CN114646700B - Detection method of (S) -pyrrolidine-2-formonitrile hydrochloride - Google Patents
Detection method of (S) -pyrrolidine-2-formonitrile hydrochloride Download PDFInfo
- Publication number
- CN114646700B CN114646700B CN202210196370.1A CN202210196370A CN114646700B CN 114646700 B CN114646700 B CN 114646700B CN 202210196370 A CN202210196370 A CN 202210196370A CN 114646700 B CN114646700 B CN 114646700B
- Authority
- CN
- China
- Prior art keywords
- pyrrolidine
- carbonitrile hydrochloride
- solution
- sample
- mobile phase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- QSJTUXCBPTVKQZ-JEDNCBNOSA-N (2s)-pyrrolidine-2-carbonitrile;hydrochloride Chemical compound Cl.N#C[C@@H]1CCCN1 QSJTUXCBPTVKQZ-JEDNCBNOSA-N 0.000 title claims abstract description 40
- 238000001514 detection method Methods 0.000 title claims abstract description 21
- 238000001212 derivatisation Methods 0.000 claims abstract description 24
- 238000010438 heat treatment Methods 0.000 claims abstract description 20
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims abstract description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 34
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 18
- SGRPQFBFOBJKQV-UHFFFAOYSA-N acetonitrile;benzoyl chloride Chemical compound CC#N.ClC(=O)C1=CC=CC=C1 SGRPQFBFOBJKQV-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000013558 reference substance Substances 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 abstract description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract description 8
- 238000011084 recovery Methods 0.000 abstract description 8
- 238000012360 testing method Methods 0.000 abstract description 7
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 abstract description 4
- 239000000377 silicon dioxide Substances 0.000 abstract description 4
- 239000000945 filler Substances 0.000 abstract description 3
- 238000004440 column chromatography Methods 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 19
- 239000000523 sample Substances 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 238000005303 weighing Methods 0.000 description 10
- 239000000203 mixture Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000005070 sampling Methods 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- YCWRPKBYQZOLCD-LURJTMIESA-N (2s)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile Chemical compound ClCC(=O)N1CCC[C@H]1C#N YCWRPKBYQZOLCD-LURJTMIESA-N 0.000 description 4
- PASDCCFISLVPSO-UHFFFAOYSA-N benzoyl chloride Chemical compound ClC(=O)C1=CC=CC=C1 PASDCCFISLVPSO-UHFFFAOYSA-N 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- KXDAEFPNCMNJSK-UHFFFAOYSA-N Benzamide Chemical compound NC(=O)C1=CC=CC=C1 KXDAEFPNCMNJSK-UHFFFAOYSA-N 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The application provides a detection method of (S) -pyrrolidine-2-carbonitrile hydrochloride, belonging to the technical field of column chromatography test or analysis materials. Adding a derivatization reagent into a sample to be detected, and heating to derivatize and introduce a chromophoric group; loading octadecylsilane chemically bonded silica as filler into chromatographic column, and adding mobile phase and derived pretreatment product for gradient elution. The application is applied to the determination of (S) -pyrrolidine-2-carbonitrile hydrochloride, and has the advantages of good linearity, good specificity, high stability and the like, and the recovery rate is 95.3-115.4%.
Description
Technical Field
The application relates to a detection method of (S) -pyrrolidine-2-carbonitrile hydrochloride, belonging to the technical field of medicine analysis and detection.
Background
In the synthesis of (S) -pyrrolidine-2-carbonitrile hydrochloride mainly from (2S) -N-chloroacetyl-2-cyanotetrahydropyrrole, amide dehydration of L-prolyl which is not completely amidated occurs under the action of TCT, and the reaction equation is shown in formula (1):
high performance liquid chromatography-ultraviolet detection (HPLC-UV) is a common means of drug quality control, however, (S) -pyrrolidine-2-carbonitrile hydrochloride method development faces an important challenge, as the molecular structure does not contain ultraviolet chromophores, which cannot be directly measured by UV detectors.
Disclosure of Invention
In view of the above, the application provides a method for detecting (S) -pyrrolidine-2-carbonitrile hydrochloride, which utilizes nucleophilic substitution reaction to introduce chromophores by liquid phase derivatization technology, so as to solve the problems that the molecular structure of the (S) -pyrrolidine-2-carbonitrile hydrochloride does not contain ultraviolet chromophores and cannot be directly detected by a UV detector.
Specifically, the application is realized by the following scheme:
a method for detecting (S) -pyrrolidine-2-carbonitrile hydrochloride, comprising the following steps:
(1) And (3) derivatization pretreatment: adding a derivatization reagent into a sample to be detected, heating at 40-90 ℃ for 20-80 min, and derivatizing and introducing a chromophoric group to obtain a derivatization pretreatment product;
(2) Gradient elution: loading octadecylsilane chemically bonded silica as filler into chromatographic column under the following chromatographic conditions: adding mobile phase and the derivative pretreatment product of the step (1) for gradient elution, wherein the flow rate is 0.9-1.2 mL/min, the column temperature is 35-45 ℃, the detection wavelength is 200-210 nm, the dissolution liquid is acetonitrile, and the temperature of a liquid chromatograph is controlled to be 2-10 ℃.
The application uses a derivatization reagent to carry out derivatization pretreatment on (S) -pyrrolidine-2-carbonitrile hydrochloride, and introduces a chromophoric group to make the chromophoric group into a product with stronger absorption in an ultraviolet region; the derivatization reaction liquid is used as a sample injection sample, and based on the principle of reverse-phase high performance liquid chromatography, the derivatization product of the (S) -pyrrolidine-2-carbonitrile hydrochloride is measured in the ultraviolet region, so that the qualitative or quantitative detection of the (S) -pyrrolidine-2-carbonitrile hydrochloride is realized.
Further, as preferable:
the concentration of the (S) -pyrrolidine-2-carbonitrile hydrochloride in the sample to be detected is 0.05-4.0 mug/ml.
The linear range of the (S) -pyrrolidine-2-carbonitrile hydrochloride is 0.19597-3.9194 mug/ml, the linear equation is y= 48.8851x-0.8166, R 2 ＞0.990。
The derivatizing agent is 0.5% -1.5% benzoyl chloride acetonitrile solution, more preferably, the derivatizing agent is 1% benzoyl chloride acetonitrile solution. The benzoyl chloride is reacted with ammonia to generate benzamide, the (S) -pyrrolidine-2-carbonitrile hydrochloride derivative is detected by liquid phase, and the related reaction equation is shown as a formula (2):
the molar ratio of the sample to be detected to the derivatization reagent is 2-4:1.
In the step (1), the heating temperature is 50-70 ℃ and the heating time is 40-60 min. More preferably, the derivatization conditions are: heating in water bath at 70deg.C for 60min.
The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is phosphoric acid aqueous solution, the volume concentration of phosphoric acid is 0.1% -0.3%, the mobile phase B is acetonitrile, and the volume of acetonitrile is 90% -100% of the total volume of the mobile phase B. More preferably, mobile phase A is a 0.2% (v/v) aqueous phosphoric acid solution and mobile phase B is an acetonitrile solution.
The mobile phase gradient elution parameters were as follows:
| time, min | 0 | 35 | 40 | 45 | 50 | 55 |
| Mobile phase,% | 15 | 15 | 80 | 80 | 15 | 15 |
。
In the step (2), the gradient elution flow rate is 1.1mL/min, the column temperature is 40 ℃, and the temperature of the liquid chromatograph is controlled to be 4 ℃.
The specification of the chromatographic column is as follows: the length is 150mm, the inner diameter is 4.6mm, and the particle size of the octadecylsilane chemically bonded silica is 3 mu m.
Using benzoyl chloride derivatization reagent, using octadecylsilane chemically bonded silica as filler chromatographic column, and adopting HPLC-UV method to determine, wherein the chromatographic conditions are as follows: the mobile phase A consists of phosphoric acid aqueous solution, wherein phosphoric acid is 0.1-0.3% (V/V), the mobile phase B is acetonitrile, acetonitrile accounts for 90-100% (V/V) of the total amount of the mobile phase B, and gradient elution is carried out, and the flow rate is 0.9-1.2 mL/min; the column temperature is 35-45 ℃; the detection wavelength is 210nm, the dissolution liquid is acetonitrile, and the temperature of the liquid chromatograph is controlled to be 2-10 ℃; the derivatization reaction condition is 0.5-1.5% (V/V) benzoyl chloride acetonitrile solution, and the mixture is heated in a water bath at 50-90 ℃ for 40-80 min.
Drawings
FIG. 1 is a chart showing HPLC detection of various amounts of derivatizing agents in example 1;
FIG. 2 is a plot of the HPLC detection at the heating temperature for the derivatization of example 2;
FIG. 3 is a graph of HPLC detection at various heating times for derivatization of example 3;
FIG. 4 is a specific HPLC detection chart of example 4.
Detailed Description
The application is further illustrated below in connection with specific examples, but is not limited to the scope of the present implementations. In the examples which follow, various processes and methods not described in detail are conventional methods well known in the art, and reagents used are not indicated as being of commercially available analytical or chromatographic purity, both in terms of source and specification.
Example 1: influence of the amount of derivatizing agent
Instrument: agilent 1260 is prepared by high performance liquid chromatography; an electronic balance, mertler XSR 105; pH meter, mertler FE28.
Reagent: phosphoric acid (analytically pure), national drug group; acetonitrile (chromatographic purity), merck; benzoyl chloride (analytically pure), national drug group.
Chromatographic conditions: mobile phase A was 0.2% (v/v) phosphoric acid in water; mobile phase B is acetonitrile; chromatographic column YMC-Triart C18.6X105 mm S-3 μm; column temperature 40 ℃; the detection wavelength is 210nm; sample injection amount is 10 μl; controlling the temperature of the liquid chromatograph to 4 ℃; the flow rate is 1.1ml/min; solvent: acetonitrile.
Mobile phase a and mobile phase B were gradient eluted according to the gradient procedure shown in table 1.
Table 1: gradient program parameter table
| Time (min) | 0 | 35 | 40 | 45 | 50 | 55 |
| Mobile phase B (%) | 15 | 15 | 80 | 80 | 15 | 15 |
The experimental steps are as follows:
derivatizing reagent: 1% (v/v) benzoyl chloride acetonitrile solution.
Selection of the amount of derivatizing agent: precisely weighing 1 mug/ml (S) -pyrrolidine-2-carbonitrile hydrochloride 2.0ml into a screw bottle, adding 1% (V/V) benzoyl chloride acetonitrile solutions with different volumes (such as 0.1ml, 0.15ml, 0.2ml, 0.3ml and 0.4 ml), shaking uniformly, heating in a water bath at 70 ℃ for 60min, sampling, recording a chromatogram with a detection wavelength of 210nm, and obtaining the best derivatization result when the dosage of the derivatization reagent is 0.2ml as shown in figure 1.
Example 2: influence of different heating temperatures
The apparatus, reagents, chromatographic conditions, etc. of this example were the same as in example 1, except for the choice of heating temperature.
The specific operation is as follows: precisely weighing 1mg/ml (S) -pyrrolidine-2-carbonitrile hydrochloride 2.0ml to a screw bottle, adding 0.2ml of 1% (v/v) benzoyl chloride acetonitrile solution (n-benzoyl chloride: n (S) -pyrrolidine-2-carbonitrile hydrochloride=2.25), shaking uniformly, heating in water bath at different temperatures (such as 40 ℃,50 ℃, 60 ℃ and 70 ℃) for 30min, sampling, recording a chromatogram with a detection wavelength of 210nm, and heating to 70 ℃ to obtain the result shown in fig. 2.
Example 3: influence of heating time
The apparatus, reagents, chromatographic conditions, etc. of this example were the same as in example 1, except for the choice of heating time.
The specific operation is as follows: precisely measuring 1 mug/ml (S) -pyrrolidine-2-carbonitrile hydrochloride 2.0ml to a sampling bottle, adding 0.2ml of 1% (v/v) benzoyl chloride acetonitrile solution, shaking uniformly, heating in a water bath at 70 ℃ for different times (such as 20min, 30min, 40min and 60 min), sampling, and recording a chromatogram (the detection wavelength is 200 nm), wherein the result is shown in fig. 3, and the derivatization reaction is the most complete when the heating time is 60min.
Example 4
The apparatus, reagents and chromatographic conditions of this example were the same as those of example 1, and the following solutions were further prepared and subjected to experiments for specificity, linearity, stability and the like.
(1) Solution preparation
(1) Blank solution: 10.0ml of acetonitrile was precisely measured and placed in a sampling bottle, 1.0ml of 1% (v/v) benzoyl chloride acetonitrile solution was added thereto, and the mixture was shaken well and heated in a water bath at 70℃for 60 minutes. 10 μl was injected for HPLC-UV analysis.
(2) Test solution: weighing a proper amount of (2S) -N-chloroacetyl-2-cyano-pyrrolidine serving as a test sample, and preparing the test sample into a mg/ml solution.
(3) Impurity stock solution: the appropriate amount of (S) -pyrrolidine-2-carbonitrile hydrochloride was formulated to contain about 10. Mu.g/ml of (S) -pyrrolidine-2-carbonitrile hydrochloride.
(4) Control solution: precisely weighing 2.0ml of impurity stock solution, placing into a 20ml volumetric flask, dissolving and diluting to scale with solvent, and shaking.
(2) Derivatization reaction conditions
(1) 1% (v/v) benzoyl chloride acetonitrile solution: precisely weighing 1.0ml benzoyl chloride, placing in a 100ml volumetric flask, dissolving and diluting to a scale with acetonitrile, and shaking uniformly.
(2) Sample solution derivatization reaction: 10.0ml of the sample solution is precisely measured and placed in a sampling bottle, 1.0ml of 1% (v/v) benzoyl chloride acetonitrile solution is added, the mixture is shaken well, and the mixture is heated in a water bath at 70 ℃ for 60min.
(3) And performing derivatization reaction of the blank solvent and the reference substance solution in the same way.
(2) Specificity experiments
And 10 mu l of each of the blank solution, the sample solution and the derivative product of the reference solution is injected into a liquid chromatogram, and the blank solvent peak and the gradient elution peak do not interfere with the (S) -pyrrolidine-2-carbonitrile hydrochloride derivative peak. The chromatogram is shown in FIG. 4 (a is blank, b is reference substance, and c is test substance).
(3) Linearity and range
For accurate determination of (S) -pyrrolidine-2-carbonitrile hydrochloride, a linear investigation, i.e. designing a relationship between peak area and concentration over a certain range, was performed, the linear concentration starting from a quantitative limit concentration, specifically as follows: respectively preparing linear solutions with the concentration of (S) -pyrrolidine-2-carbonitrile hydrochloride of 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 4.0 mug/ml, respectively taking 10.0ml of the linear solutions, respectively placing the linear solutions into different weighing bottles, respectively adding 1.0ml of 1% (v/v) benzoyl chloride acetonitrile solution, shaking uniformly, and heating in a water bath at 70 ℃ for 60min. 10 μl each was injected into HPLC-UV analysis.
And (3) carrying out linear regression analysis by taking the concentration (mug/ml) of (S) -pyrrolidine-2-carbonitrile hydrochloride as an abscissa and the peak area (A) of the derivative product as an ordinate, and calculating a linear equation and a correlation coefficient. The results show that the linear range of the (S) -pyrrolidine-2-carbonitrile hydrochloride is 0.19597-3.9194 mug/ml, the linear equation is y= 48.8851x-0.8166, R 2 > 0.990, the linear relationship is good.
(4) Limit of detection and limit of quantification
The signal to noise ratio of 3:1 is taken as the detection limit of the method, and the signal to noise ratio of 10:1 is taken as the quantitative limit of the method. The results showed that the detection limit of (S) -pyrrolidine-2-carbonitrile hydrochloride was 0.0489925. Mu.g/ml and the quantitative limit was 0.19597. Mu.g/ml.
(5) Stability of
A solution of (S) -pyrrolidine-2-carbonitrile hydrochloride (1.0. Mu.g/ml) and a solution of the test sample (1.0 mg/ml) were prepared. 10.0ml of the mixture was removed and placed in different weighing bottles, and 1.0ml of 1% (v/v) benzoyl chloride acetonitrile solution was added to each bottle, and the mixture was shaken and heated in a water bath at 70℃for 60 minutes. After standing at room temperature for 0h, 2.5h, 5.5h, 16.5h, 47.5h, 114.5h, 10.5 days, 11.5 days, 10 μl of each was injected for HPLC-UV analysis. The RSD of the peak area is less than 10.0% within 0-10.5 days of the solution at room temperature, and the stability is good.
(6) Accuracy of
To evaluate the accuracy of the method, the recovery rate of the sample in the (S) -pyrrolidine-2-carbonitrile hydrochloride impurity sample (2S) -N-chloroacetyl-2-cyanotetrahydropyrrole was calculated.
(1) Control solution: the formulation contained 1. Mu.g/ml of (S) -pyrrolidine-2-carbonitrile hydrochloride solution. Remove 10.0ml and place in a weighing flask, add 1.0ml 1% (v/v) benzoyl chloride acetonitrile solution, shake well, heat in 70℃water bath for 60min. 10 μl was injected for HPLC-UV analysis.
(2) Test solution: 1mg/ml of test solution was prepared. Remove 10.0ml and place in a weighing flask, add 1.0ml 1% (v/v) benzoyl chloride acetonitrile solution, shake well, heat in 70℃water bath for 60min. 10 μl was injected for HPLC-UV analysis.
(3) Recovery of sample solution: taking 9 parts of a test sample, respectively placing about 20mg of the test sample into a 20ml volumetric flask, precisely measuring a proper amount of (S) -pyrrolidine-2-carbonitrile hydrochloride stock solution to prepare 3 groups of recovery rate solutions containing 0.2, 1.0 and 4.0 mug/ml of (S) -pyrrolidine-2-carbonitrile hydrochloride, and 3 parts of each group of samples; 10.0ml of each was removed and placed in different weighing bottles, and 1.0ml of 1% (v/v) benzoyl chloride acetonitrile solution was added to each bottle, and the mixture was shaken well and heated in a water bath at 70℃for 60 minutes. 10 μl each was injected into HPLC-UV analysis, 3 needles were injected into each solution, and the results are shown in Table 2.
As shown in Table 2, the recovery rate of the (S) -pyrrolidine-2-carbonitrile hydrochloride (S) was 98.14-108.73%, and the recovery rate and the RSD value of the peak area per unit concentration were less than 10.0%, with good accuracy and precision.
Table 2: sample recovery rate experimental results
It can be seen in connection with the above experiments, figures 1-4 and table 2: when the method is used for measuring (S) -pyrrolidine-2-carbonitrile hydrochloride in (2S) -N-chloroacetyl-2-cyanotetrahydropyrrole, blank derivative reagent peaks and gradient elution peaks are not interfered, and the specificity is good; the detection limit of the method is 0.0489925 mug/ml, the quantitative limit is 0.19597 mug/ml, and the linear relation is good; the recovery rate is 95.3 to 115.4 percent; and the derivative product has good stability within 11.5 days.
Claims (3)
1. A method for detecting (S) -pyrrolidine-2-carbonitrile hydrochloride, comprising the steps of:
(1) And (3) derivatization pretreatment: adding a derivatization reagent into a sample to be detected, heating at 40-90 ℃ for 20-80 min, derivatizing and introducing a chromophoric group,
the concentration of the (S) -pyrrolidine-2-carbonitrile hydrochloride in the sample to be detected is 0.05-4.0 mug/ml,
the derivative reagent is 0.5 to 1.5 percent of benzoyl chloride acetonitrile solution,
the molar ratio of the sample to be detected to the derivatization reagent is 2-4:1;
(2) The chromatographic conditions are as follows: the mobile phase A is phosphoric acid aqueous solution, the volume concentration of phosphoric acid is 0.2%, the mobile phase B is acetonitrile, the chromatographic column YMC-Triart C18.6x150mm S-3 μm, the column temperature is 40 ℃, the detection wavelength is 210nm, the flow rate is 0.9-1.2 mL/min, the temperature of a liquid chromatograph is controlled to be 2-10 ℃,
gradient elution is carried out on the derivative pretreatment product in the step (1), and the gradient elution parameters are as follows:
。
2. the method for detecting (S) -pyrrolidine-2-carbonitrile hydrochloride according to claim 1, wherein: in the step (1), the heating temperature is 50-70 ℃ and the heating time is 40-60 min.
3. The method for detecting (S) -pyrrolidine-2-carbonitrile hydrochloride according to claim 1, which is characterized in thatThe method is characterized in that: the linear range of the (S) -pyrrolidine-2-carbonitrile hydrochloride is 0.19597-3.9194 mu g/ml, the concentration of a reference substance solution is x on the abscissa, the peak area is y on the ordinate, the linear equation is y= 48.8851x-0.8166, and R 2 ＞0.990。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210196370.1A CN114646700B (en) | 2022-03-01 | 2022-03-01 | Detection method of (S) -pyrrolidine-2-formonitrile hydrochloride |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210196370.1A CN114646700B (en) | 2022-03-01 | 2022-03-01 | Detection method of (S) -pyrrolidine-2-formonitrile hydrochloride |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114646700A CN114646700A (en) | 2022-06-21 |
| CN114646700B true CN114646700B (en) | 2023-10-20 |
Family
ID=81993661
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210196370.1A Active CN114646700B (en) | 2022-03-01 | 2022-03-01 | Detection method of (S) -pyrrolidine-2-formonitrile hydrochloride |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114646700B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1692115A (en) * | 2002-12-26 | 2005-11-02 | 大正制药株式会社 | Pyrrolopyrimidine and pyrrolopyridine derivatives substituted with cyclic amino group |
| CN104513188A (en) * | 2014-10-22 | 2015-04-15 | 广东药学院 | Cyano pyrrolidine derivative and preparation method and application thereof |
| CN108884068A (en) * | 2016-03-24 | 2018-11-23 | 特殊治疗有限公司 | 1- cyano-pyrolidin derivative as DUB inhibitor |
| WO2021249909A1 (en) * | 2020-06-08 | 2021-12-16 | Mission Therapeutics Limited | 1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyr role-5(1h)-carbonitrile as usp30 inhibitor for use in the treatment of mitochondrial dysfunction, cancer and fibrosis |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8338450B2 (en) * | 2007-09-21 | 2012-12-25 | Lupin Limited | Compounds as dipeptidyl peptidase IV (DPP IV) inhibitors |
-
2022
- 2022-03-01 CN CN202210196370.1A patent/CN114646700B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1692115A (en) * | 2002-12-26 | 2005-11-02 | 大正制药株式会社 | Pyrrolopyrimidine and pyrrolopyridine derivatives substituted with cyclic amino group |
| CN104513188A (en) * | 2014-10-22 | 2015-04-15 | 广东药学院 | Cyano pyrrolidine derivative and preparation method and application thereof |
| CN108884068A (en) * | 2016-03-24 | 2018-11-23 | 特殊治疗有限公司 | 1- cyano-pyrolidin derivative as DUB inhibitor |
| WO2021249909A1 (en) * | 2020-06-08 | 2021-12-16 | Mission Therapeutics Limited | 1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyr role-5(1h)-carbonitrile as usp30 inhibitor for use in the treatment of mitochondrial dysfunction, cancer and fibrosis |
Non-Patent Citations (3)
| Title |
|---|
| Integrated Synthetic, Biophysical, and Computational Investigations of Covalent Inhibitors of Prolyl Oligopeptidase and Fibroblast Activation Protein α;Jessica Plescia et al.;《J. Med. Chem.》;第62卷;全文 * |
| Synthesis of (S)-1-(2-chloroacetyl)pyrrolidine-2- carbonitrile: A key intermediate for dipeptidyl peptidase IV inhibitors;Santosh K. Singh et al.;《Beilstein Journal of organic chemistry》;全文 * |
| 维格列汀中间体(S)一1一(2一氯乙酰基) 吡咯烷一2一甲腈的合成;陶铸 等;《化学研究与应用》;第25卷(第10期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114646700A (en) | 2022-06-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110940745A (en) | Method for detecting related substances in S-2-amino-2- (2-chlorphenyl) methyl acetate or salts thereof | |
| CN107064368A (en) | The method that derivatization HPLC methods determine hydrazine hydrate | |
| CN113049699A (en) | Method for detecting biphenyl anhydride and related substances thereof and application | |
| CN113984921A (en) | Gas chromatography-mass spectrometry combined method for determining genotoxic impurity 1, 3-dichloro-2-propanol | |
| CN114646701A (en) | HPLC (high Performance liquid chromatography) test method for related substances in L-prolinamide | |
| CN112798719B (en) | Detection method of related substance N-methylpiperazine in sildenafil citrate | |
| CN114646700B (en) | Detection method of (S) -pyrrolidine-2-formonitrile hydrochloride | |
| CN114324703B (en) | Method for simultaneously detecting multiple amino acids | |
| CN106018617B (en) | Method for separating and measuring 2-chloro-1-methylpyridinium iodide content in vilazodone hydrochloride by liquid chromatography | |
| CN115684397A (en) | Method for determining content of genotoxic impurity hydroxylamine hydrochloride in parecoxib | |
| CN114280191A (en) | Method for detecting related substances in bis-cysteine and preparation thereof | |
| CN114839287A (en) | Method for detecting sodium tetradecanoate in miboplatin | |
| CN112881538B (en) | Method for detecting impurities and enantiomers in fudosteine and fudosteine tablets | |
| CN110095554B (en) | Method for analyzing milrinone related substances by high performance liquid chromatography | |
| CN114166982A (en) | Method for simultaneously determining dimer, trimer and caprolactam in amino caproic acid injection | |
| CN114544842A (en) | Method for detecting N-bromosuccinimide in voriconazole | |
| CN114518413A (en) | Method for measuring content of proline in captopril raw material medicine | |
| CN114200050B (en) | HPLC detection method for content of related substances in p-bromoanisole | |
| CN115219632B (en) | HPLC-ELSD detection method for (S) -1-amino-3-chloro-2-propanol hydrochloride | |
| CN115128184B (en) | Method for determining thiourea content in pramipexole dihydrochloride raw material by using HPLC external standard method | |
| CN117630202B (en) | Method for detecting buspirone hydrochloride intermediate impurities | |
| CN114200067B (en) | High performance liquid chromatography analysis method for 6-bromo-3-hydroxy pyrazine-2-carboxamide and impurities | |
| CN113092656B (en) | Method for detecting related substances in vecuronium bromide medicine for injection | |
| CN113447592A (en) | Method for detecting ethylene diamine tetraacetic acid disodium in metronidazole gel | |
| CN116297921A (en) | Isomer detection method of tedizolid phosphate intermediate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |