Drug impurities, as a key factor affecting drug safety and efficacy, play a vital role in drug research and quality control. Currently, drug impurities have become a key focus for drug regulatory agencies both domestically and internationally.
When detecting trace impurities in drugs, the primary concern is selecting a highly specific method so that the drug itself does not interfere with the detection of trace impurities. Therefore, drug impurity testing is primarily based on differences in the physical or chemical properties of the drug and the impurity.
Differences in physical properties between drugs and impurities primarily refer to differences in their appearance, distribution, adsorption, and light absorption. Differences in chemical properties primarily refer to differences in chemical reactions between the drug and impurity. Generally, impurities react with reagents while the drug does not. Depending on the impurity control requirements, either quantitative testing or quantitative determination of the impurities can be performed.
Methods for detecting impurities in pharmaceuticals include chemical, spectroscopic, and chromatographic methods, with different methods used depending on the drug structure and impurities. Common methods for impurity testing include chemical, chromatographic, spectroscopic, and physical methods. Chromatographic methods, particularly HPLC, are often used for detecting organic impurities.
I. Chemical Methods
When the chemical properties of an impurity differ significantly from those of the drug, appropriate reagents can be selected to react with the impurity, producing color, precipitate, or gas, thereby verifying the impurity limit. In addition to semi-quantitative testing for impurities using chemical testing methods, quantitative determination of impurities can also be achieved using titration and gravimetric methods.
1. Color Reaction Test Method
When impurities react with reagents to produce color, colorimetry is used to control the impurity limit, typically visually.
2. Precipitation Reaction Test Method
When impurities react with reagents to produce a precipitate, turbidimetry is used to control the impurity limit, allowing gravimetric determination of the impurity amount.
3. Gas Generation Test Method
When impurities react with reagents to produce gas, the corresponding gas test method is used to control the impurity limit.
4. Titration Method
The titrant reacts only with the impurity. By titrating the impurity in the drug with a titrant of a certain concentration, the impurity content can be quantitatively determined.
II. Chromatographic Methods
Organic impurities in pharmaceuticals may be known or unknown, volatile or non-volatile, and their structures and properties are often similar to those of the drug. If the drug and impurity react identically or similarly with certain reagents, or if their spectral characteristics are similar, chemical and spectroscopic methods can be difficult to detect the impurities.
Chromatography is widely used to detect impurities in pharmaceuticals because it can effectively separate and detect impurities from the drug by exploiting the differences in the chromatographic properties of the drug and impurities.
Related substances in pharmaceuticals include starting materials, intermediates, by-products, isomers, aggregates, and degradation products. Their chemical structures are often similar to or related to the drug. Chromatography is the preferred method for detecting related substances.
1. Thin-layer Chromatography
TLC is used by many national pharmacopoeias to detect impurities in drugs. It has the advantages of simple equipment, easy operation, fast separation speed, high sensitivity and resolution.
Commonly used methods include: the impurity reference method, the self-dilution control method of the test solution, a combination of these two methods, and the reference drug method. The number and limit of impurities should be specified in the quality specification.
Impurity Reference Standard Method: Applicable when impurities are known and an impurity reference standard can be prepared.
Method: Based on the impurity limit, sample the test sample solution and a certain concentration of the impurity reference standard solution. Spot samples are then applied to the same thin-layer plate, developed, and the spots located. Spots other than the main spot of the test sample solution are compared with the corresponding main spot of the impurity reference standard solution or a concentration series of impurity reference standard solutions. This method determines whether the drug meets the impurity limit.
2. High-Performance Liquid Chromatography
HPLC offers high separation efficiency, strong specificity, and excellent detection sensitivity, allowing accurate determination of the peak area of each component. Its application in impurity testing is increasing. For drugs whose content is determined using HPLC, the same chromatographic conditions can be used for impurity testing.
There are four methods for detecting impurities: the external standard method (impurity reference standard method), the principal component self-reference method with a correction factor, the principal component self-reference method without a correction factor, and the area normalization method.
3. Gas Chromatography
Gas chromatography is used to determine volatile special impurities in pharmaceuticals, particularly residual solvents. Pharmacopoeias worldwide mandate the use of gas chromatography.
Method: In addition to the same impurity testing methods as HPLC, there is also the "standard solution addition method." A predetermined amount of impurity reference solution is precisely added to the test solution. The impurity content is determined using either the external or internal standard method. The added reference solution is then subtracted to obtain the impurity content in the test solution.
4. Capillary Electrophoresis
Capillary electrophoresis can be used to test for impurities in pharmaceuticals such as peptides and enzymes. The testing method is the same as for HPLC.
III. Spectroscopic Methods
Spectroscopic methods use the differences in the selective absorption of light between a drug and its impurities to perform limit tests for impurities.
1. UV-Vis Spectrophotometry
This method utilizes the differences in UV-Vis absorption characteristics between the drug and impurities for detection. If the drug does not absorb at the impurity's maximum absorption wavelength, the absorbance of the sample solution can be measured at this wavelength. By controlling the sample solution's absorbance or transmittance, the impurity content can be controlled. Alternatively, the impurity's content can be determined in the visible light region by utilizing the color reaction between the impurity and the reagent.
2. Infrared Spectrophotometry
For certain polymorphic drugs, due to different crystalline states, the bond lengths and bond angles of some chemical intermolecular non-covalent bonds vary to varying degrees, resulting in characteristic differences in the frequency, peak shape, and intensity of certain characteristic peaks in the infrared absorption spectrum. Using infrared spectrophotometry to quantitatively measure these differences, specific crystalline impurities (those that are ineffective, ineffective, or affect quality and stability) can be detected in the drug. The method is simple and the results are reliable.
3. Atomic Absorption Spectrophotometry
Atomic absorption spectrophotometry is a highly sensitive method widely used for the analysis of trace metal elements. In impurity testing, it is primarily used to detect heavy metal impurities in pharmaceuticals.
The standard addition method is typically used to control the limits of heavy metal impurities: Prepare a test solution of the test sample according to the regulations for each product. Take an equal amount of the test sample and add a solution of the element to be measured at the limit to create a control solution. Let the reading of the control solution be a and the reading of the test solution be b. The value b should be less than (a, b). For example, consider the test for iron and copper salts in vitamin C.
IV. Other Methods
Thermal analysis is a method that precisely records the relationship between the physical and chemical properties of a substance and temperature under programmed temperature conditions. It studies physical changes such as crystal transformation, melting, evaporation, and dehydration, as well as chemical changes such as thermal decomposition, oxidation, and reduction, as well as the accompanying changes in temperature, energy, or weight. Common thermal analysis methods include thermogravimetric analysis (TG) and differential scanning calorimetry (DSC).
Notes on Testing Drug Purity by Thermal Analysis:
① The purity of the test sample is above 98.0%;
② The impurity does not react with the main component;
③ The impurity does not form a eutectic or solid solution with the main component;
④ The impurity is chemically similar to the molten sample;
⑤ The drug is chemically stable during melting;
⑥ If the drug is polymorphic, all of it must be converted to a single crystalline form.
When using the Van't Hoff equation to determine purity, it is based on the assumption that impurities do not form solid solutions. Therefore, the application of this method has certain limitations. In particular, when the test sample is a mixed crystal substance (that is, a mixture of different crystal forms has no difference in melting point) or a substance that decomposes when melted, it is difficult to accurately determine its chemical or crystal purity.
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