Enantioselective analysis of ritalinic acids in biological samples by using a protein-based chiral stationary phase.

This study was to develop and validate a new chiral HPLC-UV method for the quantitative analysis of enantiomeric ritalinic acid (RA) in human plasma. An alpha1-acid glycoprotein column was used with the mobile phase containing 0.4% acetic acid and 0.1% dimethyloctylamine, pH 3.4. The detection of enantiomeric RAs was at 220 nm. A baseline separation for d- and l-RA was achieved by a separation factor of 2.08. Methylphenidate, the precursor of RA, was eluted with the front solvent, and thus does not interfere in the analysis of RA in our method. The assay was successfully applied for the in vitro analysis of enantiomeric ritalinic acids produced by human and dog plasma and dog liver. Data demonstrated that the esterase(s) in human plasma metabolize d-methylphenidate faster than its l-isomer. The yielded intrinsic clearances (Clint) are 1.02 and 2.17 microl/min/mg protein, respectively, for d and l-methylphenidate.