the method for hydrochloric acid ammonia ketone valeric acid content and purity is analyzed according to claim 1, it is characterised in that the chromatography of use
Column is 5 μm, the anti-phase C18 chromatographic columns of 4.6 × 150 ~ 250mm.
5. according to the analysis hydrochloric acid ammonia ketone valeric acid content of claim 1 or 4 and the method for purity, it is characterised in that use
Chromatographic column is 5 μm, the anti-phase C18 chromatographic columns of 4.6 × 150mm.
6. according to the analysis hydrochloric acid ammonia ketone valeric acid content of claim 1 or 4 and the method for purity, it is characterised in that use
Chromatographic column is 5 μm, the anti-phase C18 chromatographic columns of 4.6 × 250mm.
7. according to the analysis hydrochloric acid ammonia ketone valeric acid content of claim 1 or 4 and the method for purity, it is characterised in that use
Chromatographic column is the anti-phase C18 chromatographic columns of model Waters Xbridge C18 5um, 4.6 × 150mm.
8. according to the analysis hydrochloric acid ammonia ketone valeric acid content of claim 1 or 4 and the method for purity, it is characterised in that use
Chromatographic column is the anti-phase C18 chromatographic columns of model Waters Xbridge C18 5um, 4.6 × 250mm.
9. the method for hydrochloric acid ammonia ketone valeric acid content and purity is analyzed according to claim 1, it is characterised in that step 1）Middle stream
During dynamic mutually preparation, perfluoroetane sulfonic acid sodium water solution is 90 with acetonitrile mixed volume mixing ratio:10.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112924607A (en) * | 2019-12-05 | 2021-06-08 | 湖北远大生物技术有限公司 | Method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in enzymatic reaction liquid |
| CN114264734A (en) * | 2021-11-17 | 2022-04-01 | 上海复旦张江生物医药股份有限公司 | Detection method of aminolevulinic acid hydrochloride related substances |
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|---|---|---|---|---|
| CN1178521A (en) * | 1995-03-10 | 1998-04-08 | 光治疗公司 | Esters of 5-aminolevulinic acid as photosensitizing agents in photochemotherapy |
| CA2766675A1 (en) * | 2011-02-07 | 2012-08-07 | Laboratory Corporation Of America Holdings | Methods and systems for multiplex analysis of biomolecules by liquid chromatography-mass spectrometry |
| CN103323549A (en) * | 2013-06-19 | 2013-09-25 | 哈尔滨医科大学 | Detection kit for content of delta-amino-gamma-aminolaevulini acid |
| CN104483406A (en) * | 2014-12-19 | 2015-04-01 | 精晶药业股份有限公司 | High-efficiency liquid-phase chromatography detection method of L-arginine-alpha-ketoglutarate |
| JP2015232536A (en) * | 2014-06-09 | 2015-12-24 | 株式会社Ｂｉｏｍａｔｅｒｉａｌ ｉｎ Ｔｏｋｙｏ | Separation determination analysis method of amino levulinic acid |
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2017
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1178521A (en) * | 1995-03-10 | 1998-04-08 | 光治疗公司 | Esters of 5-aminolevulinic acid as photosensitizing agents in photochemotherapy |
| CA2766675A1 (en) * | 2011-02-07 | 2012-08-07 | Laboratory Corporation Of America Holdings | Methods and systems for multiplex analysis of biomolecules by liquid chromatography-mass spectrometry |
| CN103323549A (en) * | 2013-06-19 | 2013-09-25 | 哈尔滨医科大学 | Detection kit for content of delta-amino-gamma-aminolaevulini acid |
| JP2015232536A (en) * | 2014-06-09 | 2015-12-24 | 株式会社Ｂｉｏｍａｔｅｒｉａｌ ｉｎ Ｔｏｋｙｏ | Separation determination analysis method of amino levulinic acid |
| CN104483406A (en) * | 2014-12-19 | 2015-04-01 | 精晶药业股份有限公司 | High-efficiency liquid-phase chromatography detection method of L-arginine-alpha-ketoglutarate |
Non-Patent Citations (1)
| Title |
|---|
| B. ELFSSON ET AL: "Stability of 5-aminolevulinic acid in aqueous solution", 《EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112924607A (en) * | 2019-12-05 | 2021-06-08 | 湖北远大生物技术有限公司 | Method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in enzymatic reaction liquid |
| CN114264734A (en) * | 2021-11-17 | 2022-04-01 | 上海复旦张江生物医药股份有限公司 | Detection method of aminolevulinic acid hydrochloride related substances |
| CN114264734B (en) * | 2021-11-17 | 2024-02-13 | 上海复旦张江生物医药股份有限公司 | Method for detecting related substances of aminolevulinic acid hydrochloride |
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