Open Access Rodriguez et al., J Chromatogr Sep Tech 2014, 5:5 DOI: 10.4172/2157-7064.1000244 Open Access Research Article Volume 5 • Issue 5 • 1000244 J Chromatogr Sep Tech ISSN: 2157-7064 JCGST, an open access journal Development and Validation of a Liquid Chromatography Method with Electrochemical Detection for Hydroxyurea Quantification in Human Plasma and Aqueous Solutions Anar Rodriguez 1 , Delphine Beukens 1 , Nicole Debouge 2 , Béatrice Gulbis 2 and Frédéric Cotton 1,2 * 1 Laboratory of Biological and Medical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Boulevard du Triomphe, 1050 Brussels, Belgium 2 Department of Clinical Chemistry, Erasme Hospital, Université Libre de Bruxelles, Route de Lennik 808 1070 Brussels, Belgium *Corresponding author: Frédéric Cotton, Department of Clinical Chemistry, Erasme Hospital, Université Libre de Bruxelles, 808 Route de Lennik - 1070 Brussels, Belgium, Tel: +3225553427; E-mail: fcotton@ulb.ac.be Received September 18, 2014; Accepted September 22, 2014; Published October 04, 2014 Citation: Rodriguez A, Beukens D, Debouge N, Gulbis B, Cotton F (2014) Development and Validation of a Liquid Chromatography Method with Electrochemical Detection for Hydroxyurea Quantifcation in Human Plasma and Aqueous Solutions. J Chromatogr Sep Tech 5: 244. doi:10.4172/2157- 7064.1000244 Copyright: © 2014 Rodriguez A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Hydroxyurea is the unique drug having demonstrated a signifcant effcacy 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 quantifcation 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. Keywords: Hydroxyurea; Hydroxycarbamide; Tioacetamide; Liquid chromatography; Electrochemical detection; Sickle cell disease Introduction Hydroxycarbamide, better known as hydroxyurea, is a cytostatic agent used in the treatment of myeloproliferative disorders such as essential thrombocythemia and myelofbrosis [1,2]. It is also the most active drug in sickle cell disease, a genetic disorder due to the mutation of the 6th codon of the β-globin gene, leading to the synthesis of an abnormal hemoglobin (Hb S); sickle cell disease is characterized by chronic hemolytic anaemia and vaso-occlusive crises [3]. Te main benefcial efect of hydroxyurea is to increase cellular levels of fetal hemoglobin (Hb F), which reduces Hb S polymerization [4]. Others mechanisms were demonstrated or suggested such as reduced expression of adhesion molecules, increased nitric oxide production, cation transport changes and myelosuppressive efects [5,6]. Despite hydroxyurea therapy has shown clinical improvement for sicke cell patients [7-9], diferences in response i.e., the increase in Hb F levels, are observed and accurate predictors of hydroxyurea efcacy do not currently exist [10]. When hydroxyurea is administrated orally at a dose of 20 mg/kg, plasma concentrations normally reach a peak within 2 h (T max ) with a mean maximal concentration of 26 mg/L (C max ). Nevertheless, two phenotypes have recently been described: a “fast” (T max of 15 or 30 min) and a “slow” one (T max of 60 or 120 min) [10]. Pharmacokinetics studies are therefore required to explain these diferences and to establish prediction factor. Several methods have been developed to quantify hydroxyurea in biological samples: colorimetric techniques [10], liquid chromatography coupled with UV spectrophotometry [11,12], GC-MS [13,14], reversed phase liquid chromatography coupled with mass spectrometry [15]. Colorimetric methods require sample volumes of 250-500 µL and are insensitive. GC-MS needs a preliminary derivatization of hydroxyurea. Here we describe the development and validation of a simple liquid chromatography method with electrochemical detection to quantify hydroxyurea in aqueous solutions and plasma. Material and Methods Chemicals and pre-analytical treatment A stock solution of hydroxyurea (Sigma Aldrich, Steinheim, Germany) at 1000 mg/L was prepared and kept at -20°C. Aqueous and plasma standards were prepared by dilution of the stock solution in distilled water or human plasma pool. Tiourea, methylurea, 2-thiouracil and thioacetamide (Sigma Aldrich, Steinheim, Germany) were tested as internal standard. Te solutions were prepared at a fnal concentration of 100 mg/L containing 3 g/L albumin (Behring Institut, Marburg, Germany). 10 µL of samples or standards were added with 10 µL of internal standard solution and 100 µL acetonitrile (Biosolve, Dieuze, France). Afer centrifugation at 4000 rpm during 10 minutes, 80 µL of supernatant were recovered and either diluted to 400 µL with mobile phase or dried under nitrogen and reconstituted in 400 µL of mobile phase. Two mobile phases were tested. Te frst one was described by Pujari and collaborators [16] and was composed of 0.2 M perchlorate and methanol 95/5 (V/V) (Sigma Aldrich, Steinheim, Germany). Te Journal of Chromatography Separation Techniques J o u r n a l o f C h r o m a t o g r a p h y & S e p a r a t i o n T e c h n i q u es ISSN: 2157-7064