Standardization method for measurement of hydroxyurea by Ultra High Efficiency Liquid Chromatography in plasma of patients with sickle cell disease

Abstract

Sickle cell anemia (SCA) is a recessively inherited disease characterized by chronic hemolytic anemia, chronic inflammation, and acute episodes of hemolysis. Hydroxyurea (HU) is widely used to increase the levels of fetal hemoglobin (HbF). The objective of this study was to standardize and validate a method for the quantification of HU in human plasma by using ultra high performance liquid chromatography (UPLC) in order to determine the plasma HU levels in adult patients with SCA who had been treated with HU. We used an analytical reverse phase column (Nucleosil C18) with a mobile phase consisting of acetonitrile/water (16.7/83.3). The retention times of HU, urea, and methylurea were 6.7, 7.7, and 11.4 min, respectively. All parameters of the validation process were defined. To determine the precision and accuracy of quality controls, HU in plasma was used at concentrations of 100, 740, and 1600 µM, with methylurea as the internal standard. Linearity was assessed in the range of 50-1600 µM HU in plasma, obtaining a correlation coefficient of 0.99. The method was accurate and precise and can be used for the quantitative determination of HU for therapeutic monitoring of patients with SCA treated with HU.

Hydroxyurea Scavenges Free Radicals and Induces the Expression of Antioxidant Genes in Human Cell Cultures Treated With Hemin

“…Both concentrations mentioned above are consistent with the plasma levels of HU generally observed in patients with SCA, which have been extensively used in in vitro studies ( 27 – 30 ). Accordingly, we chose these concentrations for our additional assays, in addition to combined therapy with hemin at 70 μM based on the findings from Carvalho et al ( 31 ).…”

“…This finding is of great importance, as HU could potentially confer an important protective effect against direct oxidative attacks on membrane phospholipids, as well as prevent/minimize the triggering of activation responses involved in the initiation of the oxidative cascade and establishment of inflammation (23)(24)(25)(26). Both concentrations mentioned above are consistent with the plasma levels of HU generally observed in patients with SCA, which have been extensively used in in vitro studies (27)(28)(29)(30). Accordingly, we chose these concentrations for our additional assays, in addition to combined therapy with hemin at 70 µM based on the findings from Carvalho et al (31).…”