CN108226329A - The liquid phase chromatography analytical method of L- prolineamides - Google Patents
The liquid phase chromatography analytical method of L- prolineamides Download PDFInfo
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- CN108226329A CN108226329A CN201711421154.8A CN201711421154A CN108226329A CN 108226329 A CN108226329 A CN 108226329A CN 201711421154 A CN201711421154 A CN 201711421154A CN 108226329 A CN108226329 A CN 108226329A
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- solution
- prolineamides
- ethanol solution
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- triethylamine
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
Abstract
The present invention provides a kind of liquid phase chromatography analytical methods of L prolineamides, include the following steps：Accurate respectively to measure L prolineamides solution, triethylamine ethanol solution, derivative reagent solution, vortex mixing reacts certain time, diethylamine ethanol solution is added, vortex mixing reacts certain time, trifluoroacetic acid ethanol solution is added, vortex mixing obtains L Prolinamide derivatives solution；The L Prolinamide derivatives solution is subjected to chromatography.The liquid phase chromatography analytical method of the present invention accurately can delicately detect L prolineamides and D prolineamides.
Description
Technical field
The present invention relates to field of medicaments, and in particular to a kind of liquid phase chromatography analytical method of L- prolineamides.
Background technology
L- prolineamides be synthesize vildagliptin (Vildagliptin) important chiral intermediate, enantiomter D-
Prolineamide is typically found in commercially available L- prolineamides product, and the structural formula difference of the two is as follows：
The UV absorption of L- prolineamides and D- prolineamides is weaker, the conventional high-efficient liquid phase color with UV detector
Spectrometer (HPLC) can not delicately detect the content of L- prolineamides and D- prolineamides, and there are samples to be not easy detected ask
Topic.Therefore, it is necessary to study the analysis method for the content that accurately can delicately detect L- prolineamides and D- prolineamides, with
Just the content of chiral isomer can be effectively controlled in subsequent vildagliptin synthesis technology.
Invention content
In order to overcome the problems referred above, the present invention provides a kind of liquid-phase chromatographic analysis sides of sensitive, efficient L- prolineamides
Method the described method comprises the following steps：
1) the accurate measurement L- prolineamides solution of difference, triethylamine ethanol solution, derivative reagent solution, vortex mixing, instead
Certain time to be answered, adds diethylamine ethanol solution, vortex mixing reacts certain time, adds trifluoroacetic acid ethanol solution,
Vortex mixing is to get L- Prolinamide derivatives solution；
2) L- Prolinamide derivatives solution is subjected to chromatography.
In one embodiment, the L- prolineamides solution, triethylamine ethanol solution, derivative reagent solution, diethyl
Amine ethanol solution, trifluoroacetic acid ethanol solution are prepared as follows：
L- prolineamides are dissolved in dilution to prepare L- prolineamide solution；
Derivative reagent is dissolved in acetonitrile to prepare derivative reagent solution；
Triethylamine is dissolved in ethyl alcohol to prepare triethylamine ethanol solution；
Diethylamine is dissolved in ethyl alcohol to prepare diethylamine ethanol solution；
Trifluoroacetic acid is dissolved in ethyl alcohol to prepare trifluoroacetic acid ethanol solution.
In one embodiment, it carries out the chromatographic apparatus of above-mentioned chromatography and condition is：
Detecting instrument：High performance liquid chromatograph is equipped with UV detector；
Chromatographic condition：
Chromatographic column：DAICEL CHIRALPAK IC 4.6×250mm,5um；Mobile phase：N-hexane-ethyl alcohol (40/60 (v/
v))；Detection wavelength：262nm；Flow velocity：1.0ml/min；Column temperature：30℃；Sampling volume：10μL；Sample disc temperature control：15℃；Fortune
The row time：30min；Dilution：0.1% trifluoracetic acid/alcohol mixeding liquid.
In one embodiment, the derivative reagent is selected from fluorenes methoxy dicarbonyl chloride (FMOC-Cl), and structural formula is as follows：
Further, it is corresponding to prepare its for the derivative reagent FMOC-Cl and L- prolineamides and the reaction of D- prolineamides
The principle of derivative is as follows：
On the other hand, the present invention also provides derivative reagent for the purposes of the liquid-phase chromatographic analysis of L- prolineamides.
In one embodiment, the derivative reagent is selected from fluorenes methoxy dicarbonyl chloride.
On the other hand, it the present invention also provides L- Prolinamide derivatives, has the following structure：
Another aspect, the present invention also provides compoundsPreparation method, include the following steps：
It is accurate respectively to measure L- prolineamides solution, triethylamine ethanol solution, derivative reagent solution, vortex mixing, reaction
Certain time, diethylamine ethanol solution is added, vortex mixing reacts certain time, adds trifluoroacetic acid ethanol solution, whirlpool
Mixing is revolved, obtains the compound.
In one embodiment, the L- prolineamides solution, triethylamine ethanol solution, derivative reagent solution, diethyl
Amine ethanol solution, trifluoroacetic acid ethanol solution are prepared as follows：
L- prolineamides are dissolved in dilution to prepare the L- prolineamides solution；
Derivative reagent is dissolved in acetonitrile to prepare the derivative reagent solution；
Triethylamine is dissolved in ethyl alcohol to prepare the triethylamine ethanol solution；
Diethylamine is dissolved in ethyl alcohol to prepare the diethylamine ethanol solution；
Trifluoroacetic acid is dissolved in ethyl alcohol to prepare the trifluoroacetic acid ethanol solution.
In one embodiment, the derivative reagent is selected from fluorenes methoxy dicarbonyl chloride.
The beneficial effects of the present invention are：Derivative reagent FMOC-Cl can contain L- prolineamides sample to be measured and wherein
Enantiomter D- prolineamides be fully converted to its derivative in a short time in a mild condition, the derivative is ultraviolet
Absorb larger, maximum absorption wavelength reaches 262nm, and derivative separating degree in chiral chromatogram is higher, meets sample
Derivatization requirement.
Description of the drawings
Fig. 1 is the HPLC spectrograms of test sample derivative solution.
Specific embodiment
Detecting instrument：High performance liquid chromatograph is equipped with UV detector
Chromatographic condition：
Chromatographic column：DAICEL CHIRALPAK IC 4.6×250mm,5um
Mobile phase：N-hexane-ethyl alcohol (40/60 (v/v))
Detection wavelength：262nm
Flow velocity：1.0ml/min
Column temperature：30℃
Sampling volume：10μL
Sample disc temperature control：15℃
Run time：30min
It is prepared by solution：
Dilution：1.0ml trifluoracetic acids accurately are pipetted in 1000ml ethyl alcohol, mixing.
Test solution：50mg L- prolineamides samples accurately are weighed in 25ml volumetric flasks, are dissolved with dilution and dilute
It releases to scale, shakes up.
FMOC-Cl solution：It accurately weighs 15mg FMOC-Cl samples to be dissolved in 5ml acetonitriles, mixing.(sealing, 4 DEG C of conditions
Lower preservation, matching while using).
1% triethylamine ethanol solution：1.0ml triethylamines accurately are pipetted in 100ml ethyl alcohol, mixing.
1% diethylamine ethanol solution：1.0ml diethylamine accurately is pipetted in 100ml ethyl alcohol, mixing.
1% trifluoracetic acid ethanol solution：1.0ml trifluoracetic acids accurately are pipetted in 100ml ethyl alcohol, mixing.
Blank solution：Respectively 50 μ l dilutions, 50 μ l, 1% triethylamine ethanol solutions, 200 μ l are measured with liquid-transfering gun precision
FMOC-Cl solution is placed in derivatization reaction bottle, is sealed, vortex mixing 15s, in reacting 5min under room temperature；Again with shifting
Liquid rifle precision measures 250 μ l, 1% diethylamine ethanol solutions in above-mentioned reaction bulb, seals, vortex mixing 15s, in room temperature
Under the conditions of react 1min；For accurate 300 μ l, the 1% trifluoracetic acid ethanol solutions that measure in above-mentioned reaction bulb, capping is close respectively again
Envelope, vortex mixing 15s to get.
Test sample derivative solution：Respectively 50 μ l test solutions, 50 μ l, 1% triethylamine second are measured with liquid-transfering gun precision
Alcoholic solution, 200 μ l FMOC-Cl solution are put in derivatization reaction bottle, are sealed, vortex mixing 15s, in reacting under room temperature
5min；250 μ l, 1% diethylamine ethanol solutions are measured in above-mentioned reaction bulb with liquid-transfering gun precision again, are sealed, and are vortexed mixed
Even 15s, in reacting 1min under room temperature；Precision measures 300 μ l, 1% trifluoracetic acid ethanol solutions in above-mentioned reaction respectively again
Bottle in, seal, vortex mixing 15s to get.
Blank solution and test sample derivative solution are injected separately into high performance liquid chromatograph, records chromatogram respectively.
The HPLC spectrograms of test sample derivative solution are shown in Fig. 1, and the content of wherein D- Prolinamide derivatives (impurity) is returned according to peak area
One changes calculating.
Claims (10)
1. a kind of liquid phase chromatography analytical method of L- prolineamides, includes the following steps：
1) it is accurate respectively to measure L- prolineamides solution, triethylamine ethanol solution, derivative reagent solution, vortex mixing, reaction one
It fixes time, adds diethylamine ethanol solution, vortex mixing reacts certain time, adds trifluoroacetic acid ethanol solution, is vortexed
Mixing obtains L- Prolinamide derivatives solution；
2) the L- Prolinamide derivatives solution is subjected to chromatography.
2. according to the method described in claim 1, it is characterized in that, the L- prolineamides solution, triethylamine ethanol solution, spread out
Raw reagent solution, diethylamine ethanol solution, trifluoroacetic acid ethanol solution are prepared as follows：
L- prolineamides are dissolved in dilution to prepare the L- prolineamides solution；
Derivative reagent is dissolved in acetonitrile to prepare the derivative reagent solution；
Triethylamine is dissolved in ethyl alcohol to prepare the triethylamine ethanol solution；
Diethylamine is dissolved in ethyl alcohol to prepare the diethylamine ethanol solution；
Trifluoroacetic acid is dissolved in ethyl alcohol to prepare the trifluoroacetic acid ethanol solution.
3. method according to claim 1 or 2, which is characterized in that the derivative reagent is selected from fluorenes methoxy dicarbonyl chloride.
4. method according to claim 1 or 2, which is characterized in that be selected from n-hexane/second for the mobile phase of chromatography
Alcohol, volume ratio 40:60.
5. according to the method described in claim 2, it is characterized in that, the dilution uses 0.1% trifluoroacetic ethyl alcohol
Solution.
6. method according to claim 1 or 2, which is characterized in that the ultraviolet inspection of the L- Prolinamide derivatives solution
Survey wavelength is 262nm.
7. derivative reagent is for the purposes of the liquid-phase chromatographic analysis of L- prolineamides.
8. purposes according to claim 7, the derivative reagent is selected from fluorenes methoxy dicarbonyl chloride.
9. a kind of L- Prolinamide derivatives, have the following structure：
10. compoundPreparation method, include the following steps：
Accurate respectively to measure L- prolineamides solution, triethylamine ethanol solution, derivative reagent solution, vortex mixing, reaction is centainly
Time adds diethylamine ethanol solution, and vortex mixing reacts certain time, adds trifluoroacetic acid ethanol solution, is vortexed mixed
It is even to get.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112881571A (en) * | 2021-02-22 | 2021-06-01 | 江苏沿海化学品检测技术服务有限公司 | Chiral detection method of Fmoc-L-Hyp (tbu) -OH and isomers thereof by high performance liquid chromatography |
| WO2022121383A1 (en) * | 2020-12-08 | 2022-06-16 | 海南通用三洋药业有限公司 | Method for determining contents of 3-amino-1-adamantanol and l-prolinamide in vildagliptin |
| CN114646701A (en) * | 2022-03-01 | 2022-06-21 | 浙江国邦药业有限公司 | HPLC (high Performance liquid chromatography) test method for related substances in L-prolinamide |
| CN115267021A (en) * | 2022-07-27 | 2022-11-01 | 诚达药业股份有限公司 | Liquid chromatography analysis method of L-prolinamide related substances |
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| WO2022121383A1 (en) * | 2020-12-08 | 2022-06-16 | 海南通用三洋药业有限公司 | Method for determining contents of 3-amino-1-adamantanol and l-prolinamide in vildagliptin |
| CN112881571A (en) * | 2021-02-22 | 2021-06-01 | 江苏沿海化学品检测技术服务有限公司 | Chiral detection method of Fmoc-L-Hyp (tbu) -OH and isomers thereof by high performance liquid chromatography |
| CN114646701A (en) * | 2022-03-01 | 2022-06-21 | 浙江国邦药业有限公司 | HPLC (high Performance liquid chromatography) test method for related substances in L-prolinamide |
| CN114646701B (en) * | 2022-03-01 | 2023-09-29 | 浙江国邦药业有限公司 | HPLC test method for related substances in L-prolylamide |
| CN115267021A (en) * | 2022-07-27 | 2022-11-01 | 诚达药业股份有限公司 | Liquid chromatography analysis method of L-prolinamide related substances |
| CN115267021B (en) * | 2022-07-27 | 2024-04-09 | 诚达药业股份有限公司 | Liquid chromatographic analysis method for L-prolyl amide related substances |
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