Stable-Isotope Dilution HPLC-Electrospray ionization tandem mass spectrometry method for quantifying hydroxyurea in dried blood samples

Abstract

BACKGROUND: Sickle cell anemia (SCA) is a life-Threatening blood disorder characterized by the presence of sickleshaped erythrocytes. Hydroxyurea is currently the only US Food and Drug Administration-approved treatment and there is a need for a convenient method to monitor compliance and hydroxyurea concentrations, especially in pediatric SCA patients. METHODS: We describe a novel approach to the determination of hydroxyurea concentrations in dried whole blood collected on DMPK-C cards or volumetric absorptive microsampling (VAMS) devices. Hydroxyurea was quantified by electrospray ionization LC-MS/MS using [13C15N2]hydroxyurea as the internal standard. Calibrators were prepared in whole blood applied to DMPK-C cards or VAMS devices. RESULTS: Calibration curves for blood hydroxyurea measured from DMPK-C cards and VAMS devices were linear over the range 0.5- 60 μg/mL. Interassay and intraassay CVs were <15% for blood collected by both methods, and the limit of detection was 5 ng/mL. Whole blood hydroxyurea was stable for up to 60 days on DMPK-C cards and VAMS devices when frozen at-20 °C or-80 °C. Whole blood hydroxyurea concentrations in samples collected on DMPK-C cards or VAMS devices from SCA patients were in close agreement. CONCLUSIONS: This tandem mass spectrometry method permits measurement of hydroxyurea concentrations in small volumes of dried blood applied to either DMPK-C cards or VAMS devices with comparable performance. This method for measuring hydroxyurea from dried blood permits the evaluation of therapeutic drug monitoring, individual pharmacokinetics, and medication adherence using heel/finger-prick samples from pediatric patients with SCA treated with hydroxyurea.