API Impurity Isolation

During a manufacturing process or a stability study for an Active Pharmaceutical Ingredient (API) or drug product, a sample may be found to contain impurities. These impurities might occur as degradation products, as intermediates/process impurities originating from the synthetic route, as an unwanted by-product of side-reactions or due to contamination of the original sample. Such impurities can arise at very low concentrations relative to the API, as little as 0.05% w/w, but it is important that when discovered they are isolated and identified, as per ICH Q3 guidelines for Impurities in New Drugs.

When the impurities are known impurities of the API, they can often be identified via retention time match and spectral or mass spectrometry (MS) confirmation. However, in most cases, when unknown degradation products are found, they require isolation and identification. On occasions, quantities as low as 5 mg can be sufficient for structural elucidation but it is preferable to isolate around 20-40 mg of impurity for full structural elucidation.

Preparative LC

The technique best suited to isolating impurities is preparative liquid chromatography (LC), using low or high-pressure columns. The technique requires loading of a preparative-scale LC column with repeated doses of the sample, and collecting fractions either using known time intervals or mass-based fraction collection. This allows isolation and concentration of sufficient impurity for identification purposes using highly sophisticated techniques, such as nuclear magnetic resonance (NMR) spectrometry, Fourier transform infrared spectroscopy (FT-IR) and MS, to help identify the chemical impurity. However, the process of isolating and concentrating the impurity is not always straightforward.

The specific choice of preparative LC method will rely on prior information about the chemical structure of the drug molecule: its known or anticipated impurities, stability and solubility data, earlier used chromatographic methods and other relevant details. This pre-knowledge will help determine the sort of chromatography best suited for the isolation.

Impurity isolation and identification are essential aspects of pharmaceutical development, for both existing compounds and new Chemical/Biological Entities.