Journal of Chromatography B: Biomedical Sciences and Applications
Stereoselective determination of R,S-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid and its taurine conjugates in dog urine by high-performance liquid chromatography
Introduction
R,S-2-[4-(3-Methyl-2-thienyl)phenyl]propionic acid (R,S-MTPPA) is a new, orally effective anti-inflammatory agent (Fig. 1). The S-isomer is pharmacologically active, while the R-isomer is inactive. The biological activities of S-MTPPA (code: M-5011) in animal models have been reported 1, 2. The metabolism of R,S-MTPPA is considered to involve oxidation of the thiophenyl moiety, glucuronidation and amino acid conjugation of the carboxylic group. The major urinary and fecal metabolite in dogs after oral administration of S-MTPPA was identified as the taurine conjugate of MTPPA (MTPPA-TAU) 3, 4. It is known that the enantiomers of 2-arylpropionic acid derivatives undergo chiral inversion from the inactive R-isomer to the active S-isomer 5, 6, 7, 8and the mechanism of chiral inversion is related to the mechanism of amino acid conjugation 9, 10, 11, 12. R,S-MTPPA was expected to undergo chiral inversion in the same way, but the optical form of the metabolite after dosing of each of S- and R-MTPPA is not known. A chiral separation method for R,S-MTPPA and its metabolite is needed to investigate the stereoselective metabolism and inversion of R- and S-MTPPA in animals. In this paper we report a stereoselective and quantitative assay for enantiomers of R,S-MTPPA and R,S-MTPPA-TAU in dog urine using high-performance liquid chromatography (HPLC). The method was employed to determine the enantiomers in dog urine samples after administration of S-MTPPA and R-MTPPA.
Section snippets
Chemicals and reagents
The enantiomers of R,S-MTPPA and its taurine conjugate were prepared at the Maruho Kyoto Research Laboratory (Kyoto, Japan) as described in the previous paper [4]. The optical purities of R- and S-MTPPA determined by HPLC chiral separation analysis were 99.0 and 99.8%, respectively. Other solvents and chemicals used were of analytical or HPLC grade.
Instrumentation
The HPLC system for chiral separation of R- and S-MTPPA consisted of a pump system (600E, Waters, Milford, MA, USA), an auto sample injector (715
Chromatograms
Two methods have been reported for the chiral separation of enantiomers of 2-arylpropionic acid derivatives, one is a direct method with chiral stationary phase and the other is an indirect method using chiral derivatization techniques [13]. We selected the direct method for the present assay of enantiomers of MTPPA to avoid the possibility of stereochemical conversion of enantiomers induced by derivatization. Adequate separation of enantiomers of MTPPA was obtained by using a chiral separation
Conclusion
Our newly developed chiral HPLC method for assay of R,S-MTPPA and its taurine conjugate is sensitive and accurate enough for the analysis of stereoselective pharmacokinetics in dogs.
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