Stable-Isotope Dilution HPLC-Electrospray Ionization Tandem Mass Spectrometry Method for Quantifying Hydroxyurea in Dried Blood Samples.
BACKGROUND
Sickle cell anemia (SCA) is a life-threatening blood disorder characterized by the presence of sickle-shaped 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.
Division of Hematology, Department of Pediatrics, and.
Anu
A
Marahatta
Department of Pathology and Laboratory Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
Vandana
V
Megaraj
Division of Hematology, Department of Pediatrics, and.
Patrick T
PT
McGann
Division of Hematology, Department of Pediatrics, and.
Russell E
RE
Ware
Department of Pathology and Laboratory Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, OH. kenneth.setchell@cchmc.org.
Kenneth D R
KD
Setchell
D000755:Anemia, Sickle Cell / Q000097:blood* / Q000188:drug therapy; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D059788:Dried Blood Spot Testing; D016903:Drug Monitoring; D006801:Humans; D006918:Hydroxyurea / Q000097:blood* / Q000627:therapeutic use; D011865:Radioisotope Dilution Technique; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
D006918:Hydroxyurea
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