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Drug analysis is only as good as the sample that is introduced, and chances are, poor preparation methods are the culprit in poor HPLC performance
There is nothing so frustrating as having a high-performance liquid chromatography (HPLC) analysis go awry and then trying to figure out what went wrong. Poor peak shapes, erratic results, changes over time, high background noise, or the complete lack of recovery of the analyte are all troubles that can halt the progress of an experiment in midstream. Although it is tempting to purchase a brand-new column when problems arise, or to change the solvents, the pressure, or other HPLC parameters, usually the best place to start solving a problem is sample preparation. This area of HPLC analysis is both the simplest place to make corrections and also the most likely source of problems.
Methods of sample preparation vary widely and include everything from simple dilution to high-tech Chromatographie separations that rival HPLC itself in sophistication. Lab workers typically devote a great deal of time and attention to sample preparation, using two to three preparatory steps before their main chromatography step. However, many rely on outdated techniques, use methods incompatible with their chromatography, or make critical, costly mistakes. The key is to develop simple strategies to overcome challenges in sample preparation before they ruin the entire experiment.
The molecular structure of an analyte can yield important clues about its behavior in an HPLC method. An experienced chemist can predict whether a molecule will be stable. Tendencies toward hydrophobicity or hydrophilicity are often readily apparent. Conclusions can also be drawn regarding hydrolysis, oxidation, or other chemical transformations in solution. Min Chang, PhD, section manager for the bioanalytical department at Abbott Laboratories, Abbott Park, Ill., recommends a careful study of the molecule before embarking on the first steps of HPLC method development. His laboratory assays biological samples, usually blood or plasma, for drugs and metabolites under the regulation of good laboratory practices (GLP). Blood contains approximately 7% protein, as opposed to drug concentrations that are typically in the parts-per-trillion or parts-per-quadrillion range. High background noise is a constant hurdle to be overcome in HPLC analysis.
The primary sample preparation goal in Chang's laboratory is separation of drug or metabolite from protein....