Determination of hydroxyurea in human plasma by HPLC-UV using derivatization with xanthydrol

A simple and rapid high performance liquid chromatography (HPLC) method using ultraviolet (UV) detection was developed to determine hydroxyurea (HU) concentration in plasma sample after derivatization with xanthydrol. Two hundred microliters samples were spiked with methylurea (MeU) as internal standard and proteins were precipitated by adding methanol. Derivatization of HU and MeU was immediately performed by adding 0.02 M xanthydrol and 1.5 M HCl in order to obtain xanthyl-derivatives of HU and MeU that can be further separated using HPLC and quantified using UV detection at 240 nm. Separation was achieved using a C18 column with a mobile phase composed of 20 mM ammonium acetate and acetonitrile in gradient elution mode at a flow rate of 1 mL/min. The total analysis time did not exceed 18 min. The method was found linear from 5 to 400 M and all validation parameters fulfilled the international requirements. Between- and within-run accuracy error ranged from 4.7% to 3.2% and precision was lower than 12.8%. This simple method requires small volume samples and can be easily implemented in most clinical laboratories to develop pharmacokinetics studies of HU and to promote its therapeutic monitoring.