Application of indirect detection methods in biomedical analysis.

Indirect detection is a technically simple method to follow and quantify compounds without inherent detector response in high-performance liquid chromatography. A detectable component is added to the mobile phase and peaks are obtained for injected solutes as well as for mobile phase additives (system peaks). The underlying principle can be expressed by simple equations which show how the detection sensitivity can be optimized. The detection technique can be applied to uncharged as well as charged compounds, but the sensitivity is considerably higher for ionic solutes. The response is unspecific and the technique should preferably be applied to samples with a limited number of components. Determinations in pharmaceutical products and quantification of impurities in substances are typical fields of application. Different detection principles can be used but UV absorbance is so far the dominant technique. The chromatographic system must be stable, and efficient thermostatting is essential. Indirect response effects can give rise to disturbances when the presence of a detectable component in the mobile phase is unknown or overlooked. The understanding of the underlying principle for the indirect detection is essential for tracing or preventing such disturbances.