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Development and Validation of a Liquid Chromatography Method with Electrochemical Detection for Hydroxyurea Quantification in Human Plasma and Aqueous Solutions

Anar Rodriguez, Delphine Beukens, Nicole Debouge, Beatrice Gulbis and Frederic Cotton

Hydroxyurea is the unique drug having demonstrated a significant efficacy in sickle cell disease treatment. We developed a liquid chromatography method with electrochemical detection for hydroxyurea analysis in plasma and aqueous solutions. Analytical goals included an analytical range from 2 to 50 mg/L, a total imprecision lower than 15% and a total error lower than 30%. After protein precipitation with acetonitrile, the separation was performed on a C18 Atlantis T3 column and eluted with sodium acetate 25 mM, acetonitrile 2.5%, pH 6.5. Thioacetamide was used as internal standard. The method was linear for drug concentrations ranging from 0.5 to 50 mg/L and recovery was comprised between 100 and 120%. The intra-day precision was lower than 6.0% and between-day precision was lower than 11%. The detection limit was 0.18 and 0.63 mg/L for aqueous solution and plasma, respectively and the quantification limit was 1.0 and 1.2 mg/L for aqueous solution and plasma, respectively. No interference from urea was observed. The liquid chromatography method developed can be used for pharmacokinetic studies in plasma and other biological samples such as saliva or urine. It requires low sample volumes and a simple pre-treatment and it allows a direct measure of non-derivatized hydroxyurea.