Method for determining medicine or trinitride in intermediate of medicine through derivatization HPLC (high performance liquid chromatography) method

An azide compound and derivatization technology, applied in the field of drug determination, can solve the problems of inability to achieve the detection purpose, insufficient sensitivity, and difficulty in meeting requirements, and achieve the effects of increased ultraviolet absorption intensity, high sensitivity and strong specificity

Inactive Publication Date: 2018-12-11
PRIMELONG LIFE SCI CO LTD
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  • Description
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Problems solved by technology

However, due to the weak ultraviolet absorption of some genotoxic impurities and the low detection limit, there are defects and deficiencies in the direct detection by HPLC method, and the required detection purpose cannot be achieved.
There are also reports in the literature using 3,5-dinitrobenzoyl chloride and DBA(alkyne) as derivatization reagents supplemented by HPLC for the determination of azide [7-8] , but this method often encounters two problems in the process of practical application: 1) the sensitivity is not high enough, and it is difficult to meet the demand when the detection limit of azide is relatively low; 2) 3,5-dinitrate The derivatives formed by benzoyl chloride and azide compounds are sometimes similar in polarity to other components in the sample, and the retention time in HPLC is also relatively close, and the detection results are interfered

Method used

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  • Method for determining medicine or trinitride in intermediate of medicine through derivatization HPLC (high performance liquid chromatography) method
  • Method for determining medicine or trinitride in intermediate of medicine through derivatization HPLC (high performance liquid chromatography) method
  • Method for determining medicine or trinitride in intermediate of medicine through derivatization HPLC (high performance liquid chromatography) method

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0042] Preparation of biphenyl-4-acyl chloride derivatization solution: about 100 mg of biphenyl-4-acyl chloride, accurately weighed, put in a 10 mL measuring bottle, dissolved with pure acetonitrile and diluted to the mark, and shake well.

[0043] Preparation of the test solution: Take an appropriate amount of cefamandole sodium, candesartan or zidovudine, add 100 μL of derivatization reagent, dilute to the mark with acetonitrile, shake well, and react at 25°C for 30 minutes to obtain.

[0044] Preparation of reference substance solution: take about 16 mg of sodium azide reference substance, accurately weigh it, put it in a 10mL volumetric flask, dissolve it in 2mL of water, and dilute to the mark with acetonitrile; after shaking well, accurately draw 50μL into the 10mL volumetric flask , and dilute to the mark with acetonitrile. (5 μg / mL, calculated as Azide).

[0045] Chromatographic conditions: chromatographic column is C18 (5μm, 4.6*250mm), with octadecylsilane bonded s...

Embodiment 1

[0047] In order to ensure the complete derivatization reaction, as represented by candesartan, it is necessary to investigate the amount of derivatization reagent in the system, the reaction time and the reaction temperature. For specific results, see Figure 1-Figure 3 .

[0048] Depend on figure 1 It can be seen that the peak area of ​​the derivatized product increases with the increase of the amount of derivatization reagent, until the amount of biphenyl-4-formyl chloride is 600 μL, the peak area reaches the maximum and remains unchanged. Depend on figure 2 It can be seen that increasing the reaction temperature has little effect on the peak area of ​​the derivatized product, indicating that the derivatization reaction can proceed rapidly at room temperature. Depend on image 3 It can be seen that the reaction time of 30min can complete the derivatization reaction.

Embodiment 2

[0050] Methodology Validation and Application

[0051] Exclusive experiment:

[0052] The derivatization reactions of cefamandole sodium raw material, zidovudine raw material and candesartan raw material were respectively investigated, and the investigation showed that the blank solution did not interfere with the target derivative, and the azide derivative peak and the adjacent peak Resolution ≥ 1.5, indicating that the specificity of the method is good, specific results refer to Figure 4 ~ Figure 6 shown.

[0053] Linearity and range:

[0054] Precisely pipette an appropriate amount of azide stock solution, place it in a 10mL volumetric flask, dilute to the mark with acetonitrile, prepare a series of concentration solutions, perform derivatization according to the above method, filter through a 0.22μm syringe filter, and take 20μL Inject into a liquid chromatograph, and measure the peak area of ​​the derivatized product. Taking the azide concentration (ng / ml) as the abs...

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Abstract

The invention provides a method for determining medicine or trinitride in the intermediate of the medicine through a derivatization HPLC (high performance liquid chromatography) method. The method comprises the following steps: performing derivatization reaction for 5-60 minutes on the trinitride firstly by utilizing a biphenyl acyl chloride derivatization reagent at experimental temperature, andenabling reaction liquid to serve as a sample to perform detection; taking derivation reaction liquid, which is generated above, as a sample, measuring a derivatization product with a wavelength between 220nm and 300nm by utilizing an HPLC-DAD (diode array detector) method so as to realize quantitative determination of the medicine or the trinitride in the synthesized intermediate of the medicine.The method can be used for avoiding matrix interference brought about by the medicine or the synthesized intermediate per se to establish a pre-column derivatization method which is high in sensitivity, high in specificity, simple and universal, then the HPLC method is used for measuring the residue of the medicine or the trinitride in the synthesized intermediate of the medicine, and the sensitivity and specificity of the method for determining the trinitride are improved.

Description

technical field [0001] The invention belongs to the technical field of drug determination, and in particular relates to a derivatization HPLC method for analyzing and detecting azide in drugs or their synthetic intermediates. Background technique [0002] With the gradual improvement of genotoxic impurity regulations, the national drug regulatory department has higher and higher regulatory requirements for genotoxic impurities. In recent years, there have been more and more defects in product marketing applications due to non-compliance with the control of genotoxic impurities. Sufficient research on genotoxic impurities has become the key to the successful marketing of products [1] . Genotoxic impurities may cause DNA mutation, chromosome breakage or DNA recombination, and such impurities may also lead to the occurrence of human tumors [2-4] . At the same time, genotoxic impurities are active and have poor stability. Therefore, it is necessary to establish an analytical...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/027G01N2030/062G01N2030/067
Inventor 王亚农王美玉孙浪武婕
Owner PRIMELONG LIFE SCI CO LTD
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