Page - (000124) - in Biomedical Chemistry: Current Trends and Developments

HTS sample preparation procedure is useful in initial phases of DDD but also in clinical trials including BA/BE studies, since high number of samples must be analyzed as fast as possible (Falcão 2007; Almeida 2008; Xie, 2010; Boer 2012; Kundlik, 2012). Particularly the 96-well HLB µElution SPE plate is composed of a 2 mg high-capacity SPE sorbent (divinylbenzene/N-vinylpyrrolidone polymer), exhibiting excellent wetting properties and strong hydrophobic retention. Due to these properties, the sorbent can be run to dryness without loss of recovery, which is a major drawback of SPE sorbents based on silica-C18. Moreover it allows elution of target compounds with only 25 µL of solvent and the high capacity of the sorbent also tends itself to a concentration step (up to 15 times) with no evaporation or reconstitution, which is a potentially critical factor in low dose scenarios (Mallet, 2003; Yang 2005; Murphy, 2007; Lindegardh, 2008). On the other hand, the liquid-liquid extraction (LLE) is also used due to its simplicity, ability to provide clean extracts and the lower costs required. The extraction efficiency of LLE is related with the partition coefficient that is controlled by the characteristics of the extraction solvent (e.g., viscosity, surface tension, solubility in water, etc). In general, organic solvents with low miscibility in water and are preferred for LLE. However, selecting the right solvents (and their respective quantities) for sample partitioning makes the development of LLE methods laborious. Once optimized, the application of liquid- handling workstations allows semi-automated operations and therefore few LLE performed in 96-well format have been employed for high- throughput analysis (Chapter 12). Furthermore, 96-well LLE procedures may require large volumes of extraction solvent, limiting their application during DDD programs. Instead, the automated LLE combined with LC-MS/MS has been increasingly used in recent years (Eerkes, 2003; Xue 2004) for the analysis of pharmaceutical moieties based on advantages in efficiency, cost, and throughput. It is important to bear in mind that plasma protein precipitation, dilution and shoot approaches can also be used, but only to prepare biological samples that are simple and mainly composed of water, such as urine, cerebrospinal fluid, saliva and tears. 2.1.5 Conclusions