Impurities/degradants isolation techniques

Experimental campaign bulk or in-process control samples (e.g., prior to recrystallization) are excellent sources of process-related impurities and are a vital component of the KPSS. Isolated fractions collected from a preparative or semi-preparative liquid chromatography (LC) system are also excellent KPSS samples. Sometimes small-scale synthesis of the impurity or degradant is possible and less time-consuming than the isolation techniques. 

Interpretation of the spectroscopic data from the individual spectroscopic techniques is generally done as the data are amassed. When all of the data are available, it is useful for the participating scientists to integrate their respective data, which is discussed in more detail below. The overall elapsed time for the isolation and identification of a new impurity or degradation product is quite variable. The difficulty of the actual isolation and the structural complexity of the molecule both impinge on the process. On the basis of the author s experience. 

Impurity enhancement techniques such as fraction collection and phase equilibrium purification can be used to provide enriched samples for use in the method development process.23 When using the fraction collection approach, one or more cuts (fractions) of the chromatographic separation of a bulk lot or mother liquor are isolated. The excess solvent in these fractions is then evaporated to achieve the desired concentration enhancement. These fractions typically contain extraneous peaks because of the presence of salts in the mobile phase or sample degradation during the concentration step. The salts can be removed by extraction and/or a LC cleanup step. To insure that these extraneous peaks/artifacts are not identified as key peaks for separation, the original bulk lot or mother liquor should be included in the method development sample set. The same holds true for phase-equilibrium-purification supernatants. 

Solid-phase extraction devices and applications are evolving rapidly, and novel techniques that stretch the classical definition of SPE are becoming routine. Pawliszyn introduced solid-phase micro extraction (SPME) in 1989,5,14 and a commercial apparatus is available from Supelco (Bellefonte, PA). The SPME apparatus is merely a modified syringe that houses a fused silica optical fiber coated with an immobilized polymer film. The fiber can be exposed for extraction and then retracted for insertion or removal from the sample vial or instrument. Both manual and autosampler devices are available and each can be adjusted for proper fiber depth. Several coatings are available with varying thickness including polydimethylsiloxane, polyacrylate, polydimethylsiloxane/divinylbenzene, and carbowax/divinylben-zene. In contrast to SPE, which is an exhaustive extraction approach, SPME will extract only a fraction of an available analyte, hence it is not suitable for the isolation of impurities and degradants in most applications.15... 

The final product should be free from chemical impurities, which hinder characterization of the isolated material, and should be in a form that can resist biological and chemical degradation. Many options are available for isolating and concentrating these compounds. Each method has advantages and disadvantage, and best results can be obtained by adopting a combination of techniques It is the purpose of this chapter to critically discuss and evaluate various ethods commonly used to isolate and concentrate aquatic humic substances. 

TLC is a good technique to use when normal-phase solvents provide optimum separation. Typical thin-layer separations are performed on glass plates that are coated with a thin layer of stationary phase. The stationary phases used in TLC encompass all modes of chromatography including adsorption, normal- and reverse-phase, ion-exchange, and size-exclusion." The equipment required is simple and inexpensive. TLC is an ideal technique for the isolation of compounds because of its simplicity. However, for TLC to be successful, the impurity and/or degradant level should be at or above 1%. Any component present below this level is very difficult to isolate on a TLC plate because of higher detection limits. 

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. 

Over the years a number of techniques and approaches have proved to be useful tools to successfully isolate low-level impiu ities and degradants. TLC is most useful when an impurity or degradant is identifiable by LC-MS and above the 1% level. In cases, where NMR analysis is essential for identification, semipreparative SFC, semipreparative HPLC, and flash chromatography are more suitable techniques. Please refer to the Handbook of Pharmaceutical Analysis, 1st ed. for a more in-depth explanation of TLC and flash chromatography s use for impurity isolation. As mentioned earlier, SPE and liquid-liquid extractions are at times incorporated into the process. These tools can quickly convert bulk supply materials into a more suitable form for SFC or HPLC injection. SPE is also a useful tool in dewatering and desalting final RP-HPLC isolated materials obtained from solvents containing mobile-phase additives. 

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