Patent ID: 7022528

Claim:
An assay method for high-throughput spectroscopic measurement of the membrane permeability and membrane retention values for a compound, said method not requiring knowledge or measurement of the molar absorptivity of said compound, and not requiring a calibration curve relating known concentrations of said compound to a spectroscopic property of said compound, said method comprising the steps of: a. preparing a sample solution of said compound in an aqueous buffer of known pH and separating said sample solution from any precipitate, said separated solution constituting a reference solution, b. preparing an initial donor solution of said compound, by placing an aliquot of said reference solution in a donor compartment, said donor compartment being on one side of a membrane barrier, c. placing an initial acceptor solution in an acceptor compartment, said acceptor compartment being on the second side of said membrane barrier, wherein said acceptor solution comprises a buffer of known pH and one or more sink-forming additives, wherein said sink-forming additives possess one or more of the properties selected from the group consisting of (1) high capacity to bind said compound, (2) low UV absorption, (3) high water solubility, and (4) low vapor pressure, d. preparing a donor-blank solution free of said compound, but otherwise of the same composition as said reference solution, e. preparing an acceptor-blank solution of the same composition as said initial acceptor solution, f. measuring a spectroscopic property of said compound in said reference, donor-blank, and acceptor-blank solutions at the start of the assay, g. measuring said spectroscopic property of said compound in each of said final donor and final acceptor solutions after known permeation time from the start of the assay, h. determining the relative concentration of said final donor and acceptor solutions by comparing the measured spectroscopic property of said final acceptor, final donor, reference, acceptor-blank and donor-blank solutions, and i. calculating from said determination the membrane permeability of said compound using the equation P e ( D ) = - 2.303 ⁢ ⁢ V D A ⁡ ( t - τ SS ) ⁢ ( 1 1 + r a ) ⁢ log 10 ⁡ [ - r a + ( 1 + r a 1 - R ) ⁢ C D ⁡ ( t ) C D ⁡ ( 0 ) ] where R is membrane retention, calculated from the equation R =1 −[C D ( t )+ C A ( t )· V A /V D ]/C D (0) and where r a =( V D /V A )( P e (A) /P e (D) ) and where P e (D) and P e (A) are the membrane permeabilities in the donor-to-acceptor and in the acceptor-to-donor directions, respectively; V A and V D are volumes of the acceptor and donor compartments, respectively; A is the area of the membrane barrier; t is time; τ SS is steady-state time; C A (t) and C D (t) are the acceptor and donor sample concentrations at time t, respectively; and C D (0) is the concentration of the initial donor solution.