**7. Sample preparation**

Adequate sample preparation is a key aspect of quantitative bioanalysis and it is usually the most time consuming part of analyses. Interfering matrix compounds, such as proteins, lipids, salts, other endogenous and background compounds, should be removed in sample pretreatment, not only to avoid column clogging and instrument soiling, but also to improve the sensitivity, selectivity and reliability of analyses. Selection of an appropriate preparation procedure depends upon metabolite characteristics, their expected concentrations, the sample size and matrix, and the availability of analytical techniques for analyte quantification. Insufficiently treated samples may cause interfering peaks when using spectroscopic detection techniques such as UV-absorbance or fluorescence. However, analyses by LC-MS/MS are less prone to sample matrix and therefore usually require less pretentious sample clean up. Commonly and widely applied sample preparation techniques include protein precipitation (PP), liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Manual operations associated with sample treatment may be very labor intensive and time consuming and that could be avoided with automation in 96-well plate format or direct sample injection followed by on-line extraction methods.
