Seite - (000121) - in Biomedical Chemistry: Current Trends and Developments

for the drug and metabolites at trace levels, not only in vitro but also in vivo (Table 2.1.6). For further confirmation of metabolites, the LC-Q-TOF- MS/MS technique can be applied for accurate mass measurement of the protonated molecules and their fragment ions. It must be emphasized that LC-MS/MS is also frequently employed to quantify the drug fraction unbound to plasma proteins during early screening studies (Table 2.1.6). Indeed, the plasma protein binding of a drug can dramatically affect the circulating concentration of the compound as well as its ability to be distributed and/or accumulated through different compartments of the body. It is well accepted that only the unbound drug is available to cross membrane barriers, be distributed to tissues, and exert pharmacological and/or toxicological effects. In vitro equilibrium dialysis and ultrafiltration are the techniques most frequently employed in initial phases of DDD (Table 2.1.2). Devices based on multi-well formats have been recently developed (Fung 2003; Zhang 2006; Plumb 2008; van Liempd, 2011; Zamek-Gliszczynski, 2011) and employed to investigate NCEs (Table 2.1.6). Briefly, they consist of 48 or 96-well Teflon base plates with a semi-permeable membrane that allows the passage only of the unbound drug. When equilibrium between both membrane sides is reached in the equilibrium dialysis technique, the total drug concentration is determined in the plasma compartment while the free drug concentration is determined in buffer compartment in order to calculate the percentage of drug bound to plasma proteins. On the other hand, ultrafiltration is currently emerging as a faster and simpler alternative to equilibrium dialysis once the centrifugation applied (approximately 2000 × g) accelerates the passage of the unbound drug through the membrane (Fung 2003; Zhang 2006). Particularly because of the low free drug levels that can be achieved in the buffer compartment or in the filtrate, both in vitro techniques require the application of LC-MS/MS methods to accurately quantify the drugs (Table 2.1.6). Both MRM (Zamek-Gliszczynski, 2011) and SRM (van Liempd, 2011) detection modes seem to be successful. Sample preparation prior to chromatographic analysis is very simple, and relies on protein precipitation with acetonitrile (van Liempd, 2011, Zamek- Gliszczynski, 2011) or a mixture of acetonitrile/0.1% formic acid aqueous solution (90/10, v/v) (Plumb, 2008) followed by centrifugation. It is