CN114384193A - Detection method of peramivir chiral isomer - Google Patents
Detection method of peramivir chiral isomer Download PDFInfo
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- CN114384193A CN114384193A CN202011114364.4A CN202011114364A CN114384193A CN 114384193 A CN114384193 A CN 114384193A CN 202011114364 A CN202011114364 A CN 202011114364A CN 114384193 A CN114384193 A CN 114384193A
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- phosphate
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- isoleucine
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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
Abstract
The invention discloses a detection method of peramivir enantiomer, wherein the chemical name of the enantiomer is as follows: (1R,2R,3S,4S) -3- [ (1R) -1-acetamide-2-ethylbutyl]The 4-guanidino-2-hydroxycyclopentyl-1-carboxylic acid has a structural formula shown in formula I, and is detected by reversed phase high performance liquid chromatography of mobile phase containing copper sulfate and L-isoleucine.
Description
Technical Field
The invention relates to an analytical chemistry detection method, in particular to a method for detecting peramivir enantiomer by using a high performance liquid chromatography method with copper sulfate-L-isoleucine as a mobile phase.
Background
The chemical name of peramivir trihydrate is: (1S,2S,3R,4R) -3- [ (1S) -1-acetamide-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentyl-1-carboxylic acid trihydrate; molecular formula C15H28N4O4·3H2O; the molecular weight is 382.45; the structural formula is as follows:
peramivir is a novel cyclopentane-type anti-influenza virus drug, and is another novel influenza virus NA inhibitor after Zanamivir (Zanamivir) and Oseltamivir (Oseltamivir) have been successfully developed and marketed in 1999. In 2013, 4 and 5, the national food and drug administration approves an anti-influenza drug peramivir sodium chloride injection, and the existing clinical test data prove that the injection is effective to influenza A and B.
The peramivir enantiomer is (1R,2R,3S,4S) -3- [ (1R) -1-acetamide-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentyl-1-carboxylic acid having the molecular formula C15H28N4O4Molecular weight is 328.41, and the structural formula is shown as follows:
according to the structural formula of peramivir, the peramivir has five chiral centers, and whether chiral isomers can be effectively detected directly influences the quality and the medication safety of peramivir, so that the detection and the quality control are required.
Reversed-phase high performance liquid chromatography is a common detection method, which adopts a stationary phase with non-polarity or relatively weak polarity, takes a solvent with strong polarity as a mobile phase, and is commonly used for separating and detecting compounds with non-polarity and weak polarity; reverse phase high performance liquid chromatography is most widely used in modern liquid chromatography, and statistically accounts for about 80% of the total high performance liquid chromatography.
Diastereomers can be separated by reverse phase high performance liquid chromatography, but enantiomers perform the same function as the stationary phase, and are therefore difficult to separate by reverse phase high performance liquid chromatography.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a reversed-phase high performance liquid chromatography method for detecting peramivir enantiomers by taking copper sulfate-L-isoleucine as a mobile phase.
The technical scheme is as follows: the chemical name of the peramivir enantiomer is as follows: the (1R,2R,3S,4S) -3- [ (1R) -1-acetamide-2-ethylbutyl ] -4-guanidino-2-hydroxycyclopentyl-1-carboxylic acid has a structural formula shown in a formula I, and is characterized in that the detection is carried out by adopting a reversed phase high performance liquid chromatography of a mobile phase containing copper sulfate and L-isoleucine.
In some embodiments, the mobile phase comprising copper sulfate, L-isoleucine is a mobile phase comprising a phosphate-copper sulfate-L-isoleucine buffer.
In some embodiments, the mobile phase containing copper sulfate, L-isoleucine is (phosphate-copper sulfate-L-isoleucine buffer) -acetonitrile is the mobile phase.
In some embodiments, the method for detecting an enantiomer of peramivir comprises the steps of:
a. preparing a test solution;
b. the method comprises the following steps of (1) detecting a test solution by adopting a phosphate-copper sulfate-L-isoleucine buffer solution as a mobile phase through reversed phase high performance liquid chromatography, wherein the specific detection conditions are as follows:
the column temperature of the chromatographic column is 20-50 ℃, preferably 25-40 ℃, most preferably 35 ℃,
the filler of the chromatographic column stationary phase is octadecylsilane chemically bonded silica,
the refractive index detector temperature is from 20 to 50 deg.C, preferably from 25 to 40 deg.C, most preferably 35 deg.C,
mobile phase: phosphate-copper sulfate-L-isoleucine buffer solution-acetonitrile is used as a mobile phase, the sample amount is 5-100 mu L, the preferred sample amount is 10-50 mu L, and the most preferred sample amount is 10 mu L, 20 mu L or 30 mu L.
In some embodiments, in the method for detecting an enantiomer of peramivir, the phosphate salt in step b may be selected from sodium phosphate, disodium hydrogen phosphate, sodium hexametaphosphate, ammonium dihydrogen phosphate, magnesium phosphate, barium phosphate, ammonium phosphate, calcium phosphate, potassium phosphate, ammonium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, calcium dihydrogen phosphate, magnesium hydrogen phosphate, preferably ammonium dihydrogen phosphate.
In some embodiments, the peramivir enantiomer detection method, the phosphate salt in step b is ammonium dihydrogen phosphate, and the molar concentration of the phosphate salt in the buffer is 1 to 50mmol/L, preferably 5 to 15mmol/L, and more preferably 10 mmol/L.
In some embodiments, in the peramivir enantiomer detection method, the molar concentration of copper sulfate in the buffer solution in the step b is 1-50mmol/L, preferably 3-15mmol/L, and more preferably 10 mmol/L.
In some embodiments, in the peramivir enantiomer detection method, the molar concentration of L-isoleucine in the buffer solution in step b is 1-50mmol/L, preferably 5-15mmol/L, and more preferably 10 mmol/L.
In some embodiments, the peramivir enantiomer detection method, the phosphate-copper sulfate-L-isoleucine buffer of step b, has a pH of 3.0 to 5.0, preferably 3.0 to 4.0, more preferably 3.5.
In some embodiments, the peramivir enantiomer detection method, step b (phosphate-copper sulfate-L-isoleucine buffer) and acetonitrile are performed in a volume ratio selected from the group consisting of 10-50: 1, preferably 10 to 20: 1, more preferably 10 to 11: 1. 11-12: 1. 12-13: 1. 13-14: 1. 14-15: 1. 15-16: 1. 16-17: 1. 17-18: 1. 18-19: 1. 19-20: 1, most preferably 94: 6.
In some embodiments, in the peramivir enantiomer detection method, the concentration of the test solution in step a is 0.1-100mg/mL, preferably 0.1-10mg/mL, and 5-10 mg/mL.
In some embodiments, in the method for detecting peramivir isomers, the chromatographic column is Waters Xselect CSH C18.
Has the advantages that: (1) the method for detecting the peramivir enantiomer by using the reversed-phase high performance liquid chromatography with the copper sulfate-L-isoleucine as the mobile phase can effectively separate the enantiomer peak in the API; (2) the invention optimizes and selects the concentration of the copper sulfate-L-isoleucine and the liquid chromatogram operation parameters in the detection process, shortens the detection time, and simultaneously ensures the specificity, the accuracy and the sensitivity of the detection.
Drawings
FIG. 1 is a blank solvent spectrum;
FIG. 2 is a spectrum of the mixed solution;
FIG. 3 is a diagram of a peramivir locating solution;
FIG. 4 is a diagram of a peramivir enantiomer locating solution;
FIG. 5 is a peramivir sensitivity solution chromatogram.
Detailed Description
Example 1
In the example, peramivir and its enantiomer form complexes with copper ion and L-isoleucine, respectively, and the complexes are detected by reverse high performance liquid chromatography.
The coordination principle of peramivir and enantiomer thereof is as follows:
the analysis steps for detecting the peramivir enantiomer are as follows:
a chromatographic column: waters Xselect CSH C184.6 × 150mm 3.5 μm;
mobile phase: 10mmol/L ammonium dihydrogen phosphate, 10mmol/L copper sulfate pentahydrate, 10mmol/L L-isoleucine buffer (pH adjusted to 3.5 with 50% sodium hydroxide solution) -acetonitrile 94: 6;
flow rate: 1.0 ml/min;
column temperature: 35 ℃;
refractive index detector temperature: 35 ℃;
sample introduction amount: 20 mu l of the mixture;
solvent: a mobile phase;
peramivir positioning solution: 10 mg/ml;
isomer positioning solution: 0.5 mg/ml;
mixing the solution: contains peramivir with concentration of 10mg/ml and enantiomer with concentration of 0.5 mg/ml;
sensitivity solution: 10 mug/ml;
sample introduction procedure: precisely measuring blank solvent, peramivir positioning solution, peramivir sensitivity solution, isomer positioning solution and mixed solution by 20 μ l respectively, injecting into a liquid chromatograph, and recording chromatogram as shown in figures 1-5.
The enantiomeric contents were calculated by the external standard method.
As a result:
TABLE 1 liquid phase data of peramivir and peramivir
Claims (10)
1. A method for detecting peramivir enantiomers, wherein the chemical names of the isomers are as follows: (1R,2R,3S,4S) -3- [ (1R) -1-acetamide-2-ethylbutyl ] -4-guanidino-2-hydroxycyclopentyl-1-carboxylic acid, the structural formula is shown as formula I, and the method is characterized in that the reversed phase high performance liquid chromatography of a mobile phase containing copper sulfate and L-isoleucine is adopted for detection,
2. the method for detecting peramivir enantiomers according to claim 1, wherein the mobile phase containing copper sulfate and L-isoleucine is a mobile phase containing phosphate-copper sulfate-L-isoleucine buffer, preferably phosphate-copper sulfate-L-isoleucine buffer/acetonitrile.
3. The method for detecting peramivir enantiomers according to claim 1, comprising the steps of:
a. preparing a test solution;
b. the method comprises the following steps of (1) detecting a test solution by adopting a phosphate-copper sulfate-L-isoleucine buffer solution as a mobile phase through reversed phase high performance liquid chromatography, wherein the specific detection conditions are as follows:
the column temperature of the chromatographic column is 20-50 ℃, preferably 25-40 ℃, and most preferably 35 ℃;
the filler of the chromatographic column stationary phase is octadecylsilane chemically bonded silica;
the differential refractometer temperature is 20-50 ℃, preferably 25-40 ℃, and most preferably 35 ℃;
mobile phase: the phosphate-copper sulfate-L-isoleucine buffer solution/acetonitrile is used as a mobile phase, the sample amount is 5-100 mu L, the preferred sample amount is 10-50 mu L, and the most preferred sample amount is 10 mu L, 20 mu L or 30 mu L.
4. The peramivir enantiomer detection method of claim 3, wherein the phosphate in step b is selected from sodium phosphate, disodium hydrogen phosphate, sodium hexametaphosphate, ammonium dihydrogen phosphate, magnesium phosphate, barium phosphate, ammonium phosphate, calcium phosphate, potassium phosphate, ammonium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, calcium dihydrogen phosphate, and magnesium hydrogen phosphate, preferably ammonium dihydrogen phosphate.
5. Peramivir enantiomer detection method according to claim 3, characterized in that the phosphate in step b is monoammonium phosphate at a molar concentration in the buffer of 1-50mmol/L, preferably 5-15mmol/L, more preferably 10 mmol/L.
6. The peramivir enantiomer detection method of claim 3, wherein the molar concentration of copper sulfate in the buffer of step b is 1 to 50mmol/L, preferably 3 to 15mmol/L, and more preferably 10 mmol/L.
7. The peramivir enantiomer detection method of claim 3, wherein the molar concentration of L-isoleucine in the buffer of step b is 1 to 50mmol/L, preferably 5 to 15mmol/L, and more preferably 10 mmol/L.
8. The peramivir enantiomer detection method of claim 3, wherein the phosphate-copper sulfate-L-isoleucine buffer solution of step b has a pH of 3.0 to 5.0, preferably 3.0 to 4.0, and more preferably 3.5.
9. The peramivir enantiomer detection method of claim 3, wherein the volume ratio of the (phosphate-copper sulfate-L-isoleucine buffer) to the acetonitrile in the step b is selected from 10 to 50: 1, preferably 10 to 20: 1, more preferably 10 to 11: 1. 11-12: 1. 12-13: 1. 13-14: 1. 14-15: 1. 15-16: 1. 16-17: 1. 17-18: 1. 18-19: 1. 19-20: 1, most preferably 94: 6.
10. The method for detecting peramivir isomers according to claim 3, wherein the chromatographic column is Waters XSelect CSH C18.
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| CN103543230A (en) * | 2012-07-09 | 2014-01-29 | 北大方正集团有限公司 | Method for separating and measuring moxifloxacin hydrochloride and enantiomer thereof |
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| CN111208216A (en) * | 2018-11-21 | 2020-05-29 | 太景生物科技股份有限公司 | Method for separating optical isomers of nemonoxacin or salt thereof |
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2020
- 2020-10-19 CN CN202011114364.4A patent/CN114384193B/en active Active
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| CN105021730A (en) * | 2015-07-17 | 2015-11-04 | 江西富祥药业股份有限公司 | Method for detecting Tenofovir optical antimers by high performance liquid chromatography |
| CN105859648A (en) * | 2016-05-05 | 2016-08-17 | 青岛辰达生物科技有限公司 | Method for preparing peramivir intermediate |
| CN111208216A (en) * | 2018-11-21 | 2020-05-29 | 太景生物科技股份有限公司 | Method for separating optical isomers of nemonoxacin or salt thereof |
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