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 235/450 nm 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. 10 mg kg(-1)). The metabolite was identified as nor-L-arginine using mass spectrometry. Validated method was successfully used for pilot pharmacokinetic experiment on rats.

Charles University Prague Faculty of Medicine in Hradec Králové Hradec Králové Czech Republic

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