ABSTRACT
The increasing signi‰cance of oligonucleotides as therapeutic agents necessitates a high level of quality control. In such protocols, chromatographic analysis of crude and ‰nal active pharmaceutical ingredients (API) is necessary to ensure the detection of contaminants at concentration levels down to trace amounts relative to the drug. A combination of chromatographic techniques, in particular reverse-phased high-performance liquid chromatography (RP-HPLC), anion-exchange (AEX) HPLC (Chapter 2), and mass spectrometry (Chapters 4 and 5), is needed for the identi‰cation and structural elucidation of by-products and degradation products resulting from the production
1.1 Introduction ..............................................................................................................................1 1.2 Historical Aspects .....................................................................................................................2 1.3 Reverse-Phased High-Performance Liquid Chromatography Columns...................................2 1.4 Stationary Phases ......................................................................................................................7 1.5 Mobile Phases ......................................................................................................................... 10 1.6 Retention Time and Separation Selectivity Prediction Models .............................................. 14 1.7 Selected Practical Examples of IP-HPLC .............................................................................. 17
1.7.1 Column Inªuence on Chromatography ...................................................................... 17 1.7.2 Mobile Phase Inªuence on Chromatography .............................................................24 1.7.3 HPLC versus UPLC ....................................................................................................25 1.7.4 Temperature Inªuences of Chromatography ..............................................................27 1.7.5 Sample Preparation .....................................................................................................28
