A drug in plasma is bound to plasma proteins, and the bound and unbound drug concentrations reach a rapid and reversible binding equilibrium. It has been known that the unbound drug concentration in plasma is more closely related to the pharmaceutical activity and side effects than to the total (bound+unbound) drug concentration. Several important pharmacokinetic properties, such as the hepatic metabolism rate, renal excretion rate, biomembrane permeation rate and steady state distribution volume, are functions of the unbound drug fraction. [1][2][3] Albumin is the major protein in plasma responsible for protein binding of many kinds of drugs. Human serum albumin (HSA) has two major binding sites: warfarin site (site I) and benzodiazepin site (site II); three additional specific binding sites are also proposed in the case of digitoxin, bilirubin and fatty acids.4,5 Coadministration of drugs which share the same binding site can cause an increase in the unbound drug concentrations due to binding competition, and may result in undesirable side-effects or unexpected changes in drug disposition. Qualitative and quantitative binding studies to estimate the binding parameters and to identify the binding site on a protein molecule are essential for effective drug development and safety in clinical use. In addition, the protein binding of a chiral drug is potentially different between the enantiomers 6,7 because of the intrinsic chirality of proteins, which may cause enantioselectivity in the drug disposition.So far, equilibrium dialysis and ultrafiltration methods have been widely used to determine unbound drug concentrations. However, these conventional methods have problems, such as drug adsorption onto the membrane and leakage of bound drug through the membrane, which often make it difficult, or even impossible, to determine the unbound concentrations in the case of hydrophobic and strongly bound drugs. To overcome these problems, we proposed a novel chromatographic method, named high-performance frontal analysis (HPFA). [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] HPFA has several unique features. Since HPFA does not use any membrane, this method is free from undesirable drug adsorption onto the membrane, or leakage of the bound drug. During the frontal analysis, bound drug is transformed into the unbound form, and all drug molecules are eluted as a single zonal peak of unbound drug. If the zonal drug peak is completely separated from the protein peak, both unbound and total drug concentrations are determined simultaneously from the plateau height and peak area, respectively. In addition, HPFA can be easily incorporated into an on-line HPLC system. The detectability can be improved by on-line preconcentration of the unbound drug in the plateau zone. The unbound concentrations of a racemic drug can be determined enantioselectively by the on-line coupling of a HPFA column with a chiral HPLC column.Semotiadil and levosemotiadil (chemical structure, see Fig. 1) are an enantiomeric pair of drugs wh...