Method for measuring content of enantiomer in test sample
By using 2,4-dinitrobenzaldehyde as a derivatizing agent and a specific mobile phase, the enantiomer content of S2005M3A was detected in a high-performance liquid chromatography-mass spectrometry system. This solved the detection problem in the prior art, achieved detection results with high specificity and good separation, and controlled the chirality of intermediates and final products.
Patent Information
- Application Number
- CN202511374349.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-25
- Publication Date
- 2025-12-19
AI Technical Summary
Existing technologies are insufficient for the efficient and accurate detection and quantification of S2005M3A enantiomers, especially since hydrazine compounds are difficult to detect directly by conventional detectors, and traditional methods are cumbersome and inefficient.
2,4-Dinitrobenzaldehyde was used as a derivatizing agent, combined with a specific ratio of A and B mobile phases, and derivatization-HPLC detection was performed using a high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) instrument. The content of S2005M3A was determined by calculating the peak area by recording the mass spectrum.
The method achieves high specificity and good separation for the detection of enantiomer content, effectively controls the chirality of intermediate S2005M3A, and thus indirectly controls the chirality of the final product ruxolitinib phosphate. The method is simple and efficient.
Smart Images
Figure CN121164480A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of analytical chemistry and relates to a method for determining the enantiomer content in a test sample. Background Technology
[0002] Ruxolitinib phosphate is a first-in-class small molecule drug developed by Incyte Corp. It is a JAK2 and JAK1 inhibitor. Currently, the highest stage of development for this drug is market approval for the treatment of vitiligo, graft-versus-host disease, atopic dermatitis, myelofibrosis, essential thrombocythemia, and polycythemia vera.
[0003] S2005M3A (molecular formula shown below) is a key intermediate in the synthesis of ruxolitinib phosphate. The chirality of ruxolitinib phosphate is introduced by it, therefore the content of the enantiomers of S2005M3A needs to be strictly controlled.
[0004]
[0005] S2005M3A is a hydrazine compound, which is typically difficult to detect directly by conventional detectors such as UV and flame ionization detectors (FID). Common methods include derivatization-TLC or HPLC. However, TLC methods suffer from low specificity and sensitivity, making accurate quantification difficult. For example, current methods for detecting hydrazine hydrate involve derivatization with benzaldehyde, salicylaldehyde, or p-dimethylaminobenzaldehyde (PDAB), followed by limit or quantitative detection using TLC or HPLC. However, these methods are cumbersome and inefficient. Summary of the Invention
[0006] The purpose of this invention is to provide a method for determining the enantiomeric content in a test sample, so as to solve the problems mentioned in the background art.
[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for determining the enantiomeric content in a test sample, comprising the following steps:
[0008] (a) Dissolve S2005M3A in the derivatization solution and seal it; keep it at 40-60℃ for 20-40 min and then cool it; the derivatization solution is an ethanol solution of 2,4-dinitrobenzaldehyde;
[0009] (b) Prepare S2005M3AP01 impurity stock solution, system suitability solution and test solution;
[0010] (c) Take appropriate amounts of the S2005M3AP01 impurity stock solution, system suitability solution and test solution respectively, inject them into the high performance liquid chromatography-mass spectrometry instrument, and record the mass spectra respectively;
[0011] (d) Calculate the content of S2005M3A based on the peak area of each mass spectrum;
[0012] In step (c), the mobile phase of the high performance liquid chromatography-mass spectrometry instrument is divided into phase A and phase B. Phase A is a 0.1% (v / v) aqueous solution of diethylamine n-hexane, and phase B is ethanol. The volume ratio of phase A to phase B is 60%:40%.
[0013] Optimally, in step (a), the derivatized solution is prepared by the following steps: weigh 50 mg of 2,4-dinitrobenzaldehyde, place it in a 20 ml volumetric flask, add an appropriate amount of ethanol to dissolve it, and dilute to the mark, then shake well to obtain the solution.
[0014] Optimally, in step (b), the S2005M3AP01 impurity stock solution is prepared by the following steps: weigh 10 mg of S2005M3AP01 reference standard, place it in a 100 ml volumetric flask, add an appropriate amount of diluent to dissolve it, dilute it to the mark with diluent, and shake well to obtain the solution.
[0015] Optimally, in step (b), the system suitability solution is prepared by the following steps: Weigh about 10 mg of S2005M3A reference standard, place it in a 20 ml volumetric flask, add an appropriate amount of diluent to dissolve it, transfer 1 ml of S2005M3AP01 impurity stock solution into the volumetric flask, add diluent to the mark, and shake well to obtain the solution.
[0016] Optimally, in step (b), the test solution is prepared by the following steps: take 2 mg of the test sample and place it in a 20 ml headspace vial, accurately add 4 ml of derivatizing solution to dissolve it, and seal it; place it at 60 °C for half an hour, take it out and let it cool before directly injecting the sample.
[0017] Ideally, in step (c), the high-performance liquid chromatography-mass spectrometry instrument uses a CHRALPAK AD-H4.6×250mm, 5μm column, a flow rate of 1-1.2ml / min, and a column temperature of 25-30℃.
[0018] Furthermore, in step (c), the high-performance liquid chromatography-mass spectrometry instrument is equipped with an ultraviolet detector or a diode array detector.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The method for determining the enantiomeric content in the test sample of the present invention uses 2,4-dinitrobenzaldehyde as a derivatizing agent in combination with specific A phase and B phase as mobile phase, and then achieves the result through derivatization-HPLC method. It has the characteristics of strong specificity and good separation, and can effectively control the chirality of intermediate S2005M3A, and indirectly control the chirality of the final product ruxolitinib phosphate. Attached Figure Description
[0020] Figure 1 This is a high-performance liquid chromatogram of the blank solution of this invention;
[0021] Figure 2 This is a high-performance liquid chromatogram of the test solution of the present invention;
[0022] Figure 3 The chemical formula of the impurity S2005M3AP01 of this invention;
[0023] Figure 4 The chromatograms of the test solution of the present invention at different temperatures are shown below.
[0024] Figure 5 The chromatogram of the derivatives obtained by using Fmoc-Cl as a derivatizing agent in this invention is shown below.
[0025] Figure 6 The chromatogram for this invention uses 0.1% diethylamine-n-hexane-isopropanol (70:30) as the mobile phase. Detailed Implementation
[0026] The method of the present invention will be described below through specific embodiments. It should be understood that these embodiments are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited to the scope of the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to specific requirements. Implementation conditions not specified are generally the conditions in the following conventional experiments.
[0027] The method for determining the enantiomer content in the test sample of the present invention includes the following steps:
[0028] (a) Dissolve S2005M3A in the derivatization solution and seal it; keep it at 40-60℃ for 20-40 min and then cool it; the derivatization solution is an ethanol solution of 2,4-dinitrobenzaldehyde;
[0029] (b) Prepare S2005M3AP01 impurity stock solution, system suitability solution and test solution;
[0030] (c) Take appropriate amounts of the S2005M3AP01 impurity stock solution, system suitability solution and test solution respectively, inject them into the high performance liquid chromatograph, and record the mass spectra respectively;
[0031] (d) Calculate the content of S2005M3A based on the peak area of each mass spectrum;
[0032] In step (c), the mobile phase of the high-performance liquid chromatograph is divided into phase A and phase B. Phase A is a 0.1% (v / v) aqueous solution of diethylamine-n-hexane, and phase B is ethanol. The volume ratio of phase A to phase B is 60%:40%. The chromatographic column used in the high-performance liquid chromatograph is a CHRALPAK AD-H 4.6×250mm, 5μm column, with a flow rate of 1–1.2 ml / min and a column temperature of 25–30℃. In step (c), the high-performance liquid chromatograph is equipped with an ultraviolet detector or a diode array detector, as detailed in Table 1.
[0033] Table 1. Parameters of High Performance Liquid Chromatography (HPLC)
[0034] instrument High-performance liquid chromatograph (with UV detector or diode array detector) chromatographic column CHRALPAK AD-H 4.6×250mm 5μm Detection wavelength 395nm Flow rate 1ml / min Column temperature 30℃ mobile phase Contains 0.1% diethylamine-n-hexane-ethanol (60:40) runtime 30min Injection volume 10μl
[0035] In step (a), the derivatized solution is prepared according to the following steps: Weigh 50 mg of 2,4-dinitrobenzaldehyde, place it in a 20 ml volumetric flask, add an appropriate amount of ethanol (i.e., diluent) to dissolve it, dilute to the mark, and shake well. In step (b), the S2005M3AP01 impurity (such as...) Figure 3 The stock solution (shown) is prepared as follows: Weigh 10 mg of S2005M3AP01 reference standard, place it in a 100 ml volumetric flask, add an appropriate amount of diluent to dissolve it, dilute to the mark with diluent, and shake well. In step (b), the system suitability solution is prepared as follows: Weigh approximately 10 mg of S2005M3A reference standard, place it in a 20 ml volumetric flask, add an appropriate amount of diluent to dissolve it, transfer 1 ml of the S2005M3AP01 impurity stock solution to the same volumetric flask, add diluent to the mark, and shake well. In step (b), the test sample solution is prepared as follows: Take 2 mg of the test sample, place it in a 20 ml headspace vial, accurately add 4 ml of derivatizing solution to dissolve it, seal the vial; incubate at 60°C for half an hour, remove and cool, then inject directly. The specific preparation process of the above solutions is listed in Table 2.
[0036] Furthermore, the results of derivatization experiments at 40℃ for half an hour were examined. The peak area at 40℃ was significantly smaller than that at 60℃, therefore 60℃ was chosen for derivatization (e.g., ...). Figure 4 (As shown).
[0037] Table 2. Preparation methods for each solution
[0038]
[0039] Experiments were conducted using blank solutions and system suitability solutions under the chromatographic conditions in Table 1, such as... Figure 1 and Figure 2As shown, the conclusions are as follows: (1) Blank solution: There are no obvious interfering peaks near the target peak position. (2) System suitability solution: The peak separation degree between S2005M3A and S2005M3AP01 is 8.9, which meets the acceptable standard.
[0040] The detection method of the present invention has the characteristics of no blank baseline interference, strong specificity and good separation. It is suitable for the detection of S2005M3A enantiomer content. The chirality of this intermediate can be controlled by using the derivatization-HPLC method, thereby controlling the chirality of the final product ruxolitinib phosphate.
[0041] This application also uses Fmoc-Cl as a derivatizing agent, and compares it with the aforementioned 2,4-dinitrobenzaldehyde, under the following specific conditions:
[0042] Derivatizer 1: 2,4-Dinitrobenzaldehyde;
[0043] Derivatizer 2: Fmoc-Cl;
[0044] Experimental steps:
[0045] Derivative solution 1: Weigh about 50 mg of 2,4-dinitrobenzaldehyde, place it in a 20 ml volumetric flask, add an appropriate amount of acetonitrile:water = 1:1 (volume ratio) to dissolve and dilute to the mark, shake well to obtain the solution.
[0046] Derivatized solution 2: Weigh approximately 50 mg of Fmoc-Cl, place it in a 20 ml volumetric flask, add an appropriate amount of dichloromethane to dissolve and dilute to the mark, and shake well to obtain the solution.
[0047] Test solution 1: Take about 2 mg of the test sample and place it in a 20 ml headspace vial. Accurately add 4 ml of derivatizing solution to dissolve it, and seal the vial. Incubate at 60°C for half an hour, then remove and cool before injecting the sample directly.
[0048] Test Solution 2: Place approximately 2 mg of the test sample in a 20 ml headspace vial, accurately add 4 ml of derivatizing solution to dissolve, and seal. Let stand at room temperature for half an hour, then remove and cool before injecting directly. Add 0.6 ml of 5% K₂CO₃ (mass percentage) solution and shake well. After separation, discard the aqueous layer, evaporate to dryness, and reconstitute with acetonitrile:water = 1:1 before injecting.
[0049] Detection: Inject test solution 1 and test solution 2 into the high-performance liquid chromatograph (HPLC) for analysis and record the chromatograms. When 2,4-dinitrobenzaldehyde is used as a derivatizing agent, the derivatized product is simple; when Fmoc-Cl is used as a derivatizing agent, the derivatized product is complex (e.g., ...). Figure 5 (As shown).
[0050] This application also uses 0.1% diethylamine-n-hexane-isopropanol (70:30) as the mobile phase for comparison, under the following specific conditions:
[0051] Derivatized solution: Weigh about 50 mg of 2,4-dinitrobenzaldehyde, place it in a 20 ml volumetric flask, add an appropriate amount of acetonitrile:water = 1:1 (volume ratio) to dissolve and dilute to the mark, shake well to obtain the solution.
[0052] Test solution: Place approximately 2 mg of the test sample into a 20 ml headspace vial, accurately add 4 ml of derivatizing solution to dissolve, and seal. Incubate at 60°C for half an hour, then remove and allow to cool before injecting the sample directly.
[0053] Detection: Inject the above test solution into a high-performance liquid chromatograph and perform the determination and comparison under two different mobile phase conditions, and record the chromatograms.
[0054] Conclusion: The mobile phase containing 0.1% diethylamine-hexane-ethanol (60:40) provides better separation, while the mobile phase containing 0.1% diethylamine-hexane-isopropanol (70:30) provides poorer separation (e.g., ...). Figure 6 (As shown).
[0055] This application also conducts accuracy experiments, linearity experiments, and solution stability experiments on the above-mentioned detection method, as detailed below:
[0056] (I) Accuracy test (chromatographic conditions are shown in Table 1, the same below)
[0057] (1) Solution preparation: Add 0.40% level to the test sample solution to prepare 6 parallel solutions.
[0058]
[0059] (2) Detection: Inject the above accuracy solution into a high performance liquid chromatograph for determination and record the chromatogram.
[0060]
[0061]
[0062]
[0063] In the formula:
[0064] Measured quantity (i) : The content of S2005M3AP01 in the accuracy solution.
[0065] blank (i) The content of S2005M3AP01 in the test solution.
[0066] Amount added:
[0067]
[0068] In the formula:
[0069] W1: The amount of S2005M3AP01 added to the accuracy solution.
[0070] P1: Content of S2005M3AP01.
[0071] W2: The amount of S2005M3A sample weighed in the accuracy solution.
[0072] P2: Content of S2005M3A.
[0073] ee%: S2005M3A content in S2005M3AP01.
[0074] As shown in the table above, the average recovery rate of the six accuracy solutions was 82.8%, with a recovery RSD of 1.6%, which meets the acceptable standard.
[0075] (II) Accuracy test (chromatographic conditions are shown in Table 1, the same below)
[0076] (1) Solution preparation:
[0077]
[0078] (2) Detection: Inject the standard series solutions of the above concentrations into the high performance liquid chromatograph for determination and record the chromatogram.
[0079] Based on the above chromatographic conditions, the peak areas at each concentration were measured. A standard curve was plotted with peak area on the ordinate and concentration on the abscissa. The correlation coefficient R, Y-intercept, and sum of squared residuals were calculated. The results are shown in the table below. The results show that the linearity meets the acceptable criteria.
[0080] S2005M3A linearity determination results
[0081]
[0082] S2005M3AP01 linearity determination results
[0083]
[0084]
[0085] (III) Solution Stability Experiment
[0086] The changes in the test solution over time were investigated. After being left at room temperature for a period of time, the sample was injected at different times, and the chromatograms were recorded. The results are shown in the table below, indicating that the test solution was stable at room temperature for 12 hours.
[0087] Results of stability study of the test sample
[0088]
[0089] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.
Claims
1. A method for determining the content of an enantiomer in a test sample, said test sample being S2005M3A, characterized in that, It comprises the following steps: (a) dissolving S2005M3A in a derivatization solution, sealing; incubating at 40-60℃ for 20-40 min, and cooling; the derivatization solution is an ethanol solution of 2,4-dinitrobenzaldehyde; (b) preparing S2005M3AP01 impurity stock solution, system suitability solution and test sample solution; (c) taking the S2005M3AP01 impurity stock solution, system suitability solution and test sample solution respectively, and injecting them into a high performance liquid chromatograph, and recording mass spectra respectively; (d) calculating the content of S2005M3A according to the peak area of each mass spectrum; In step (c), the mobile phase of the high performance liquid chromatograph is divided into phase A and phase B, phase A is a 0.1% diethylamine n-hexane aqueous solution by volume concentration, and phase B is ethanol, and the volume ratio of phase A and phase B is 60:
40.
2. The method according to claim 1, wherein In step (a), the derivatization solution is prepared by the following steps: weighing 2,4-dinitrobenzaldehyde 50 mg, placing it in a 20 ml volumetric flask, adding ethanol to dissolve, and diluting to the mark, and shaking to obtain the derivatization solution.
3. The method according to claim 1, wherein In step (b), the S2005M3AP01 impurity stock solution is prepared by the following steps: weighing S2005M3AP01 reference substance 10 mg, placing it in a 100 ml volumetric flask, adding diluent to dissolve, diluting to the mark with diluent, and shaking to obtain the S2005M3AP01 impurity stock solution.
4. The method according to claim 1, wherein In step (b), the system suitability solution is prepared by the following steps: weighing S2005M3A reference substance about 10 mg, placing it in a 20 ml volumetric flask, adding diluent to dissolve, transferring 1 ml of S2005M3AP01 impurity stock solution to the volumetric flask, adding diluent to the mark, and shaking to obtain the system suitability solution.
5. The method according to claim 1, wherein In step (b), the test sample solution is prepared by the following steps: taking 2 mg of test sample and placing it in a 20 ml headspace bottle, precisely adding 4 ml of derivatization solution to dissolve, sealing; placing it in a 60℃ oven for half an hour, taking it out and cooling, and directly injecting it into the sample.
6. The method according to claim 1, wherein: In step (c), the high performance liquid chromatograph uses a CHRALPAK AD-H 4.6*250 mm, 5 μm column, a flow rate of 1-1.2 ml / min, and a column temperature of 25-30℃.
7. The method according to claim 6, wherein In step (c), the high performance liquid chromatograph is equipped with an ultraviolet detector or a diode array detector.