CP-122288

Turbulent flow chromatog. coupled to tandem mass spectrometry (TFC-MS-MS) has been widely used within bioanal., because of the ability to inject directly neat biol. samples with no prior pretreatment.TFC-MS-MS removes the need for time consuming sample preparation procedures such as protein precipitation, liquid-liquid extraction or solid-phase extractionThere are two standard configurations that are commonly adopted for the use of TFC, namely fast elute and focus mode.In this paper, a new micro TFC column which has the advantages of reducing solvent consumption and improving mass sensitivity, was used to develop a fast elute method.A wide range of pharmaceutical compounds with various physicochem. properties was used to assess the system performance.Carry-over has often been identified as a potential source of inaccuracy in a fast elute mode.This paper shows how by using a systematic approach, carryover was eliminated.The parameters influencing the robustness of the micro TFC column are also discussed.This method was fully validated for a Pfizer development compound in human plasma using a new TFC parallel platform allowing two systems to be operated in parallel.Multiplexing the sample anal. allowed a 2-fold increase in throughput and provided an efficient use of the mass spectrometer.

[(3)H]LY334370 was developed as a radioligand to study the characteristics of this compound's interaction with the 5-HT(1F) receptor. Monovalent or divalent cations did not enhance the binding of [(3)H]LY334370 to the cloned human 5-HT(1F) receptor. In the presence of MgCl(2), the time to reach equilibrium was approximately 2 h, while in its absence equilibrium was reached in less than 1 h. [(3)H]LY334370 had high affinity for the cloned human 5-HT(1F) receptor (K(d)=0.446 nM) and the 5-HT(1F) receptor in rat brain (K(d)=0.388 nM). The expression density of 5-HT(1F) receptors, as determined by binding to homogenates of cortical regions from rat, was low (B(max)=79.1 fmol/mg protein). There was a statistically significant correlation between the apparent pK(i) for inhibition of [(3)H]LY334370 binding and the pEC(50) for stimulation of [(35)S]GTPgammaS binding to homogenates of cells expressing the cloned human 5-HT(1F) receptor. In addition, there was a statistically significant correlation between the apparent pK(i) for inhibition of [(3)H]LY334370 binding to the cloned human 5-HT(1F) receptor and the pID(50) for inhibition of trigeminal nerve stimulated dural plasma protein extravasation in the guinea pig. The conclusion from these studies is that [(3)H]LY334370 is a high affinity radioligand which can be used for the study of the 5-HT(1F) receptor in rat brain or in cells transformed with the human 5-HT(1F) receptor.