Method for detecting impurities in vincristine sulfate by liquid chromatography-mass spectrometry

Abstract

The application provides a method for detecting impurities in vinblastine sulfate by liquid chromatography-mass spectrometry, and belongs to the technical field of drug analysis. Ammonium acetate is used as mobile phase A, and acetonitrile is used as mobile phase B, the traditional eluent diethylamine and phosphoric acid are abandoned, the negative influence on material performance and chromatographic resolution is avoided, the separation effect of vinblastine sulfate and each impurity is better, and the resolution is higher. Meanwhile, by combining ultraviolet detection and mass spectrometry detection, vinblastine sulfate and impurities A, C, D, E, F, G and H can be all separated and detected, qualitative and quantitative analysis of each impurity can be carried out, so that each impurity prepared can be accurately evaluated, and the content of impurities in the product can be accurately controlled.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for detecting impurities in vincristine sulfate using liquid chromatography-mass spectrometry. Background Technology

[0002] Vincristine sulfate is an antitumor drug used to treat acute leukemia, Hodgkin's disease, malignant lymphoma, and other malignant tumors. Its chemical structure is as follows:

[0003] The quality standards for vincristine sulfate are included in the Chinese Pharmacopoeia, the United States Pharmacopoeia, and the European Pharmacopoeia. The European Pharmacopoeia includes the structures of impurities A, B, C, D, E, F, G, and H. Chromatograms of mixed solutions of vincristine sulfate and these impurities can be found in the Knowledge Database. Figure 1 As shown.

[0004] Vincristine is the raw material for the synthesis of vincristine sulfate, and its main source is the periwinkle, a plant belonging to the Apocynaceae family. Due to its natural origin, the extraction process of vincaristine inevitably introduces numerous impurities, thus affecting the quality of the target product, vincristine sulfate. To achieve product quality control, it is necessary to control impurities A, C, D, E, F, G, and H in the product. Therefore, it is necessary to prepare impurities A, C, D, E, F, G, and H to achieve qualitative and quantitative analysis of impurities in vincristine sulfate.

[0005] However, due to the large number of impurities in the sample and the dense peaks, it is difficult to determine the accuracy of the obtained impurities based solely on the relative retention times of each impurity in the reference figure attached to the European Pharmacopoeia Knowledge Database. Therefore, combining the peak characteristics of the impurities in the mass spectrometer is crucial for judging the accuracy of the prepared impurities.

[0006] According to the European Pharmacopoeia, the mobile phase used for the detection of vincristine sulfate impurities is 1.5% diethylamine aqueous solution (adjusted to pH 7.5 with phosphoric acid) and methanol. However, this detection method has significant drawbacks: (1) phosphoric acid is a non-volatile acid and cannot be used for mass spectrometry; (2) high concentrations of diethylamine can adversely affect the properties of materials in contact with the solvent in the mass spectrometer, thereby affecting the chromatographic resolution. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for detecting impurities in vincristine sulfate using liquid chromatography-mass spectrometry, so as to evaluate the accuracy of the prepared impurities A, C, D, E, F, G, and H, thereby more accurately controlling the content of impurities in the product.

[0008] The structures of each impurity are shown below:

[0009] To achieve the above and other related objectives, this invention provides a method for detecting impurities in vincristine sulfate using liquid chromatography-mass spectrometry (LC-MS). The method simultaneously employs both ultraviolet (UV) and mass spectrometry detectors to detect impurities A, C, D, E, F, G, and H in vincristine sulfate. Ammonium acetate solution is used as mobile phase A, and acetonitrile is used as mobile phase B. Gradient elution is employed with the following gradient ratios: .

[0010] In the technical solution of this invention, the chromatograph selected is a Waters ACQUITY Arc high-performance liquid chromatograph, equipped with a 2489 UV / Vis detector and a QDA mass spectrometer detector, which simultaneously performs ultraviolet detection and mass spectrometry detection.

[0011] In the technical solution of this invention, the mass spectrometer detector is in scanning mode, simultaneously performing positive and negative ion scanning. The mass spectrometer has a cone voltage of 15V, a capillary voltage of 0.8kV, and a probe temperature of 600℃.

[0012] In the technical solution of the present invention, the chromatographic column used is Welch Ultimate XB-C18, with the following specifications: inner diameter 4.6 mm, length 250 mm, and packing particle size 5 μm.

[0013] In the technical solution of the present invention, the column temperature of the chromatographic column is set to 28℃~32℃, preferably 30℃.

[0014] In the technical solution of the present invention, the concentration of ammonium acetate in mobile phase A is 4 mmol / L to 6 mmol / L. Preferably, the concentration of ammonium acetate in mobile phase A is 5 mmol / L.

[0015] In the technical solution of the present invention, the flow rate of the mobile phase is set to 1.8 mL / min to 2.2 mL / min, preferably 2.0 mL / min.

[0016] In the technical solution of this invention, the ultraviolet detection wavelength is 297nm.

[0017] In the technical solution of the present invention, the injection volume is 40~60μL, preferably 50μL.

[0018] As described above, the liquid chromatography-mass spectrometry (LC-MS) method for detecting impurities in vincristine sulfate of the present invention has the following beneficial effects: The present invention provides a novel gradient elution procedure using ammonium acetate and acetonitrile, eliminating the need for diethylamine and phosphoric acid in traditional eluents. This avoids negative impacts on material properties and chromatographic resolution, and provides better separation of vincristine sulfate and various impurities. Furthermore, by combining ultraviolet detection and mass spectrometry, the present invention can separate vincristine sulfate from impurities A, C, D, E, F, G, and H, enabling qualitative and quantitative analysis of each impurity. This allows for accurate assessment of the impurities in the prepared product and precise control of impurity content. Attached Figure Description

[0019] Figure 1 Chromatograms of vincristine sulfate and various impurities are provided in the European Pharmacopoeia Knowledge Database.

[0020] Figure 2 The chromatogram is shown for the test solution in Comparative Example 1.

[0021] Figure 3 This is a UV overlay image of vincristine sulfate and impurities A, C, D, E, F, G, and H in Example 1.

[0022] Figure 4 The image shows the UV spectrum of the impurity in Example 1 (taking impurity A as an example).

[0023] Figure 5 The images show the negative ion mass spectra and matching diagrams of the impurities in Example 1 (taking impurity A as an example).

[0024] Figure 6 The images show the positive ion mass spectra and matching diagrams of the impurities in Example 1 (taking impurity A as an example). Detailed Implementation

[0025] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0026] Comparative Example 1 This comparative example references the chromatographic conditions for related substances in the European Pharmacopoeia, reduces the concentration of diethylamine to 0.2%, and uses formic acid instead of phosphoric acid to adjust the pH value for the detection of vincristine sulfate test sample.

[0027] (1) Chromatographic conditions Column: Merck Lichrospher 100 RP8, 250 × 4.6 mm, 5 μm; Mobile phase A: 0.2% diethylamine (adjusted to pH 7.5 with formic acid); Mobile phase B: Methanol; Gradient elution procedure:

[0028] Flow rate: 2.0 ml / min Column temperature: 30℃ Injection volume: 50µl (2) Detection method Preparation of test solution: Take an appropriate amount of vincristine sulfate test sample, accurately weigh it, dissolve it in water and dilute it quantitatively to prepare a test solution containing approximately 0.5 mg of vincristine sulfate per ml.

[0029] Under the chromatographic conditions described above, measure 50 μl of the test solution, inject it into the liquid chromatography-mass spectrometry (LC-MS) instrument, and record the chromatogram.

[0030] (3) Conclusion: according to Figure 2 The chromatographic results showed that impurities were trapped within the main peak, and the peak shape was poor. The poor peak shape of the impurities indicated that they were not effectively separated from the main peak, and the separation between the impurities was also poor. The inventors adjusted the gradient multiple times (using the same gradient as the one used in this invention), but without significant improvement.

[0031] Example 1 This embodiment uses liquid chromatography-mass spectrometry to detect impurities in vincristine sulfate. At the same time, ultraviolet detectors and mass spectrometers are used to detect impurities A, C, D, E, F, G, and H in vincristine sulfate.

[0032] (1) Chromatographic conditions Column: Welch Ultimate XB-C18, 250 × 4.6 mm, 5 μm Mobile phase A: 5 mmol / L ammonium acetate buffer Mobile phase B: Acetonitrile Gradient elution procedure:

[0033] Flow rate: 2.0 ml / min Column temperature: 30℃ Injection volume: 50µl (2) Detection method Preparation of test solution: Take an appropriate amount of vincristine sulfate test sample, accurately weigh it, dissolve it in water and dilute it quantitatively to prepare a test solution containing approximately 0.5 mg of vincristine sulfate per ml.

[0034] Preparation of impurity solution: Take appropriate amounts of impurities A, C, D, E, F, G, and H respectively, dissolve them in water, and dilute quantitatively to prepare a solution containing approximately 0.1~0.5 mg of impurities per 1 ml.

[0035] According to the chromatographic conditions of this invention, 50 μl each of the test solution and the impurity solution are injected into the liquid chromatography-mass spectrometry (LC-MS) instrument, and the chromatogram is recorded.

[0036] (3) Conclusion according to Figure 3 , 4 The UV spectra show that the peaks of each impurity are well-shaped and well-separated, indicating that the method has good specificity. All impurities can be effectively separated from vincristine sulfate, and vincristine sulfate can be separated from impurities A, C, D, E, F, G, and H for detection and quantification. (Reference) Figure 5 , 6 The mass spectrum of the impurities is clearly visible, and their molecular weight and structure can be accurately identified, so as to achieve the purpose of qualitative and quantitative analysis of impurities in vincristine sulfate and thus achieve the purpose of drug quality control.

[0037] In summary, this invention uses ammonium acetate and acetonitrile as mobile phases, eliminating the need for diethylamine and phosphoric acid in traditional eluents. This avoids negative impacts on material properties and chromatographic resolution, and provides better separation of vincristine sulfate and various impurities with higher resolution. Furthermore, by combining UV detection and mass spectrometry, this invention can separate vincristine sulfate from impurities A, C, D, E, F, G, and H, enabling qualitative and quantitative analysis of each impurity. This allows for accurate assessment of the impurities in the prepared product and precise control of their content. Therefore, this invention effectively overcomes the shortcomings of existing technologies and possesses high industrial applicability.

[0038] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for detecting impurities in vincristine sulfate using liquid chromatography-mass spectrometry, characterized in that, Simultaneously, ultraviolet (UV) and mass spectrometry (MS) detectors were used to detect impurities A, C, D, E, F, G, and H in vincristine sulfate. Ammonium acetate solution was used as mobile phase A, and acetonitrile was used as mobile phase B. Gradient elution was employed with the following gradient ratios: ； The mass spectrometer detector is in scanning mode, simultaneously performing positive and negative ion scans; the cone voltage of the mass spectrometer is 15V, the capillary voltage is 0.8kV, and the probe temperature is 600℃; the chromatographic column used is a Welch Ultimate XB-C18 with an inner diameter of 4.6mm, a length of 250mm, and a packing particle size of 5μm. The concentration of ammonium acetate in mobile phase A is 4 mmol / L to 6 mmol / L.

2. The method for detecting impurities in vincristine sulfate using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The chromatograph used was a Waters ACQUITY Arc high-performance liquid chromatograph, equipped with a 2489 UV / Vis detector and a QDA mass spectrometer detector, which simultaneously performed UV detection and mass spectrometry detection.

3. The method for detecting impurities in vincristine sulfate using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The column temperature was set to 28℃~32℃.

4. The method for detecting impurities in vincristine sulfate using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The flow rate of the mobile phase was set to 1.8 mL / min to 2.2 mL / min.

5. The method for detecting impurities in vincristine sulfate using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The ultraviolet detection wavelength is 297nm.

6. The method for detecting impurities in vincristine sulfate using liquid chromatography-mass spectrometry according to claim 1, characterized in that, The injection volume is 40~60μL.

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