HPLC determination of arginases inhibitor N-(ω)-hydroxy-nor-l-arginine using core–shell particle column and LC–MS/MS identification of principal metabolite in rat plasma

Abstract

For the purpose of in vivo pharmacokinetic studies, an HPLC method was developed and validated for the quantification of N-(ω)-hydroxy-nor-l-arginine, l-arginine and N-(ω)-ethyl-l-arginine (internal standard) in rat plasma. Sample processing involved a solid-phase extraction on the Waters MCX cartridges and on-line pre-column derivatization of the analytes with o-phthaldialdehyde and 3-mercaptopropionic acid. Separation of the derivatives was carried out on a core–shell Kinetex C18 column in a gradient elution mode with a mobile phase consisting of methanol and water (pH=3.00 adjusted with formic acid). Fluorimetric detection with the excitation/emission wavelengths of 235/450nm was used. The method was validated according to the FDA guidelines and applied to pilot pharmacokinetic experiments. An unknown metabolite was extracted from the plasma of Wistar rats after a single bolus of N-(ω)-hydroxy-nor-l-arginine (i.v. 10mgkg−1). The metabolite was identified as nor-l-arginine using mass spectrometry. Validated method was successfully used for pilot pharmacokinetic experiment on rats.