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

Abstract

The invention provides a method for detecting impurities in vincristine sulfate through liquid chromatography-mass spectrometry, and belongs to the technical field of pharmaceutical analysis. According to the method, ammonium acetate is used as a mobile phase A, acetonitrile is used as a mobile phase B, diethylamine and phosphoric acid of a traditional eluent are abandoned, negative effects on material performance and chromatographic resolution are avoided, the effect of separating vincristine sulfate and various impurities is better, and the resolution is higher. Meanwhile, by combining ultraviolet detection and mass spectrum detection, vincristine sulfate and impurities A, C, D, E, F, G and H can be detected separately, and qualitative and quantitative analysis is performed on the impurities, so that the prepared impurities can be evaluated accurately, and the content of the impurities in the product can be controlled accurately.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting impurities in vincristine sulfate by using liquid chromatography-mass spectrometry. Background Art

[0002] Vincristine sulfate is an anti-tumor 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 of 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. The chromatograms of the mixed solutions of vincristine sulfate and various impurities can be found in the Knowledge Database, such as Figure 1 shown.

[0004] Vinblastine is the raw material for the synthesis of vincristine sulfate, and its main source is the Apocynaceae plant Catharanthus roseus. Due to its natural source, more impurities are bound to be introduced during the extraction process of vinblastine, thereby affecting the quality of the target product, vincristine sulfate. In order to achieve the purpose of 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 in order to achieve the qualitative and quantitative purpose 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 whether the obtained impurities are accurate based only on the relative retention time of each impurity in the reference diagram attached to the European Pharmacopoeia Knowledge Database. Therefore, it is crucial to judge the accuracy of the prepared impurities based on the peak conditions of the impurities in the mass spectrum.

[0006] According to the European Pharmacopoeia, the mobile phase used for the detection of vincristine sulfate impurities is a 1.5% diethylamine aqueous solution (adjusted to pH 7.5 with phosphoric acid) and methanol. However, this detection method has major drawbacks: (1) phosphoric acid is a non-volatile acid and cannot be used for mass spectrometry; (2) high concentrations of diethylamine will adversely affect the performance 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 object of the present invention is to provide a method for detecting impurities in vincristine sulfate by liquid chromatography-mass spectrometry 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 the impurities are shown below:

[0009] To achieve the above-mentioned object and other related objects, the present invention provides a method for detecting impurities in vincristine sulfate by liquid chromatography-mass spectrometry, wherein an ultraviolet detector and a mass spectrometer are used to detect impurities A, C, D, E, F, G, and H in vincristine sulfate, and an ammonium acetate solution is used as a mobile phase A, and acetonitrile is used as a mobile phase B. Gradient elution is used, and the gradient ratio is as follows: .

[0011] In the technical solution of the present invention, the chromatograph is a Waters ACQUITY Arc high performance liquid chromatograph, and is equipped with a 2489 UV / Vis detector and a QDA mass spectrometer detector, and simultaneously performs ultraviolet detection and mass spectrometry detection.

[0012] In the technical solution of the present invention, the mass spectrometer detector is in scanning mode, and performs positive and negative ion scanning simultaneously. The cone voltage of the mass spectrometer detector is 15V, the capillary voltage is 0.8kV, and the probe temperature is 600°C.

[0013] In the technical solution of the present invention, the chromatographic column used is Welch Ultimate XB-C18, and its specifications are: inner diameter 4.6 mm, length 250 mm, and filler particle size 5 μm.

[0014] In the technical solution of the present invention, the column temperature of the chromatographic column is set to 28°C~32°C, preferably 30°C.

[0015] 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.

[0016] In the technical solution of the present invention, the flow rate of the mobile phase is set to 1.8 mL / min~2.2 mL / min, preferably 2.0 mL / min.

[0017] In the technical solution of the present invention, the ultraviolet detection wavelength is 297nm.

[0018] In the technical solution of the present invention, the injection volume is 40-60 μL, preferably 50 μL.

[0019] As described above, the method for detecting impurities in vincristine sulfate by liquid chromatography-mass spectrometry of the present invention has the following beneficial effects: the present invention provides a new gradient elution procedure, using ammonium acetate and acetonitrile, abandoning diethylamine and phosphoric acid of the traditional eluent, which will not have a negative impact on material properties and chromatographic resolution, and has a better separation effect on vincristine sulfate and various impurities. At the same time, the present invention can detect vincristine sulfate and impurities A, C, D, E, F, G, and H separately by combining ultraviolet detection and mass spectrometry detection, and perform qualitative and quantitative analysis on each impurity, so as to accurately evaluate each prepared impurity and accurately control the content of impurities in the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the chromatogram of vincristine sulfate and its impurities attached to the European Pharmacopoeia Knowledge Database.

[0021] Figure 2 It is the chromatogram of the test solution in Comparative Example 1.

[0022] Figure 3 It is the UV superposition image of vincristine sulfate and impurities A, C, D, E, F, G, and H in Example 1.

[0023] Figure 4 The UV spectrum of the impurities in Example 1 (taking impurity A as an example).

[0024] Figure 5 The negative ion mass spectrum and matching diagram of the impurities in Example 1 (taking impurity A as an example).

[0025] Figure 6 The positive ion mass spectrum and matching diagram of the impurities in Example 1 (taking impurity A as an example). DETAILED DESCRIPTION

[0026] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0027] Comparative Example 1 This comparative example refers to the chromatographic conditions of the relevant 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, and detects the vincristine sulfate test sample.

[0028] (1) Chromatographic conditions Chromatographic column: Merck Lichrospher 100 RP8, 250 × 4.6 mm, 5 μm; Mobile phase A: 0.2% diethylamine (pH adjusted to 7.5 with formic acid); Mobile phase B: methanol; Gradient elution program:

[0029] Flow rate: 2.0ml / min Column temperature: 30°C Injection volume: 50µl (2) Detection method Preparation of test solution: Take an appropriate amount of vincristine sulfate test sample, weigh it accurately, dissolve it in water and quantitatively dilute it to make a test solution containing about 0.5 mg of vincristine sulfate per 1 ml.

[0030] According to the above chromatographic conditions, 50 μl of the test solution was measured and injected into the LC-MS instrument to record the chromatogram.

[0031] (3) Conclusion: according to Figure 2 The chromatographic results show that the main peak is wrapped with impurities and the peak shape is poor. The impurity peak shape is poor, indicating that the impurities are not effectively separated from the main peak, and the separation between the impurities is also poor. The inventor has adjusted the gradient several times (the same gradient as the present invention is used in the adjustment process), but there is no obvious improvement.

[0032] Example 1 This embodiment adopts a method for detecting impurities in vincristine sulfate by liquid chromatography-mass spectrometry, and simultaneously uses an ultraviolet detector and a mass spectrometer to detect impurities A, C, D, E, F, G, and H in vincristine sulfate.

[0033] (1) Chromatographic conditions Column: Welch Ultimate XB-C18, 250×4.6mm, 5μm Mobile phase A: 5 mmol / L ammonium acetate buffer Mobile phase B: acetonitrile Gradient elution program:

[0034] Flow rate: 2.0ml / min Column temperature: 30°C Injection volume: 50µl (2) Detection method Preparation of test solution: Take an appropriate amount of vincristine sulfate test sample, weigh it accurately, dissolve it in water and quantitatively dilute it to make a test solution containing about 0.5 mg of vincristine sulfate per 1 ml.

[0035] Preparation of impurity solution: Take appropriate amount of impurities A, C, D, E, F, G, and H respectively, dissolve them in water and quantitatively dilute them to make a solution containing about 0.1~0.5 mg of impurities per 1 ml.

[0036] According to the chromatographic conditions of the present invention, 50 μl of the test solution and the impurity solution were measured and injected into the liquid chromatography-mass spectrometer to record the chromatogram.

[0037] (3) Conclusion according to Figure 3 , 4 From the UV spectrum, we can see that the peaks of each impurity are good and the separation is good. It can be seen that the method has good specificity. Each impurity can be effectively separated from vincristine sulfate, and vincristine sulfate and impurities A, C, D, E, F, G, and H can be detected and quantified separately. Figure 5 , 6 The mass spectrum of the impurity is clearly identifiable, and its molecular weight and structure can be accurately confirmed, so as to achieve the qualitative and quantitative purpose of the impurities in vincristine sulfate and achieve the purpose of drug quality control.

[0038] In summary, the present invention adopts ammonium acetate and acetonitrile as mobile phase respectively, abandons the diethylamine and phosphoric acid of traditional eluent, will not have a negative impact on material performance and chromatographic resolution, and is better to the separation effect of vincristine sulfate and each impurity, and has higher resolution. Meanwhile, the present invention can detect vincristine sulfate and impurity A, C, D, E, F, G, H separately by coupling ultraviolet detection and mass spectrometry detection, and each impurity is qualitatively and quantitatively analyzed, so that each prepared impurity can be accurately evaluated, and the content of impurities in the product can be accurately controlled. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has a high industrial utilization value.

[0039] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for detecting impurities in vincristine sulfate by liquid chromatography-mass spectrometry, characterized in that: At the same time, an ultraviolet detector and a mass spectrometer 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, acetonitrile was used as mobile phase B, and gradient elution was used. The gradient ratio was as follows: 。 2. The method for detecting impurities in vincristine sulfate by 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 for simultaneous UV and mass spectrometry detection.

3. The method for detecting impurities in vincristine sulfate by liquid chromatography-mass spectrometry according to claim 2, characterized in that: The mass spectrometer detector is in scanning mode, and performs positive and negative ion scanning simultaneously; the cone voltage of the mass spectrometer detector is 15V, the capillary voltage is 0.8kV, and the probe temperature is 600°C.

4. The method for detecting impurities in vincristine sulfate by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The chromatographic column used was Welch Ultimate XB-C18, with specifications of inner diameter 4.6 mm, length 250 mm, and filler particle size 5 μm.

5. The method for detecting impurities in vincristine sulfate by liquid chromatography-mass spectrometry according to claim 4, characterized in that: The column temperature of the chromatographic column was set at 28°C~32°C.

6. The method for detecting impurities in vincristine sulfate by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The concentration of ammonium acetate in mobile phase A is 4 mmol / L~6 mmol / L.

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

8. The method for detecting impurities in vincristine sulfate by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The UV detection wavelength is 297nm.

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

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