Research on Impurities in APIs

Research on Impurities in APIs

In an API, any substance that affects the purity of the API is called an impurity. ICHQ3A defines an impurity in a new API as any component that is present in the new API but whose chemical structure is different from that of the new API. In general, impurities are classified into organic impurities, inorganic impurities and residual solvents according to their physical and chemical properties. According to the source, impurities can be divided into: process impurities (reactants and reagents, intermediates, by-products, etc.), degradation products and various impurities introduced from reactants or reagents, etc.; From the perspective of drug toxicity, impurities can be divided into genotoxic impurities and common impurities. Impurity is a key attribute of drug quality and safety, which runs through the whole drug research and development process. Therefore, it is directly related to the quality and safety of marketed drugs to conduct standardized impurity research and control it within a safe and reasonable limit.

Method for Separation of Impurities from Active Substances

  1. The Crystallization
    Crystallization is a method to separate and purify impurities and target compounds by using their solubility in the same solvent or their solubility changing trend with temperature. Due to the convenience of crystallization operation, low requirements for equipment, less investment and high purity of the product, it is the first method to consider in the preparation of laboratory impurities.
  2. Preparative Chromatography
    Chromatography uses different distribution ratios of various substances between stationary and mobile phases to achieve the purpose of separation. Depending on the stationary phase and mobile phase, there are many types of chromatography, such as normal phase chromatography, reverse phase chromatography, ion chromatography, gel chromatography, etc. According to the different operating pressure of the system, it can be divided into low pressure chromatography, medium pressure chromatography, high pressure chromatography, etc. Chromatography for biological macromolecules such as protein, nucleic acid and other complex mixtures of organic matter separation analysis has a very high power, after the selection of suitable packing, the success of the chance has reached more than half. At present, when the separation is difficult and the preparation of a small amount of impurities cannot be obtained by crystallization and other processes, most of them consider the use of medium and high pressure preparation liquid to achieve.
  3. The Precipitation
    The difference in chemical properties between impurity and target compound is used to make the impurity or target compound react with appropriate reagents to form precipitation, which is separated by filtration and other methods. In the development of the separation and extraction process for API, once this method is found, the improvement of the process is often revolutionary, saving a lot of equipment, shortening the process and improving the quality of the product.
  4. High Speed Countercurrent Chromatography
    High-speed-counter-current chromatography (HSCC) is a kind of countercurrent color separation method based on unidirectional fluid dynamic and flat system. Since the composition and ratio of solvent system can be infinite, it can be applied to the separation of samples in any polar range in theory, and has its unique feature in the separation of natural compounds.

Identification of Drug Impurities

  1. UV Identification (HPLC-UV)
    Most of the drug impurities can be quantitatively analyzed by the ultraviolet identification (HOLC-UV) technique, but the accurate quantification of very and trace impurities cannot be achieved due to the low sensitivity of the ultraviolet detection. However, mass spectrometry technology has the advantages of high sensitivity and high resolution, and has been rapidly developed in recent years due to its excellent quantitative and qualitative analysis ability. At the same time, reducing sample consumption is also one of the driving forces for the development of NMR technology.
  2. Mass Spectrometry (MS)
    • Quantitative Analysis
      Mass spectrometry can be used as an alternative method for the quantification of UV-insensitive impurities. At the same time, because of its high detection sensitivity, it can accurately determine trace impurities that cannot be quantified by UV single wavelength detection. However, in the determination of some special impurities, it is still necessary to obtain the mass spectral response by derivatization or adding alkali metal ions in the mobile phase due to the weak ionization ability.
    • Structure Identification
      It takes a long time to identify the structure of impurity monomer. Therefore, liquid mass spectrometry can be used to identify the structure of impurity quickly in the impurity spectrum analysis of drugs. In this method, the molecular ion peak determined by the primary mass spectrometry is used for the secondary mass spectrometry fragmentation analysis.
  3. Nuclear Magnetic Resonance (NMR)
    The qualitative and quantitative application of NMR technology in impurities mainly depends on the acquisition of impurity monomers. In addition, quantitative NMR can be used for the standardization of impurity reference materials in the establishment of quality standards for special impurities. NMR technology is also a quality-related detection technology. Using NMR technology to calculate correction factors and then calibrate other detectors, the reaction process can be monitored. The sensitivity of NMR is dependent on the performance of the probe.
    Therefore, in order to improve the sensitivity of NMR detection, some researchers invented a cooling probe, which can detect compounds with only microgram level of compound monomer, which makes the sample consumption of NMR detection realize a leap from milligram to microgram. Simultaneous on-line liquid phase-nuclear magnetic resonance (LC-NMR) technology can also achieve rapid structure identification of drug impurities.
  4. New Analytical Techniques
    With the need of rapid impurity analysis and accurate structure identification, some methods have been used for impurity structure identification, such as direct determination of drug impurities, establishment of database of similar impurity fragments by molecular imprinting (MIP) and single crystal X-ray diffraction.

Control of Impurities in Active Substances

Related laws and regulations related to impurity control of commonly used apis:

  1. ICH Q3A: Guidelines for Impurity Research in new apis
  2. ICH Q3B: Guidelines for the study of impurities in new drug preparations
  3. ICH Q3C: Guidelines for residual solvent research
  4. ICH Q3D: Guiding Principles for elemental impurity research
  5. ICH M7: Guidelines for the study of genotoxic impurities
  6. 2020 edition of Chinese Pharmacopoeia IV 9102 Guidelines for drug impurity analysis

Impurity Classification Control Strategy

  1. Starting Material and Introducing Impurities
    In API impurity study, need to focus on the potential of part of the reaction can be introduced impurities, because such impurities have similar structure and starting material, may enter the subsequent reaction with the starting material, and the physical and chemical properties of this kind of potential impurities and starting material is relatively close, in the subsequent process steps on its ability to remove co., LTD. The starting material itself is also a process impurity in the finished product, so it is particularly important to control and regulate the impurities that need to be controlled at the source.
    Based on the concept of "source-FIRST" impurity research, strict supplier screening and audit should be carried out. Obtain the production process of the supplier's starting material, analyze the key quality attributes of the starting material based on the process characteristics, especially whether the starting material and its impurities are removed or transformed in the subsequent process, so as to establish the internal control quality standard of the starting material.
  2. Intermediates and By-products
    For impurities such as intermediates and by-products, the concept of process control should be fully reflected. In the study of API, process control should be carried out at each reaction step to ensure the quality of finished products by controlling the purity of intermediates and the conversion rate of reactants at a reasonable limit level. For the by-products that may be produced in each step, it is necessary to track the whereabouts of impurities in the subsequent process, and according to the accumulated data of multiple batches, reasonable development of in-process control and intermediate standards for each step of the reaction.
  3. Degradation of Impurities
    Starting materials, intermediates and final products can produce degradation impurities if stored improperly. We can design different strong degradation tests according to the stability test guidelines, so that a large amount of information about degradation impurities can be obtained in a short time. ICH Q1A clearly points out the content of strong degradation test, including high temperature, oxidation, acid-base destruction, light and other destruction methods. The test sample can be destroyed in the solid state of API itself or in the way of solution. In general, suitable strong degradation conditions were selected through different test conditions, and the degradation of principal components was about 10%. It is not necessary to degrade compounds that are stable under severe test conditions.
  4. Organic Reagents and Their Conversion
    A large number of organic reagents will be used in the research of APIs. In the process of research, the whereabouts of these organic reagents and their transformation products should be strictly tracked and analyzed. Data should be accumulated in multiple batches and reasonable internal control standards should be formulated.


Impurity is the key and difficult point in the quality research process of active drug. Whether it is a new drug or a generic drug, impurity research runs through the whole research and development. Our study on impurities should be based on the actual process route, refer to the pharmacopoeia and literature of various countries, and reasonably and fully study all kinds of impurities that affect the quality of API, so as to ensure the source control, process control and end point control, and each step has a targeted target, so as to realize the safety and reliability of drugs.

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