Page - (000110) - in Biomedical Chemistry: Current Trends and Developments

predominantly analyzed by LC (Table 2.1.6). Indeed, chromatography is critical to the success of any bioanalytical assay as it allows the separation of the analytes from endogenous substances and metabolites. Resolution of the peaks correspondent to analytes and metabolites is essential to prevent an overestimation of the concentration of parent compound and subsequently incorrect determination of pharmacokinetic parameters and drug exposure. Even using highly selective MS or MS/MS detection, a prior chromatographic separation (although simpler than those required with other detection methods) is essential because the co-elution of endogenous or exogenous compounds may cause ion suppression or ion enhancement, both detrimental to the development of a sensitive method. Reversed- phase LC is the chromatographic technique most widely employed in bioanalysis of drugs and their metabolites during DDD due to its application to small molecules, which are separated by their different affinity to the hydrophobic stationary phase in relation to the mobile phase. However, compounds with low octanol-water partition coefficients and the majority of common metabolites may hamper the development of a reliable reversed-phase LC method, as they present a very short or non-existent retention time. Thus, the demand for analytical laboratories to increase sample throughput provided the impetus for HPLC column manufacturers to introduce new stationary phases and column geometries in order to increase speed and sensitivity. In this context, monolithic columns were created and have been employed with fast gradients and high flow rates for the direct analysis of several pharmaceutical compounds in biological samples (Zhou, 2005; Huang, 2006), but these columns have not been very frequently employed during DDD. Indeed, ultra-high pressure liquid chromatography (UHPLC) seems to be the most important achievement in LC evolution over the last decade and its application is increasing in the pharmaceutical field (Table 2.1.6). This technology retains the practicality and principles of traditional HPLC system but it is capable of providing liquid flow at pressures higher than 10,000 psi and columns packed with small particles (< 2 µm) that can withstand these pressures (Guillarme, 2013; Rodriguez-Aller, 2013; Fekete, 2014; Nishi, 2014). It is well-known that the theoretical plate height is proportional to the particle size, so using particle sizes down to 1.7 µm decreases theoretical