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Method using derivatization HPLC-DAD method to determine small-molecule halogenated carboxylic acid in medicine

A halogenated carboxylic acid and small molecule technology, applied in the field of drug analysis and detection, can solve the problems of inability to suppress the dechlorination reaction, high baseline noise, and unreported system comparison.

Active Publication Date: 2016-07-13
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

The main drawback of this method is: on the one hand, high temperature is easy to produce degradation impurities [37] ; On the other hand, the detection baseline noise at low wavelength is large and the interference is serious [38]
2-Nitrophenylhydrazine (2-NPH) [38-43] , 3-nitrophenylhydrazine (3-NPH) [44] , 4-nitrophenylhydrazine (4-NPH) [45] and 2,4-dinitrophenylhydrazine (2,4-DNPH) [43] It is a commonly used commercial nitrophenylhydrazine derivatization reagent reported at present, but there is no report on the systematic comparison of such reagents
Although the probability of chlorination reaction can be reduced by reducing the amount of hydrochloride, it cannot prevent the occurrence of dechlorination reaction
In addition, the current commercial reagents all contain hydrochloride, and the solubility of EDC and 2-NPH without hydrochloride is poor. Taking into account, it is impossible to effectively improve the stability of derivatives only by reducing the concentration of activators and derivatization reagents. sex

Method used

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  • Method using derivatization HPLC-DAD method to determine small-molecule halogenated carboxylic acid in medicine
  • Method using derivatization HPLC-DAD method to determine small-molecule halogenated carboxylic acid in medicine
  • Method using derivatization HPLC-DAD method to determine small-molecule halogenated carboxylic acid in medicine

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Experimental program
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Effect test

preparation example Construction

[0042] 1.3. Solution preparation

[0043] Each halogenated carboxylic acid stock solution (hereinafter referred to as HCA): take an appropriate amount of HCA, accurately weigh it, place it in a 10mL measuring bottle, dilute to the mark with 70% acetonitrile, and shake well. Each stock solution has good stability within 8 hours at room temperature.

[0044] 2-nitrophenylhydrazine hydrochloride, 3-nitrophenylhydrazine hydrochloride, 4-nitrophenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine test solution (hereinafter referred to as 2-NPH·HCl, 3-NPH·HCl, 4-NPH·HCl and DNPH): Take about 100 mg of each reagent, weigh it accurately, place it in a 10 mL measuring bottle, dilute to the mark with 80% acetonitrile, and shake well. Must be freshly prepared.

[0045] 1-Ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution (hereinafter referred to as EDC·HCl): take 1-ethyl-(3-dimethylaminopropyl) carbodiimide 200mg of amine hydrochloride, accurately weighed, placed ...

Embodiment 1

[0049] Taking chloroacetic acid as a representative, it was derivatized with four kinds of nitrophenylhydrazine derivatization reagents at the same concentration. See 1.4 for derivatization experimental conditions. Under the same chromatographic conditions, use DAD to scan each derivatized product, record the spectrum and chromatogram of the product, and evaluate the pros and cons of the derivatized reagent with the maximum absorption wavelength and absorption intensity of the product.

[0050] figure 1It shows that except for 3-nitrophenylhydrazine, the ultraviolet absorption bands of the products generated by the other three derivatization reagents and chloroacetic acid all present a certain red-shift effect, and the maximum absorption wavelengths are 392nm (2-nitrophenylhydrazine) and 356nm ( 4-nitrophenylhydrazine) and 337nm (2,4-dinitrophenylhydrazine). The ideal situation in this experiment is to measure in the near visible light region (400nm), which can minimize back...

Embodiment 2

[0053] Preparation of the test solution: Accurately weigh an appropriate amount of the drug, place it in a 5mL volumetric bottle, add 500 μL of derivatization reagent and 500 μL of DC·HCl solution, add 70% acetonitrile to dilute to the mark, vortex for 10 seconds, shake well, and react in the dark for 2 hours , Shake well to obtain the test solution. After filtration, 20 μL was taken for direct injection.

[0054] Preparation of reference solution: Precisely pipette 100 μL of stock solutions of halogenated carboxylic acids with different concentrations, place them in a 5 mL volumetric flask, add 500 μL of derivatization reagent and 500 μL of DC·HCl solution, add 70% acetonitrile to dilute to the mark, vortex for 10 seconds, shake Evenly, avoid light and react for 2h, shake well to obtain the reference substance solution. After filtration, 20 μL was taken for direct injection.

[0055] Chromatographic conditions: Shimadzu LC20AT liquid chromatograph (including online vacuum d...

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Abstract

The invention discloses a method using a derivatization HPLC-DAD method to determine small-molecule halogenated carboxylic acid in medicine.The method includes: under normal temperature, using nitrobenzene hydrazine derivatization reagent to derivatize the small-molecule halogenated carboxylic acid so as to generate products with high absorption in the ultraviolet visible region; using the reaction liquid after the derivatization reaction as the feeding sample, and using the HPLC-DAD method to determine the derivatization products of halogenated carboxylic acid in the feeding sample in the ultraviolet visible region on the basis of the reversed phase partition chromatography principle so as to achieve the qualitative or quantitative detection of the small-molecule halogenated carboxylic acid.Due to the fact that the ultraviolet absorption band of the carboxylic acid derivatization products of the nitrobenzene hydrazine derivatization reagent has evident redshift effect due to the existence of nitro electron-withdrawing group on benzene ring and most medicine and impurities thereof are weak in absorption in the ultraviolet visible region (300-450 nanometers), the simple and universal method using pre-column derivatization HPLC-DAD to determine the small-molecule halogenated carboxylic acid is built.Methodology validation shows that the method is good in specificity.

Description

technical field [0001] The invention belongs to the field of drug analysis and detection, and relates to a method for determining small-molecule halogenated carboxylic acids by a derivatization HPLC-DAD method, in particular to a method for determining small-molecule halogenated carboxylic acids in medicines by a derivatization HPLC-DAD method. Background technique [0002] Small molecule halogenated carboxylic acids (Halogenatedcarboxylic acids, HCAs), as the main disinfection by-products in the water environment, due to their potential genotoxicity and carcinogenicity [1,2] , has long been subject to many environmental regulators including the U.S. Environmental Protection Agency (USEPA) [3] , World Health Organization (WHO) [4] etc., and is listed as one of the routine monitoring indicators for water quality research. In the pharmaceutical industry, these compounds have high reactivity and special physiological activity (introduced by halogen elements) [5] , often used...

Claims

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Application Information

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IPC IPC(8): G01N30/89
CPCG01N30/89
Inventor 郑枫柳文媛侯德胜冯锋
Owner CHINA PHARM UNIV
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