Impurities/degradants preparative HPLC

Preparative HPLC is typically the technique of choice for impurity purification. It is often necessary to enrich the impurity before preparative HPLC purification. Various techniques such as solid-phase extraction can be used to enrich the low-level impurity. To ultimately confirm the structure of a new impurity, it may be necessary to synthesize the compound and compare its spectroscopic characteristics to those observed in the original sample. A very effective means of getting useful structural information is to conduct a degradation study on the purified impurity. 

This multidisciplinary team approach toward impurity identification was successfully applied to the identification of this impurity. Each and every discipline played a very important role. Without SPE enrichment to remove the drug substance from the impurities, the impurity could not be purified by preparative HPLC. The novel acidic degradation study of the impurity provided very valuable information of the structure of the impurity. Mass spectrometry and nuclear magnetic resonance spectroscopy were the ultimate tools in this structure elucidation. Furthermore, the formation mechanisms were concluded by a careful examination of the process. 

Developing an isolation approach is an activity that is frequently overlooked or addressed as an afterthought. However, solubility and stability data may dictate the development of a chromatographic method that requires the elaboration of the isolation, that is, it is more complicated than a simple evaporation of the mobile phase. The development of the chromatographic process should be linked to and interactively codeveloped with the isolation. Ideally, the isolated impurity sample should not contain other compounds or artifacts, such as solvents, mobile-phase additives or particulate matter from the preparative chromatography, as they may interfere with the structure elucidation effort or adversely affect the stability of the impurity during the isolation process. Therefore, it is preferable to avoid or minimize the use of mobile-phase additives. However, should this prove to be impossible, the additive used should be easy to remove. The judicious choice of mobile phase in the HPLC process increases the ability to recover the compound of interest without or with minimum degradation. The most common... 

The columns are generally packed with silica gel. For the separation to be successful, the size of the silica gel particles should be 40-63 pm. A concentrated solution of the sample is prepared. The sample solution is applied at the top of the column, and the walls of the column are washed with a few milliliters of eluent. Solvent is added to the column, and air pressure is applied at a flow rate of 2 in./min to rapidly elute the desired impurity and/or degradant. Separation is based upon the differential interactions between the solute molecules and the adsorbent surface of the silica gel. Fractions are continuously collected and monitored by chromatographic techniques (HPLC with UV detection, GC, or TLC). The fractions containing the compound of interest are combined and evaporated to dryness. The isolated material is cleaned (post isolation cleanup, such as small-scale column or analytical HPLC reinjection, is essential) and submitted for LC-MS and NMR analysis. 

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