Initial method development experiments for both neutral and ionic samples are best carried out with reversed-phase high-performance liquid chromatography (HPLC) using acetonitrile-methanol-buffer mobile phases. The preceding paper (Part I) suggests the use of an acetonitrile-buffer gradient to start method development. In this paper, optimum conditions for this first separation are discussed. Sequential method development experiments plus computer simulations are then used to obtain a final HPLC method. In this connection, we have examined how many experiments are required for reliable predictions of ternary-solvent retention. Three experiments are sufficient to predict isoeluotropic retention for methanol-acetonitrile-buffer ternaries where solvent strength does not vary, but five experiments are required for ternaries that contain tetrahydrofuran.

A complex pharmaceutical raw material was characterized by means of reversed-phase gradient elution. By varying gradient steepness and mobile-phase pH, it was possible to optimize band spacing so as to separate 16 impurities or degradation products from the drug substance. Computer simulation was useful in interpreting these complex chromatograms and determining the maximum number of peaks that could be separated in this way. A marginal separation of all 17 sample components could be obtained, but the resulting method was quite pH-sensitive and therefore not very rugged. As an alternative, a rugged method was developed that separates the drug substance from all other sample components. The present study also describes how present computer simulation software for isocratic separation can be used to predict resolution for gradient elution runs as a function of pH.

Isocratic optimization of coumarin, potassium sorbate, ascorbic acid, sodium benzoate, vanillin, ethylvanillin, methylparaben, ethylparaben, sodium saccharin

Computer simulation by DryLab G/plus has proved to be an invaluable tool in the rapid development of analytical HPLC methods for antibiotics. A glycopeptide antibiotic under study at this Research Center was taken into consideration as a case study. Retention data from two preliminary experiments have allowed us to perform several simulations which greatly shortened the time normally required for the identification of the optimum gradient conditions. The “key steps” in the simulation process have been experimentally verified and a more than satisfactory agreement between calculated and experimental retention times was consistently found.

A complex mixture of arachidonic acid metabolites was separated by reversed-phase HPLC using a multi-step gradient, which was modelled by computer-assisted HPLC method development. The metabolites were extracted on-line on a precolumn connected to the analytical column in the same HPLC system. The predictions of the resolution and also the retention times calculated by computer simulation were very accurate when compared with the corresponding experimental run (maximum deviation 0.86%). An appropriate HPLC method and additionally an on-line extraction procedure could be developed with just three experimental HPLC runs. This method could be useful for evaluating the concentrations of arachidonic acid metabolites involved in inflammatory diseases.