Application of antibody-mediated extraction for the stereoselective determination of the active metabolite of loxoprofen in human and rat plasma.
Antibody-mediated extraction followed by chiral high-performance liquid chromatography (HPLC) was applied to stereoselective determination in human and rat plasma of the active metabolite [(2S,1'R,2'S)-trans-alcohol] with three chiral centers of Loxoprofen, a 2-arylpropionic acid antiinflammatory agent after oral administration. Antiserum against the (1'R,2'S)-cyclopentanol moiety was obtained from a rabbit immunized with bovine serum albumin conjugate linked to the propionic acid moiety, in which another chiral center is located. Then, the immunoglobulin G purified by a protein A column was coupled to BrCN-activated Sepharose 4B. Plasma samples were applied to the immobilized antibody column. After washing the column to remove unrequired stereoisomers, a mixture of two diastereomers whose configurations were 1'R,2'S in the cyclopentanol moiety was extracted with 95% methanol. The solvent was evaporated and the residue was derivatized with (+)-(R)-1-(1-naphthyl)ethylamine as a chiral reagent to separate the diastereomers by HPLC. This combined analytical method showed the stereoselective metabolism of Loxoprofen in human, that is, 64% of the total amount of four trans-alcohol stereoisomers was in the 2S,1'R,2'S form, which is the active metabolite. This phenomenon was also observed in rats given Loxoprofen and its (2S)- and (2R)-isomers, and is explained by stereoselective ketone reduction of Loxoprofen to the (1'R,2'S)-trans-alcohol and inversion from 2R to 2S in the propionic acid moiety. Antibody-mediated extraction should be a selective and simple clean-up method for determining haptens with complicated structures, combined with an appropriate analytical method.
Takasaki
W
W
Analytical and Metabolic Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan.
Tanaka
Y
Y
D000284:Administration, Oral; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000097:blood*; D000906:Antibodies; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007124:Immunoenzyme Techniques; D010666:Phenylpropionates / Q000008:administration & dosage / Q000097:blood* / Q000378:metabolism; D051381:Rats; D013237:Stereoisomerism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D000906:Antibodies; D010666:Phenylpropionates; C040656:loxoprofen