Detailed Explanation of Drug Impurity Research Methods

The impurity analysis in medicine is a very important part. The analysis of the important role of impurity source analysis focused on the development of analytical technology in the process of impurity research, especially the development of mass spectrometry in structure identification. At the same time, According to the guiding principles of foreign toxic impurities research, the method of impurity toxicity research is clarified. In recent years, with the continuous deepening of drug research and the continuous improvement of impurity research requirements, the analysis techniques and research methods of impurities are undergoing important changes. When establishing an analytical method for impurities, a clear impurity research process is the basis for method establishment, and the selection of appropriate analytical techniques is also crucial.

Part 1 Source Analysis Of Impurities

Impurities in drugs may come from various links such as drug production and sales. According to the ICH guidelines, pharmaceutical impurities can be divided into organic impurities, inorganic impurities, residual solvents and other impurities. This paper mainly discusses the organic impurities.

The concept of “Quality by Design (QbD)” is introduced into the research on drug impurities, which can preliminarily outline the impurities of the product according to the synthesis mechanism of the specific process, the basic structure of the starting material and each intermediate before drug production. mass spectrum. The analysis of impurity sources is the basis for formulating drug impurity control strategies, especially when analyzing the sources of toxic impurities, reagents, intermediates, and by-products in all synthesis and production processes should be analyzed, potential impurities that may be produced, and actual existing impurities should be analyzed. Impurities. After the synthesis of the raw material drug is completed, although the active compound of the drug does not contain the “alerting structure” (alerting structure) after toxicity analysis, the genotoxicity of the compound containing the alerting structure still needs to be considered during the production process.

Part 2 Research Methods Of Impurities

In the process of drug development, the analysis of drug impurities is the key. Therefore, clear impurity structure research ideas and appropriate impurity analysis techniques in impurity research can greatly shorten the time for impurity research and promote the rapid development of drug research.

1 Impurity Pre-treatment Technology

The pretreatment of impurities is accompanied by the pretreatment of pharmaceutical active ingredients. However, the content of impurities in drugs is low and their structures are quite different from the main components. Therefore, the pretreatment and detection methods of conventional pharmaceutical active ingredients (such as initial mobile phase dissolution HPLC-UV analysis directly afterwards) is not necessarily applicable to drug impurities, and different pretreatment techniques should be selected for different samples.

(1) Samples With Low Detection Sensitivity

For samples with low detection sensitivity, derivatization pretreatment methods are usually used, such as introducing chromophores to generate UV responses, or adding easily ionizable groups to increase ionization efficiency, etc. Although conventional derivatization methods can meet the needs of daily detection, in order to achieve rapid screening and quantification of low-concentration genetic impurities, traditional derivatization reagents can be changed to increase their specificity and sensitivity, and gas-solid Derivatization to make up for the lack of liquid-solid derivatization.

(2) Low concentration of impurities

The choice of pretreatment method for low-concentration impurities is determined by the type of impurities. For example, the concentration of degradation products is increased by forced degradation, etc., but conventional degradation methods often introduce other impurities, which will interfere with the impurity profile of special impurities. , in order to obtain a single impurity research mechanism, Ueya-ma et al. proposed a new oxidative degradation platform for solid drugs, which excludes hydrolysis, solvolysis or thermal effects caused by common oxidation methods (such as H2O2-dominated), and can be used for oxidation Specific studies of degradation mechanisms.

(3) Samples Prone To Contaminating Instruments

Different instruments have different conditions of use, so the pretreatment of complex samples can prolong the service life of the instrument. For example, the mass spectrometry detector cannot use mobile phases containing non-volatile salts, so two The two-dimensional liquid chromatography technology separates the peaks in the first dimension and retains the sample in the sample loop, and the second-dimensional liquid phase uses a mobile phase acceptable to mass spectrometry and a desalting column to elute the sample in the sample loop to achieve Analytes are “desalted” to preserve the mass spectrum.

2 Impurity Separation Technology

The content of impurities in the drug is low, and the qualitative and quantitative analysis of impurities cannot be achieved by direct determination. Therefore, impurities should be separated to obtain a single component of impurities, so as to realize the detection of impurities. In recent years, liquid chromatography technology and supercritical fluid technology have developed rapidly.

• Liquid Chromatography Technology

High Performance Liquid Chromatography (HPLC) HPLC is still the most traditional method used in the separation of impurities. The vast majority of compounds can be separated and analyzed by various chromatographic column technologies and combined technologies. In order to solve the problem of low sensitivity in combination with ultraviolet detectors, the new two-dimensional high-performance liquid chromatography (2D-HPLC) utilizes the characteristics of separation and enrichment of liquid chromatography technology to improve the detection ability of low-concentration impurities. At present, HPLC consumes a large amount of organic solvent no matter whether it adopts normal phase elution or reverse phase elution, which brings great pollution to the environment. Phase chromatography techniques are being extensively researched. Ultra-High Performance Liquid Chromatography (UHPLC) In order to meet the needs of drug research and development, UHPLC, as a rapid separation chromatography technology, has appeared in the development of various drugs. Dong et al. discussed how to establish a stable UHPLC method for different samples according to the UHPLC usage method. In terms of chiral separation, UHPLC currently does not have a large number of commercial chiral chromatographic columns with a particle size of less than 2 μm, but chiral impurities can also be separated by using the traditional chiral mobile phase addition method. In terms of laboratory construction, HPLC is still the main research instrument in analytical laboratories, but in order to obtain separation performance similar to UHPLC, many researchers have achieved rapid separation by optimizing the HPLC system and using core-shell chromatographic columns.

• Supercritical Fluid Chromatography (SFC)

The SFC technology with liquid CO2 as the main mobile phase has been developing very slowly due to its low detection sensitivity when combined with ultraviolet light. However, the popularity of mass spectrometry detectors (MS) and the needs of an environmentally friendly society enable SFC to be used for separation chiral impurities. Although the use of SFC and mass spectrometry for impurity analysis has been reported, commercial SFC-MS instruments have not appeared in large numbers.

3 Impurity Preparation And Purification Technology

In order to achieve the quantitative and qualitative analysis of impurities, it is necessary to obtain high-purity single-component impurities. However, the content of impurities in drugs is low. It takes a lot of time to prepare impurities by analytical liquid chromatography. Using preparative liquid chromatography, etc. The method can increase the speed of obtaining high-purity impurities and accelerate the research work of impurities.

(1) Preparative Liquid Chromatography Technology And Preparative SFC Technology

Purified impurities can obtain higher-quality spectra, but the problem of low impurity content in drugs has always restricted the acquisition speed of impurity monomers. The source and simple structure of the impurity can be obtained by liquid chromatography-mass spectrometry, and then the impurity monomer can be prepared by forced degradation, crystallization mother liquor or direct synthesis. In order to overcome the shortcomings of conventional one-dimensional preparative liquid chromatography and preparative SFC chromatography, such as difficulty in establishing methods and long preparation time, a preparative two-dimensional liquid chromatography (Prep 2D-LC) and two-dimensional SFC chromatography were proposed. Technology has been increasingly used.

The new Prep 2D-LC firstly separates the sample through one-dimensional liquid chromatography, and uses mass spectrometry to monitor the relative molecular mass and heart cutting to save the target in the sample loop (Sample loop), and then use the column elution ( At column dilution) the sample in the sample loop is injected into the second-dimensional liquid chromatography, and the second-dimensional liquid chromatography is further separated by using the same or different mobile phase samples as the first dimension and using mass spectrometry monitoring method This new method not only replaces one-dimensional preparative liquid chromatography, but also solves the problems of high pressure and peak broadening in traditional preparative 2D-LC systems. Similar to 2D-LC, the research group also proposed 2D-SFC preparation technology for the screening of chiral and achiral compounds.

(2) Other Preparation Techniques

The content of impurities in the drug is low, and the method of liquid-solid adsorption separation will cause the loss of impurities and prolong the preparation time. Counter-current chromatography (counter-current chromatography-phy, CCC) minimizes the adsorption of impurities through liquid-liquid extraction, and the sample reproducibility can reach 100%, and it is reported that the preparative liquid chromatography technology is compared with CCC, and the results show that CCC The loading capacity and high-throughput capability of samples with low solubility are superior to that of preparative liquid chromatography. Centrifugal partition chromatography (CPC), which is similar to CCC, uses hydrostatic distribution to achieve the purification of drugs, but CPC will have problems with stationary phase loss and unstable flow pulses during use. Amarouche et al. introduced The co-current elution method solved the above problems and successfully purified insoluble cyclosporin A. In order to achieve rapid preparation and purification, flash chromatography (FC) which accelerates liquid-solid separation by air pressure is also a method for preparing compounds in large quantities.

4 Impurity Detection Technology

Although HPLC-UV technology can quantify most pharmaceutical impurities, it cannot accurately quantify extremely trace impurities due to the low sensitivity of UV detection. However, mass spectrometry 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 capabilities. At the same time, reducing sample consumption is also one of the driving forces for the continuous development of NMR technology.

(1) Mass Spectrometry (MS)

Quantitative analysis: Mass spectrometry can be used as an alternative quantitative method for impurities that do not respond to ultraviolet light. At the same time, because of its high detection sensitivity, it can accurately determine trace impurities that cannot be quantified by single-wavelength ultraviolet detection. However, in the determination of some special impurities, due to the weak ionization ability, methods such as derivatization or adding alkali metal ions to the mobile phase are still required to obtain mass spectral responses. Conventional MS detectors have low quantitative accuracy for trace impurities due to the limitation of resolution, while high-resolution mass spectrometry (HRMS) can reduce the relative error of the charge-to-mass ratio (m/z) detection of analytes by 1×10 -6~2×10-6, which improves the quantitative accuracy of trace analytes in selected ion scanning quantification. Another advantage of HRMS high resolution is that mass distinction is more accurate, which can be used to distinguish various compounds with similar relative molecular masses. Using this advantage in combination with UHPLC can realize the separation and quantification of various impurities in a short time.

Structural identification: It takes a long time to identify the structure of impurity monomers, so the method of liquid chromatography-mass spectrometry can be used in the impurity profile analysis of drugs to quickly identify the impurity structure. In this method, the molecular ion peak determined by the first-order mass spectrometry is used for the fragmentation analysis of the second-order mass spectrometry. However, the insufficient resolution of mass spectrometry often makes the determination of the m/z of the parent ion inaccurate, resulting in unclear elemental composition of the impurity. At the same time, the insufficient information of the secondary fragments also hinders the further analysis of the structure of the impurity. Therefore, techniques such as HRMS, multi-stage mass spectrometry (MS n ) and hydrogen/deuterium (H/D) exchange can accurately analyze the structure of impurities with their respective advantages.

The improvement of mass spectrometry resolution increases the accuracy of relative molecular mass information and can accurately predict elemental composition. At the same time, the scores of different molecular formulas can be calculated by using the built-in software of mass spectrometry or other calculation software. The improvement of mass spectrometry resolution also has the function of distinguishing different isotopes. For example, for molecules with a relative molecular mass of about 500, only when the resolution reaches 400,000 can the two isotopic peaks of 34S and 37Cl in the mass spectrogram be separated.

(2) Nuclear Magnetic Resonance (NMR)

The application of NMR technology in the qualitative and quantitative application of impurities mainly depends on the acquisition of impurity monomers. In addition, the standardization of impurity reference substances can be carried out by quantitative NMR when the quality standards of special impurities are established. NMR technology is also a mass-related detection technology. Using NMR technology to calculate the correction factor and then calibrate other detectors can realize the monitoring of the reaction process. The detection sensitivity of NMR technology depends on the performance of the probe. Therefore, in order to improve the sensitivity of NMR detection, some researchers have invented cryogenic probes, which can detect compounds only in micrograms of compound monomers, which makes the consumption of NMR detection samples from milligrams to micrograms leap. Simultaneously on-line liquid phase-nuclear magnetic (LC-NMR) technology can also realize rapid structure identification of drug impurities.

5 New Analytical Techniques

With the need for rapid impurity analysis and structural identification accuracy, some methods are also used for the structural identification of impurities, such as the direct determination of pharmaceutical impurities, the establishment of similar impurity fragment databases by molecular imprinting (MIP), and single crystal X-ray diffraction techniques. Wait.

Part 3 Analysis of Genotoxic Impurities

Genotoxic impurities can directly act on DNA in the human body, causing DNA damage and having carcinogenic, teratogenic or mutagenic properties. A small amount of genotoxic impurities can also cause great damage to the human body, and the research on genotoxic impurities has attracted widespread attention of pharmaceutical researchers.

1 Quantitative Analysis

Due to the extreme danger of genotoxic impurities, the US FDA and the European Medicines Agency (EMA) have stipulated a toxicological concern threshold (TTC): when a person takes long-term medication, the daily intake of potentially toxic impurities should not exceed 1.5 μg. According to the above regulations, if the daily dosage of the drug is 200mg, the mass fraction of toxic impurities should not exceed 7.5-×10-6. Therefore, the quantitative analysis of genotoxic impurities requires a trace analysis method. Commonly used ultraviolet detectors can detect impurities with a mass fraction of 5×10-4 at least, but with the invention of 2D-HPLC and the use of high-sensitivity ultraviolet detectors, trace impurities in drugs are separated by one-dimensional liquid chromatography, Enrichment by solid-phase extraction technology and high-sensitivity detection by two-dimensional liquid chromatography can also achieve accurate quantification.

However, this method requires multiple high-concentration injections and is not suitable for impurity determination. Therefore, mass spectrometry detection technology is an important prerequisite for the establishment of quantitative analysis methods for genotoxic impurities. Kakasaheb et al. used the GC-MS method to quantitatively determine the genotoxic impurities in the starting materials for the synthesis of candesartan. The method verification results can meet the requirements of ICH regulations. This method is suitable for the content of genotoxic impurities in marketed drugs Determination. In the process of establishing a method for trace impurities, the pretreatment method of the sample is very important, and the loss of a small amount may have a great impact on the results. Devenport et al. innovatively used mass spectrometry under atmospheric pressure to directly quantify the genotoxicity in simulated drugs. Impurities, this method makes sample determination more convenient and enables high-throughput detection of drugs.

2 Determination Of Toxic Impurities

In 2006, EMA released the guidelines for genotoxicity limits, and since then ICH, FDA, and my country’s Drug Evaluation Center have also proposed research and control programs for genotoxic impurities. According to the relevant guidelines, even if the content of the impurity is below the specified quantitative requirement, the toxicity assessment of the impurity is required. The toxicity evaluation of compounds is achieved through in vitro bacterial toxicity experiments, and the guidelines recommend the use of impurity monomers for research. However, toxic impurities in drugs are present in trace or trace amounts, and it takes a long time and high cost to obtain impurity monomers through preparation and other methods. To this end, the FDA and other companies have developed computer software to assess the toxicity of compounds. This software does not need to obtain the monomer. After the structure of the impurity is identified, the toxicity assessment software can be used to evaluate whether there is a warning structure in the structure of the impurity. In terms of software, FDA recommends using MC4PC, MDL-QSAR and Derk for Window for evaluation.

In order to evaluate the toxicity accurately, each software has a special algorithm and scope of application: MC4PC software splits the structure to be evaluated into structural fragments of 2~3 (non-hydrogen) atoms, and then combines these fragments with the existing Compared with the fragment database of known toxic compounds, the cumulative toxicity score of the fragments in the impurity structure is made and the molecular characteristics of the structure are described, and a comprehensive and professional report is finally generated to evaluate the structural toxicity of the impurity. Although MC4PC can evaluate the toxicity of drugs from the perspective of the fine structure of impurities, it can only search the same fragments in the existing database for comparison, and cannot comprehensively evaluate the structure of more than one unknown fragment. The research results of toxic compounds show that there are electrophilic groups or structures that can be activated as electrophilic groups in the structure of toxic compounds. The MDL-QSAR toxicity assessment software is based on the correlation between the toxicity of the compound and the electrophilic ability of the compound. To make an assessment, the software calculates the quantitative relationship between a compound’s structure and its toxicity, and predicts the results of in vitro bacterial toxicity experiments.

However, in order to achieve the most accurate assessment of the toxicity of impurities with known structures, it is necessary to find the most suitable model for calculation when using MDL-QSAR software. report on their assessment. Although some impurities are evaluated by computer as potentially toxic impurities with warning structures, the evaluation software will over-estimate the toxicity of impurities during the calculation process, so in vitro bacterial reverse mutation experiments are still required to determine whether drug impurities are toxic impurities (Ames Experiment) or mammalian cell analysis, the FDA gives detailed instructions on the process of the experiment.

3 Control Method

According to the EMA guidelines and the answers to the questions about the guidelines, all compounds present in the drug should pass the compound toxicity assessment or control methods that meet the guidelines, and Ames experiments are required to determine whether potential toxic impurities are toxic impurities.

Part 4 Outlook

Impurity research occupies an extremely important position in the drug development process, which not only affects the speed of new drugs being launched, but also affects the safety of human medication. As analytical techniques continue to evolve, strategies for impurity studies have become increasingly diverse. With the improvement of detector resolution and sensitivity, the quantification of impurities has achieved a leap from trace to trace, and the qualitative work of impurities has also realized the transformation from offline to online detection. However, the accelerated development of new drugs, the continuous emergence of biological drugs and the requirements for rapid qualitative and quantitative analysis will bring great challenges to researchers.