Pickering Laboratories, Inc.

An overview of post-column derivatization methods from a pharmaceutical applications perspective

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High performance liquid chromatography (HPLC) is the technique of choice for separating analytes pursuant to their detection, identification, quantification and/or purification. Most organic substances, and certainly the majority of pharmaceuticals, are polar, nonvolatile or thermally labile molecules that are amenable to HPLC separation. Moreover, the availability of a large variety of LC column media, particle sizes and near-infinite gradation of mobile phase types and polarities greatly empowers the discriminatory capabilities and ease of adaptation of HPLC. Once separated, analytes of interest must be registered by detecting a characteristic physical property such as the absorption of light, emission of fluorescence, mass, etc.

The detected signal, in conjunction with a reproducible retention time for a chromatographic method, is usually sufficient to identify and (with an appropriate set of calibration standards) quantify each analyte of interest. Other things being equal, factors that increase analyte signal strength or the ability to discriminate the signal from background noise or other interferences will lower the limit of detection and quantitation.

This is a highly desirable objective for analytical methods used in pharmaceutical applications where analytes of interest are generally active and often monitored at low physiological concentrations. Since much of the effort in chromatographic sample preparation is focused on isolating analytes from sample constituents that may confound detection, techniques that bolster analyte signal strength and selectivity often permit simpler and more efficient sample preparation, with the time saved translated into increased throughput.

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