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

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Abstract

Two high-performance liquid chromatographic methods for the stereoselective determination of R,S-2-[4-(3-methyl-2-thienyl)-phenyl]propionic acid (R,S-MTPPA), a new anti-inflammatory agent, and its taurine conjugates (R,S-MTPPA-TAU) in dog urine have been developed and validated. The urine samples were subjected to solid extraction or TLC preparation, then R,S-MTPPA and R,S-MTPPA-TAU were separated on Chiralcel OD and Chiral AGP columns, respectively, with ultraviolet absorbance detection at 272 nm. The dose–response relationships for enantiomers of R,S-MTPPA and R,S-MTPPA-TAU were linear in the concentration ranges of 0.5–50 (r>0.9993) and 5–200 μg/ml (r>0.9982), respectively. Recoveries of all tested enantiomers from dog urine were roughly 90% within the above concentration ranges. Intra- and inter-day reproducibilities were sufficient for metabolic studies. These methods were applied to stereoselective determination of the enantiomers in dog urine after administration of either S- or R-MTPPA.

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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