Biological matrices consisting of high molecular weight matrices i.e. endogenous substances, metabolites, proteins, blood cells and coexisting drugs are often difficult to handle for chromatographic analysis. To purify and enrich the target analytes and drugs in this complex matrices an effective sample pretreatment step is essential. Biological sample pretreatment has always been a forgotten part of the biopharmaceutical analysis. Among the sample pretreatment techniques traditional off-line sample processing like liquid-liquid extraction, protein precipitation and solid phase extraction are gradually becoming a limiting bottleneck in the chromatographic analysis. As an on-line and fully automated technique, coupling SPE column with HPLC so called on-line SPE-LC, leads to complete automation improving the analytical quality due to enhanced reproducibility, elimination of human errors and the possibility of multiple step elutions for clean-up of complex samples, reducing the cost and analysis time required (1–5).
In this presentation, importance of sample pretreatment in chromatography, requirements of coupling SPE to LC, chromatographic method development steps and achievement of complete depletion of matrix components will be discussed. Some applications of this technique to drug determination in complex bio-fluids will be presented in consideration of current publications. Also, different applications for LC systems equipped with MS/MS and UV detectors and examples of two and multidimensional separations will be shown, obtained results will be presented.