The first molecule has a cis-double bond, a chiral center in α-position to aromatic ring and a labile ester bond. The product and starting materials for its synthesis (which are also its dominant degradation products) have extremely different polarity and lipophilicity. The peak of one of them, which is very polar, elutes before the solvent front. The other cannot be seen using UV detector due to lack of chromophore. This caused a need in developing complementary methods: the method for “Polar Impurities” developed using a mixed-mode HPLC column and the method for lipophilic “UV-invisible” – using non-silica based RP column and evaporative detector (ELSD or Corona CAD). In addition, enantioselective separation under normal phase chromatographic conditions has been developed.

Principles of work of evaporative detectors are explained and the benefits of using alternative (non-UV) detection techniques discussed. Data processing for non-linear detection in HPLC is explained. Specific case of MS detection following normal phase liquid chromatography is also addressed.

The second molecule is strongly lipophilic and poorly soluble in water. RP HPLC method for lipophilic API requires a high content of organic solvent both in eluent and diluent.

Due to very low water solubility of API, a lipid-based formulation has been prepared, which demanded to either develop a complex extraction procedure to be used with RP method, or chose an alternative analytical technique – normal phase (NP) HPLC; where the entire drug product may be dissolved in eluent-compatible organic solvent.

Principles of HPLC method development, both for RP and NP chromatography, are discussed in terms of various retention mechanisms and their combinations in chromatographic separations. Special attention is given to available tools in development of NP HPLC methods.

Since NP HPLC provides fewer possibilities in mobile phase composition for optimization of separation, a “scouting of columns” attitude had to be applied. Usefulness of less volatile solvents in NP HPLC is explained.
Real cases of unpredictable troubleshooting sessions illustrate the material. Influence of HPLC hardware and its compatibility with HPLC methods is also pointed out.

Questions of compliance of analytical methods for pharmaceutical industry to ICH (FDA, etc.) guidances are briefly reviewed.

This knowledge sharing seminar (Master Classes) is intended for the analytical chemists having experience in HPLC or related techniques, working in Quality Control or Research and Development laboratories, as well as for university students and post-graduates in the field of modern instrumental methods of analysis.

The aim of this seminar is to elaborate a mindset of a developer of liquid chromatography methods and to converse the need in complementary “orthogonal” chromatographic procedures and diverse detection techniques for a complex coverage of all the characteristics of a pharmaceutical material