Method for detecting 5-pyrimidine carbaldehyde compounds by liquid chromatography

Abstract

The application discloses a liquid chromatography analysis and detection method for 5-pyrimidine formaldehyde compounds, adopts a Hypercarb chromatographic column, uses a formic acid aqueous solution and a formic acid acetonitrile solution as a mobile phase, and through exploration of chromatographic conditions such as column temperature, elution mode and detection wavelength, obtains the liquid chromatography analysis and detection method for 5-pyrimidine formaldehyde compounds with good peak shape, high precision and excellent reproducibility, can detect various 5-pyrimidine formaldehyde compounds such as 5-pyrimidine formaldehyde, 2-methyl-5-pyrimidine formaldehyde, 2-amino-5-pyrimidine formaldehyde and 4, 6-dichloro-5-pyrimidine formaldehyde, and has strong specificity, good durability, high reproducibility and precision.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a liquid chromatography method for the detection of 5-pyrimidine carboxaldehyde compounds. Background Technology

[0002] 5-Pyrimidine carboxaldehyde compounds are commonly used raw materials in the chemical and pharmaceutical fields, and have wide applications in chemical synthesis and biopharmaceuticals. Therefore, the qualitative and quantitative analysis of 5-pyrimidine carboxaldehyde compounds plays an important role in the fields of drug production and analytical technology.

[0003] Pyrimidine aldehydes are unstable in water and alcohol and have high boiling points. Currently, common qualitative and quantitative analyses using ordinary liquid chromatography columns such as C18 or T3 columns produce peak shapes that are difficult to meet the requirements of qualitative and quantitative analysis. For example, the existing technology using a C18 column to analyze and detect 4,6-dichloro-5-pyrimidine formaldehyde produces the following results: Figure 1 As shown in the figure, conventional techniques for analyzing and detecting 4,6-dichloro-5-pyrimidine formaldehyde produce peaks with saddle-shaped peaks and inverted peaks, and the separation is poor, making it impossible to obtain true and effective results on purity and content.

[0004] Therefore, there is an urgent need in this field for an analytical detection method for 5-pyrimidine carboxaldehyde compounds that is easy to operate, widely applicable, has good separation effect, high accuracy, strong specificity, excellent reproducibility, and low cost. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a liquid chromatography method for the detection of 5-pyrimidine carboxaldehyde compounds, the method comprising the following steps:

[0006] S1. Preparation of sample solution: Take the 5-pyrimidine carboxaldehyde compound to be tested into a volumetric flask, add N,N-dimethylformamide, and after the 5-pyrimidine carboxaldehyde compound to be tested is dissolved, dilute to volume with acetonitrile and mix well for later use.

[0007] S2. Preparation of sensitivity solution: Take 5 mL of the sample solution prepared in S1 into a 100 mL volumetric flask, dilute to the mark with diluent and mix well. Then take 1 mL of the diluted solution into a 100 mL volumetric flask, dilute to the mark with diluent and mix well for later use.

[0008] S3. Take samples of the sample solution and the sensitivity solution respectively, inject them into a liquid chromatograph for liquid chromatography analysis and detection, record the chromatogram and analyze the detection results, and perform quantitative analysis on the 5-pyrimidine carboxaldehyde compound to be tested;

[0009] The liquid chromatography analysis and detection method uses a high-performance liquid chromatograph (HPLC), and the liquid chromatography conditions of the method are as follows:

[0010] The chromatographic column is a Hypercarb porous graphite carbon column, the column temperature is 40-50℃, the wavelength for liquid chromatography detection is 250-260nm, the mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B, mobile phase A is an aqueous formic acid solution, and mobile phase B is an acetonitrile formic acid solution, the diluent for liquid chromatography detection is N,N-dimethylformamide acetonitrile solution, the flow rate for liquid chromatography detection is set to 0.8-1.0mL / min, the injection volume for liquid chromatography detection is 4-6μL, and the elution method for liquid chromatography detection is isocratic elution.

[0011] Specifically, the 5-pyrimidinecarboxaldehyde compounds include 5-pyrimidinecarboxaldehyde, 2-methyl-5-pyrimidinecarboxaldehyde, 2-amino-5-pyrimidinecarboxaldehyde, and 4,6-dichloro-5-pyrimidinecarboxaldehyde. The concentration of the sample solution of 5-pyrimidinecarboxaldehyde is 0.25 mg / mL, the concentration of the sample solution of 2-methyl-5-pyrimidinecarboxaldehyde is 0.5 mg / mL, and the concentrations of the sample solutions of 2-amino-5-pyrimidinecarboxaldehyde and 4,6-dichloro-5-pyrimidinecarboxaldehyde are both 1 mg / mL.

[0012] Specifically, the column temperature of the chromatographic column is 45°C.

[0013] Specifically, the Hypercarb porous graphite carbon chromatography column has the following specifications: column length 150 mm, column inner diameter 4.6 mm, and packing particle size 5 μm.

[0014] Specifically, the wavelength for detection in the liquid chromatography analysis is 254 nm.

[0015] Specifically, the ratio of mobile phase A to mobile phase B is 5:95.

[0016] Specifically, the volume percentage of formic acid in mobile phase A is 0.1%, the volume percentage of formic acid in mobile phase B is 0.1%, and the concentration of the diluent is 10%.

[0017] Specifically, the flow rate for the liquid chromatography analysis was set to 0.8 mL / min.

[0018] Specifically, the injection volume for the liquid chromatography analysis is 5 μL.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. This invention employs a Hypercarb column and uses formic acid aqueous solution and formic acid acetonitrile solution as the mobile phase. Through exploration of chromatographic conditions such as column temperature, elution mode, and detection wavelength, a liquid chromatography method for the analysis and detection of 5-pyrimidine carboxaldehyde compounds with good peak shape, high precision, and excellent reproducibility has been obtained. This method can detect a variety of 5-pyrimidine carboxaldehyde compounds, including 5-pyrimidine carboxaldehyde, 2-methyl-5-pyrimidine carboxaldehyde, 2-amino-5-pyrimidine carboxaldehyde, and 4,6-dichloro-5-pyrimidine carboxaldehyde. Furthermore, it exhibits strong specificity and robustness, using only N,N-dimethylformamide acetonitrile solution as a diluent to accurately analyze and detect a wide range of 5-pyrimidine carboxaldehyde compounds, demonstrating the broad applicability of the method provided by this invention.

[0021] 2. The detection method provided by this invention overcomes the shortcomings of poor peak shape and low accuracy in the prior art. It can complete the quantitative analysis and detection of multiple 5-pyrimidine formaldehyde compounds with only one detection method. It is simple to operate, low in cost, and ensures the sensitivity and accuracy of detection. Attached Figure Description

[0022] Figure 1 This is a chromatogram of 4,6-dichloro-5-pyrimidine formaldehyde analyzed and detected using a C18 column in existing technologies;

[0023] Figure 2 This is a superimposed graph of blank, limit of quantitation and control solutions for 2-methyl-5-pyrimidinecarboxaldehyde, 2-amino-5-pyrimidinecarboxaldehyde and 4,6-dichloro-5-pyrimidinecarboxaldehyde;

[0024] Figure 3 This is a superimposed graph of the blank, limit of quantitation, and control solutions for 5-pyrimidinecarboxaldehyde.

[0025] Figure 4 This is a graph showing the linear range of 2-methyl-5-pyrimidinecarboxaldehyde.

[0026] Figure 5 This is a graph showing the linear range of data for 4,6-dichloro-5-pyrimidinecarboxaldehyde.

[0027] Figure 6 This is a graph showing the linear range of 2-amino-5-pyrimidinecarboxaldehyde.

[0028] Figure 7 This is a graph showing the linear range of 5-pyrimidinecarboxaldehyde. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the materials, instruments, and reagents used in the following embodiments are all commercially available.

[0030] In the following examples, DMF is N,N-dimethylformamide, ACN is acetonitrile, and FA is acetic acid. The reagents used were sourced as follows: 5-pyrimidinecarboxaldehyde, 2-methyl-5-pyrimidinecarboxaldehyde, 2-amino-5-pyrimidinecarboxaldehyde, and 4,6-dichloro-5-pyrimidinecarboxaldehyde were purchased from Bidex Pharmaceuticals Co., Ltd., DMF was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., ACN was purchased from Merck Chemical Technology (Shanghai) Co., Ltd., and FA was purchased from Shanghai Anpu Experimental Technology Co., Ltd. The liquid chromatograph used was a Waters Arc or equivalent instrument.

[0031] Example 1

[0032] In this embodiment, four 5-pyrimidine carboxaldehyde compounds were analyzed and detected by liquid chromatography, namely 5-pyrimidine carboxaldehyde, 2-methyl-5-pyrimidine carboxaldehyde, 2-amino-5-pyrimidine carboxaldehyde, and 4,6-dichloro-5-pyrimidine carboxaldehyde. The specific steps included:

[0033] S1. The procedures for preparing sample solutions are as follows:

[0034] 5-Pyrimidine formaldehyde sample solution: Weigh about 25 mg of sample into a 100 mL volumetric flask, add 10 mL of DMF, and after the sample is completely dissolved, dilute to the mark with acetonitrile, mix well and take the sample.

[0035] 2-Methyl-5-pyrimidinecarboxaldehyde sample solution: Weigh about 50 mg of sample into a 100 mL volumetric flask, add 10 mL of DMF, and after the sample is completely dissolved, dilute to the mark with acetonitrile, mix well and take the sample.

[0036] 4,6-Dichloro-5-pyrimidinecarboxaldehyde sample solution: Weigh approximately 100 mg of sample into a 100 mL volumetric flask, add 10 mL of DMF, and after the sample has completely dissolved, dilute to the mark with acetonitrile, mix well, and then take the sample.

[0037] 2-Amino-5-pyrimidine carboxaldehyde sample solution: Weigh about 100 mg of sample into a 100 mL volumetric flask, add 10 mL of DMF, and after the sample is completely dissolved, dilute to the mark with acetonitrile, mix well and take the sample.

[0038] S2. The procedures for preparing the sensitivity solutions are as follows:

[0039] Take 5 mL each of the 5-pyrimidine formaldehyde sample solution, 2-methyl-5-pyrimidine formaldehyde sample solution, 4,6-dichloro-5-pyrimidine formaldehyde sample solution, and 2-amino-5-pyrimidine formaldehyde sample solution prepared in S1 into a 100 mL volumetric flask. Dilute to the mark with diluent and mix well. Then take 1 mL of the diluted solution into a 100 mL volumetric flask, dilute to the mark with 10% N,N-dimethylformamide acetonitrile solution, and mix well for later use.

[0040] S3. Take samples of the sample solution and the sensitivity solution respectively, inject them into a Waters Arc liquid chromatograph for liquid chromatography analysis and detection, record the chromatograms and analyze the detection results, and perform quantitative analysis of the 5-pyrimidine carboxaldehyde compounds to be tested. The chromatographic conditions are as follows:

[0041] Chromatographic column: Hypercarb (150×4.6mm, 5μm)

[0042] Wavelength: 254nm

[0043] Column temperature: 45℃

[0044] Flow rate: 0.8 mL / min

[0045] Injection volume: 5 μL

[0046] Mobile phase A: 0.1% FA in Water (v / v)

[0047] Mobile phase B: 0.1% FA in ACN (v / v)

[0048] gradient:

[0049] Time (min) A％ B％ 0.00 5 95 15.00 5 95

[0050] Diluent: 10% DMF in ACN

[0051] The chromatogram of the test results is as follows: Figure 2 and Figure 3 As shown, Figure 2 This is a superimposed graph of blank, limit of quantitation, and control solutions for 2-methyl-5-pyrimidinecarbaldehyde, 2-amino-5-pyrimidinecarbaldehyde, and 4,6-dichloro-5-pyrimidinecarbaldehyde. Figure 3 This is a superimposed graph of the blank, limit of quantitation, and control solutions of 5-pyrimidinecarboxaldehyde. The retention times of the four samples are shown in the table below:

[0052] Chemical name CAS number Retention time 5-Pyrimidinecarboxaldehyde 10070-92-5 3.70min 2-Methyl-5-pyrimidinecarboxaldehyde 90905-33-2 4.46min 4,6-Dichloro-5-pyrimidinecarboxaldehyde 5305-40-8 6.77min 2-Amino-5-pyrimidinecarboxaldehyde 120747-84-4 8.43min

[0053] As can be seen from the chromatogram results, the detection method provided by this invention can perform liquid chromatography detection of the above four 5-pyrimidine carboxaldehyde compounds with good peak shape, good resolution, high sensitivity, and strong specificity.

[0054] Following the same procedure described above, five injections were performed, and the relative standard deviation (RSD) was calculated. The results are as follows: Figure 4-7 As shown.

[0055] In the detection results of 2-methyl-5-pyrimidinecarboxaldehyde, the signal-to-noise ratio (S / N) was 49.6, and the linear range was 0.00025 mg / mL to 1.0 mg / mL (0.05% to 200%, r = 0.9998). Figure 4 As shown, the RSD of the peak area of ​​the five needles is 0.10%, and the RSD of the retention time of the five needles is 0.03%.

[0056] In the detection results of 4,6-dichloro-5-pyrimidine formaldehyde, the signal-to-noise ratio (S / N) was 21.8, and the linear range was 0.0005 mg / mL to 2.0 mg / mL (0.05% to 200%, r = 1.0000). Figure 5 As shown, the RSD of the peak area of ​​the five needles is 0.06%, and the RSD of the retention time of the five needles is 0.03%.

[0057] In the detection results of 2-amino-5-pyrimidinecarboxaldehyde, the signal-to-noise ratio (S / N) was 53.1, and the linear range was 0.0005 mg / mL to 2.0 mg / mL (0.05% to 200%, r = 0.9993). Figure 6 As shown, the RSD of the peak area of ​​the five needles is 0.15%, and the RSD of the retention time of the five needles is 0.03%.

[0058] In the detection results of 5-pyrimidine formaldehyde, the signal-to-noise ratio (S / N) was 24.3, and the linear range was 0.000125 mg / mL to 0.5 mg / mL (0.05% to 200%, r = 1.0000). Figure 7 As shown, the RSD of the peak area of ​​the five needles is 0.15%, and the RSD of the retention time of the five needles is 0.05%.

[0059] The results above show that the detection method provided by this invention has a small relative standard deviation and good precision and reproducibility for the detection of the above four 5-pyrimidine carboxaldehyde compounds by liquid chromatography.

[0060] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A liquid chromatography method for the analysis and detection of 5-pyrimidine carboxaldehyde compounds, characterized in that, The method includes the following steps: S1. Preparation of sample solution: Take the 5-pyrimidine carboxaldehyde compound to be tested into a volumetric flask, add N,N-dimethylformamide, and after the 5-pyrimidine carboxaldehyde compound to be tested is dissolved, dilute to volume with acetonitrile and mix well for later use. S2. Preparation of sensitivity solution: Take 5 mL of the sample solution prepared in S1 into a 100 mL volumetric flask, dilute to the mark with diluent and mix well. Then take 1 mL of the diluted solution into a 100 mL volumetric flask, dilute to the mark with diluent and mix well for later use. S3. Take samples of the sample solution and the sensitivity solution respectively, inject them into a liquid chromatograph for liquid chromatography analysis and detection, record the chromatogram and analyze the detection results, and perform quantitative analysis on the 5-pyrimidine carboxaldehyde compound to be tested; The liquid chromatography analysis and detection method uses a high-performance liquid chromatograph (HPLC), and the liquid chromatography conditions of the method are as follows: The chromatographic column is a Hypercarb porous graphite carbon column, the column temperature is 40-50℃, the wavelength for liquid chromatography detection is 250-260nm, the mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B, mobile phase A is a 0.1% formic acid aqueous solution, mobile phase B is a 0.1% formic acid acetonitrile solution, the ratio of mobile phase A to mobile phase B is 5:95, the diluent for liquid chromatography detection is N,N-dimethylformamide acetonitrile solution, the flow rate for liquid chromatography detection is set to 0.8-1.0mL / min, the injection volume for liquid chromatography detection is 4-6μL, and the elution method for liquid chromatography detection is isocratic elution; The 5-pyrimidine carboxaldehyde compounds are 5-pyrimidine carboxaldehyde, 2-methyl-5-pyrimidine carboxaldehyde, 2-amino-5-pyrimidine carboxaldehyde, and 4,6-dichloro-5-pyrimidine carboxaldehyde.

2. The liquid chromatography method for the detection of 5-pyrimidine carboxaldehyde compounds according to claim 1, characterized in that, The concentration of the 5-pyrimidinecarboxaldehyde sample solution is 0.25 mg / mL, the concentration of the 2-methyl-5-pyrimidinecarboxaldehyde sample solution is 0.5 mg / mL, and the concentrations of the 2-amino-5-pyrimidinecarboxaldehyde and 4,6-dichloro-5-pyrimidinecarboxaldehyde sample solutions are both 1 mg / mL.

3. The liquid chromatography method for the detection of 5-pyrimidine carboxaldehyde compounds according to claim 1, characterized in that, The column temperature of the chromatographic column is 45℃.

4. The liquid chromatography method for the detection of 5-pyrimidine carboxaldehyde compounds according to claim 1, characterized in that, The Hypercarb porous graphite carbon chromatography column has the following specifications: column length 150 mm, column inner diameter 4.6 mm, and packing particle size 5 μm.

5. The liquid chromatography method for the detection of 5-pyrimidine carboxaldehyde compounds according to claim 1, characterized in that, The wavelength for the liquid chromatography analysis was 254 nm.

6. The liquid chromatography method for the detection of 5-pyrimidine carboxaldehyde compounds according to claim 1, characterized in that, The concentration of the diluent is 10%.

7. The liquid chromatography method for the detection of 5-pyrimidine carboxaldehyde compounds according to claim 1, characterized in that, The flow rate for the liquid chromatography analysis was set to 0.8 mL / min.

8. The liquid chromatography method for the detection of 5-pyrimidine carboxaldehyde compounds according to claim 1, characterized in that, The injection volume for the liquid chromatography analysis was 5 μL.

Images