CN114646701A - HPLC (high Performance liquid chromatography) test method for related substances in L-prolinamide - Google Patents
HPLC (high Performance liquid chromatography) test method for related substances in L-prolinamide Download PDFInfo
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Abstract
The application provides an HPLC (high performance liquid chromatography) test method for related substances in L-prolinamide, belonging to the technical field of column chromatography test or analysis materials. The L-prolinamide related substances refer to impurities introduced or generated by degradation in the L-prolinamide synthesis process, the mobile phase is a solution obtained by dissolving sodium octane sulfonate and sodium dihydrogen phosphate dihydrate in water, adding acetonitrile, mixing uniformly, and performing chromatographic column separation by using high performance liquid chromatography and octadecylsilane chemically bonded silica as a filler, and the mobile phase is used as a solvent to perform isocratic elution on the impurities. The method is applied to the impurity determination of L-prolinamide related substances, and has the advantages of stable chromatographic condition system, good linearity, high precision, high chromatographic condition sensitivity, good specificity, stability in a determination solution within 35 hours and the like.
Description
Technical Field
The application relates to an HPLC (high performance liquid chromatography) test method for related substances in L-prolinamide, belonging to the technical field of column chromatography test or analysis materials.
Background
L-prolinamide is an important optical activity pyrrole derivative, and is an important chiral intermediate of a hypoglycemic medicament Vildagliptin (Vildagliptin). L-prolinamide of the formula C5H10N2OThe structural formula of (A) is shown as formula (1):
in the process of synthesizing the compound, impurities possibly introduced mainly comprise L-proline, proline dimer, L-hydroxyproline, isomer D-prolinamide and the like, the isomer D-prolinamide can be effectively detected by a chiral chromatographic column, the structures of the L-prolinamide, the impurities of the L-proline, the proline dimer and the L-hydroxyproline have no conjugated system and weak ultraviolet absorption, and the impurities of the L-prolinamide, the L-proline dimer and the L-hydroxyproline have short retention time and small response value and cannot be effectively detected by a conventional HPLC (high performance liquid chromatography) reverse phase chromatographic analysis method, and the impurity structural formulas are respectively shown as a formula (2):
the L-prolinamide is used as a vildagliptin chiral intermediate, and the effective detection of the purity and impurities of the L-prolinamide has important practical significance on the quality control in the subsequent vildagliptin synthesis process, so that a method for simply and accurately determining related substances in the L-prolinamide is urgently needed to be developed.
Disclosure of Invention
In view of the above, the present application provides an HPLC method for testing related substances in L-prolinamide, which comprises adding a certain amount of ion-pair reagent and adjusting the pH of the mobile phase to determine the reverse phase ion-pair chromatography method of related substances in L-prolinamide, so as to increase the retention time and enhance the response of L-prolinamide and related substances, thereby achieving effective separation and detection.
Specifically, the method is realized through the following scheme:
an HPLC test method for related substances in L-prolinamide, wherein the related substances of the L-prolinamide refer to impurities introduced or generated by degradation in a synthesis process of the L-prolinamide, and the test comprises the following two parts:
(1) preparing a mobile phase: the mobile phase is a solution obtained by adding water to dissolve sodium octane sulfonate and sodium dihydrogen phosphate dihydrate, then adding acetonitrile and mixing uniformly, wherein the concentration of the sodium octane sulfonate is 0.02-0.03 mol/L, the concentration of the sodium dihydrogen phosphate dihydrate is 0.005-0.015 mol/L, the adding volume of the acetonitrile is 10-20% of the total volume of the mobile phase, the pH value of the mobile phase is 2.5-3.5,
(2) elution and separation: performing chromatographic column separation by adopting a high performance liquid chromatography and using octadecylsilane chemically bonded silica as a filler, wherein the chromatographic conditions are as follows: the flow rate is 0.4-0.6 mL/min, the column temperature is 25-35 ℃, the detection wavelength is 190-400nm, and the mobile phase in the step (1) is used as a solvent to perform isocratic elution on impurities.
The method adopts a high performance liquid chromatography, uses octadecylsilane chemically bonded silica as a filler chromatographic column to carry out separation, uses a UV detector to carry out quantitative analysis, and uses a mobile phase as a solvent to carry out isocratic elution. Octadecylsilane chemically bonded silica is used as a filling agent to endow the chromatographic column with good acid resistance, and then sodium octane sulfonate, sodium dihydrogen phosphate dihydrate buffer salt and an organic phase in a certain proportion are used as mobile phases for isocratic elution to quantitatively detect the content of L-prolinamide and related substances thereof, so that the quality of the L-prolinamide is effectively controlled, and the method has the advantages of simplicity, sensitivity, accuracy, reliability, good reproducibility and the like.
Further, as preferable:
the impurities comprise at least one of L-proline, cyclo (proline-proline) dipeptide (namely proline dimer) and L-hydroxyproline, and high-precision and sensitive measurement is realized aiming at the impurities which are not suitable for measurement.
The linear range of the L-hydroxyproline is 1.00575-160.92 mu g/ml, the linear equation is that y is 0.9861x-0.5863, and R is21.0000; the linear range of L-proline is 1.0015-160.2400 mu g/ml, the linear equation is that y is 1.1214x-0.2115, and R is21.0000; the linear range of the cyclo (proline-proline) dipeptide is 0.0501-40.0800 mu g/ml, the linear equation is that y is 67.5073x +1.1843, and R is2＝1.0000。
The mobile phase comprises 0.026mol/L sodium octane sulfonate, 0.011mol/L sodium dihydrogen phosphate dihydrate and acetonitrile accounting for 15 percent of the total volume of the mobile phase, thereby realizing the preparation of the optimal mobile phase.
The pH of the mobile phase was 3.0. More preferably, the pH of the mobile phase is adjusted with phosphoric acid.
The filling length of the octadecylsilane chemically bonded silica is 250mm, the inner diameter of the octadecylsilane chemically bonded silica is 4.6mm, and the particle size of a filler is 5 micrometers.
The column temperature is 28-32 ℃, the recovery rate is 80-120% in the temperature stage, the RSD value of the recovery rate is not more than 10.0%, and the method is high in accuracy.
In the chromatographic condition, the flow rate is 0.5mL/min, the column temperature is 30 ℃, the absorption wavelength is 200nm, all impurity peaks are effectively separated under the chromatographic condition, and a main peak is completely separated from adjacent impurity peaks.
The result of methodology verification shows that the test method has stable chromatographic condition system, good linearity, high precision, high sensitivity of chromatographic conditions, good specificity, stability in 35h of the measured solution and durability test result that the small changes of flow rate, column temperature, pH and mobile phase proportion do not influence the measurement of the L-prolinamide related substances.
Drawings
FIG. 1 is a scanning HPLC detection spectrum of the maximum absorption wavelength of L-prolinamide in example 1 of the present application;
FIG. 2 is a scanning HPLC detection spectrum of the maximum absorption wavelength of L-hydroxyproline in example 1 of the present application;
FIG. 3 is a scanning HPLC detection spectrum of maximum absorption wavelength of proline dimer in example 1 of the present application;
FIG. 4 is a scanning HPLC detection spectrum of the maximum absorption wavelength of L-proline in example 1 of the present application;
FIG. 5 is a HPLC detection profile of a test solution in the system suitability test of example 1;
FIG. 6 is a HPLC detection profile of a system suitability solution in the system suitability test of example 1;
FIG. 7 is a HPLC detection profile of a solution suitable for use in the system of example 2;
FIG. 8 is a HPLC detection profile of a solution suitable for use in the system of example 3;
FIG. 9 is a HPLC detection profile of a solution suitable for use in the system of example 4;
FIG. 10 is a HPLC detection profile of a solution suitable for use in the system of example 5.
Detailed Description
The present invention is further illustrated below with reference to specific examples, but is not limited to the scope of the implementation. In the following examples, the procedures and methods not described in detail are conventional methods well known in the art, and reagents used are commercially available analytically or chromatographically pure, without source or specification.
Example 1
(1) Instruments and reagents
The instrument is as follows: high performance liquid chromatography, Agilent 1260 is adopted; electronic balance, mettler XSR 105; pH meter, Mettler FE 28.
Reagent II: sodium octane sulfonate (chromatographically pure), Scharlau; sodium dihydrogen phosphate dihydrate (analytically pure), group of national drugs; acetonitrile (chromatographically pure); phosphoric acid (analytically pure), national medicine group.
③ chromatographic conditions: chromatography column Agilent TC-C18(2), 5 μm; 4.6 is multiplied by 250mm, and the flow rate is 0.5 ml/min; the detection wavelength is 200 nm; the sample volume is 10 mu L; the column temperature was 30 ℃.
Fourthly, mobile phase: weighing 5.13g of sodium octane sulfonate and 1.48g of sodium dihydrogen phosphate dihydrate, adding 850ml of water for dissolving, adding 150ml of acetonitrile, uniformly mixing, and adjusting the pH value to 3.0 by using phosphoric acid.
Solvent: a mobile phase.
(2) Preparation of Each solvent
Locating solution of each impurity: respectively weighing appropriate amount of impurities of L-hydroxyproline, L-proline and proline dimer to prepare 0.5mg/ml solution.
System applicability solution: respectively weighing appropriate amount of L-prolinamide, impurities of L-hydroxyproline, L-proline and proline dimer to prepare 4mg/ml of L-prolinamide solution and 0.02mg/ml of impurities solution.
③ test solution: the test sample was weighed to make a 4mg/ml solution.
Fourthly, comparison solution: precisely transferring 1.0ml of the sample solution into a 100ml volumetric flask, diluting to the scale with a solvent, shaking up to obtain a solution containing about 0.04mg per 1 ml.
(3) Maximum absorption wavelength sweep
Precisely measuring the sample and 10 μ l of each impurity solution, injecting into a liquid chromatograph, scanning the peak purity, and determining the maximum absorption wavelength of each impurity at the wavelength of 190-400nm, wherein the results are shown in FIG. 1, FIG. 2, FIG. 3 and FIG. 4.
The L-proline, the L-hydroxyproline and the proline dimer are absorbed at the tail ends, other obvious maximum characteristic absorption peaks do not exist, and the detection result is influenced by large base line fluctuation due to the fact that the selected detection wavelength is too low, so that the detection wavelength of related substances of the product is 200 nm.
(4) System suitability test
Respectively taking 10 mu l of test solution, control solution, system applicability solution, impurity positioning solutions and salt solution, injecting into a liquid chromatograph, and inspecting the separation degree. The results are shown in Table 1 below and FIGS. 5 to 6, Cl-peak, SO2The-peak, L-hydroxyproline, L-proline and proline dimer are well separated from the L-prolinamide.
Table 1: results of System suitability test
(5) Linearity and range
In order to accurately measure the product and each impurity, linear investigation is carried out, namely, the relation between peak areas and concentrations of each substance in a certain range is designed, and each linear concentration starts to be tested from a quantitative limit concentration, and the method comprises the following steps: and (3) taking the L-prolinamide and each impurity linear solution for sample injection analysis, and drawing a regression equation of the L-prolinamide and the dimer of L-hydroxyproline, L-proline and proline.
The result shows that the standard curve equation of the concentration range of the L-hydroxyproline in the range of 1.00575-160.92 mu g/ml is that y is 0.9861x-0.5863, R21.0000; the standard curve equation of the concentration range of the L-proline in the range of 1.0015-160.2400 mu g/ml is that y is 1.1214x-0.2115, and R is21.0000; the standard curve equation of the concentration range of the proline dimer in the range of 0.0501-40.0800 mu g/ml (0.25% -200%) is that y is 67.5073x +1.1843, R21.0000; the standard curve equation of the concentration range of the L-prolinamide in 0.5030-40.2400 mu g/ml (2.5% -200%) is that y is 12.1167x +2.9260, and R is2＝1.0000。
(6) Accuracy and precision experiment
Firstly, preparing a recovery rate solution: precisely transferring 10ml of 8000 mu g/ml of sample stock solution, respectively precisely transferring 1.0ml, 2.0ml and 4.0ml to 20ml of proline dimer impurity stock solution, 400 mu g/mlL-hydroxyproline impurity stock solution and 800 mu g/ml of L-hydroxyproline impurity stock solution into volumetric flasks, diluting the volumetric flasks to a scale by using a mobile phase, shaking the volumetric flasks uniformly, and preparing solutions with recovery rates of 50%, 100% and 200%.
② comparison product solution: and transferring 1.0ml to 10ml of impurity stock solutions respectively, and diluting with a solvent to a constant volume to a scale.
Sampling the sample, the reference solution and the recovery solution, and recording the chromatogram. The recovery rate of each impurity is calculated according to a main component self-contrast method added with a correction factor and an external standard method, the recovery rate is between 80% and 120%, the RSD value of the recovery rate is not more than 10.0%, and the method is high in accuracy.
Fourthly, the solution spectra of 50 percent, 100 percent and 200 percent of recovery rate are taken, the peak areas of the solution of 50 percent, 100 percent and 200 percent of recovery rate of each impurity are calculated, the RSD values of the peak areas of the solution of unit concentration of recovery rate are not more than 10.0 percent, and the method has good precision.
(7) Stability of
Taking the system applicability solution, the test solution and the control solution, standing at room temperature for 0, 5, 10, 15, 25 and 35 hours, precisely measuring 10 mu l, injecting into a liquid chromatograph, and recording the chromatogram. The peak area RSD value of L-prolinamide and each impurity is not more than 10% in 35 hours. Each solution was stable over 35 hours.
Example 2
Chromatographic conditions in this example: the flow rate was 0.4ml/min, and other chromatographic conditions were the same as in example 1.
And (5) taking 10 mu L of the system applicability solution, injecting the solution into a liquid chromatograph, and recording a chromatogram.
The detection results of related substances of the system applicability solution are shown in table 2, the obtained HPLC chromatogram is shown in table 7, and as can be seen from table 2, all impurity peaks are effectively separated, and the main peak is completely separated from the adjacent impurity peaks.
Table 2: system applicability solution-related substance detection results
| Name of impurity | Retention time (min) | Degree of separation | Number of theoretical plate |
| L-hydroxyproline | 8.292 | 1.7 | 28718 |
| L-proline | 9.665 | 1.96 | 17762 |
| Proline dimer | 11.089 | 4.96 | 23867 |
| L-prolinamides | 28.625 | 16.4 | 17367 |
Example 3
Chromatographic conditions in this example: the flow rate was 0.6ml/min, and other chromatographic conditions were the same as in example 1.
And (5) taking 10 mu L of the system applicability solution, injecting the solution into a liquid chromatograph, and recording a chromatogram.
The detection results of related substances of the system applicability solution are shown in table 3, the obtained HPLC chromatogram is shown in table 8, and it can be seen from table 3 that all impurity peaks are effectively separated, and the main peak is completely separated from the adjacent impurity peaks.
Table 3: system applicability solution-related substance detection results
| Name of impurity | Retention time (min) | Degree of separation | Number of theoretical plate |
| L-hydroxyproline | 5.502 | 1.71 | 22619 |
| L-proline | 6.407 | 1.91 | 18082 |
| Proline dimer | 7.384 | 5 | 21316 |
| L-prolinamides | 18.907 | 16.96 | 18078 |
Example 4
The chromatographic conditions of the example are as follows: the column temperature was 28 ℃ and other chromatographic conditions were the same as in example 1.
And (4) taking 10 mu L of the system applicability solution, injecting the solution into a liquid chromatograph, and recording a chromatogram.
The detection results of related substances of the system applicability solution are shown in table 4, the obtained HPLC chromatogram is shown in table 9, and as can be seen from table 4, all impurity peaks are effectively separated, and the main peak is completely separated from the adjacent impurity peaks.
Table 4: system applicability solution-related substance detection results
Example 5
Chromatographic conditions in this example: the column temperature was 32 ℃ and other chromatographic conditions were the same as in example 1.
And (5) taking 10 mu L of the system applicability solution, injecting the solution into a liquid chromatograph, and recording a chromatogram.
The detection results of related substances of the system applicability solution are shown in a table 5, the obtained HPLC chromatogram is shown in a table 10, and the table 5 shows that all impurity peaks are effectively separated, and a main peak is completely separated from adjacent impurity peaks.
Table 5: system applicability solution-related substance detection results
| |
Retention time (min) | Degree of separation | Number of theoretical plate |
| L-hydroxyproline | 6.571 | 1.96 | 23814 |
| L-proline | 7.598 | 1.64 | 19103 |
| Proline dimer | 8.821 | 5.42 | 23261 |
| L-prolinamides | 22.117 | 17.39 | 17649 |
In combination with the above experiments, the figures and tables 1-5 can be seen: the method comprises 0.02-0.03 mol/L sodium octane sulfonate, 0.005-0.015 mol/L sodium dihydrogen phosphate dihydrate, 10-20% acetonitrile and a mobile phase with the pH value of 2.5-3.5, when octadecylsilane chemically bonded silica is used as a filler for chromatographic column separation, the method can realize long retention time of various impurities, has high responsiveness, can realize specific effective separation and response of the impurities, has recovery rates of 80-120%, and has the RSD value of the recovery rate of not more than 10.0%.
Claims (9)
1. An HPLC test method for related substances in L-prolinamide, which is characterized in that the related substances of the L-prolinamide refer to impurities introduced or generated by degradation in the synthesis process of the L-prolinamide, and the test comprises the following two parts:
(1) preparing a mobile phase: the mobile phase is a solution obtained by adding water to dissolve sodium octane sulfonate and sodium dihydrogen phosphate dihydrate, then adding acetonitrile to mix uniformly, wherein the concentration of the sodium octane sulfonate is 0.02-0.03 mol/L, the concentration of the sodium dihydrogen phosphate dihydrate is 0.005-0.015 mol/L, the acetonitrile accounts for 10-20% of the total volume of the mobile phase, the pH value of the mobile phase is 2.5-3.5,
(2) elution and separation: performing chromatographic column separation by adopting a high performance liquid chromatography and using octadecylsilane chemically bonded silica as a filler, wherein the chromatographic conditions are as follows: the flow rate is 0.4-0.6 mL/min, the column temperature is 25-35 ℃, the detection wavelength is 190-400nm, and the mobile phase in the step (1) is used as a solvent to perform isocratic elution on the impurities.
2. An HPLC test method for related substances in L-prolinamide according to claim 1, wherein the HPLC test method comprises the following steps: the impurities comprise at least one of L-proline, cyclo (proline-proline) dipeptide and L-hydroxyproline.
3. An HPLC test method for related substances in L-prolinamide according to claim 2, wherein the HPLC test method comprises the following steps: respectively taking the concentration of the L-prolinamide and an impurity reference substance solution thereof as an abscissa x, the peak area as an ordinate y, the linear range of L-hydroxyproline is 1.00575-160.92 μ g/m, the linear equation is y =0.9861x-0.5863, and R = 1.0000; the linear range of L-proline is 1.0015-160.2400 μ g/ml, the linear equation is y =1.1214x-0.2115, and R = 1.0000; the linear range of the cyclic (proline-proline) dipeptide is 0.0501-40.0800 μ g/ml, the linear equation is y =67.5073x +1.1843, and R = 1.0000.
4. An HPLC test method for related substances in L-prolinamide according to claim 1, wherein the HPLC test method comprises the following steps: the mobile phase comprises 0.026mol/L sodium octane sulfonate, 0.011mol/L sodium dihydrogen phosphate dihydrate and acetonitrile accounting for 15 percent of the total volume of the mobile phase.
5. The HPLC test method for related substances in L-prolinamide according to claim 1, wherein the HPLC test method comprises the following steps: the pH of the mobile phase was 3.0.
6. An HPLC test method for related substances in L-prolinamide according to claim 1, wherein the HPLC test method comprises the following steps: the pH of the mobile phase was adjusted with phosphoric acid.
7. An HPLC test method for related substances in L-prolinamide according to claim 1, wherein the HPLC test method comprises the following steps: the octadecylsilane bonded silica gel had a packing length of 250mm, an inner diameter of 4.6mm, and a filler particle diameter of 5 μm.
8. An HPLC test method for related substances in L-prolinamide according to claim 1, wherein the HPLC test method comprises the following steps: the column temperature is 28-32 ℃.
9. An HPLC test method for related substances in L-prolinamide according to claim 1, wherein the HPLC test method comprises the following steps: in the chromatographic conditions, the flow rate is 0.5mL/min, the column temperature is 30 ℃, and the absorption wavelength is 200 nm.
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