Colorimetric determination of hydroxyurea in human serum using high-performance liquid chromatography.
A high-performance liquid chromatography assay for hydroxyurea in human serum was developed based on a commercial colorimetric assay kit for urea (Sigma Diagnostics). Serum (0.5 ml), spiked with methylurea as an internal standard, was treated with 70% perchloric acid. Supernatant (0.2 ml) was combined with 0.7 ml of BUN acid reagent and 0.6 ml of BUN color reagent. The resulting colored reactant (100 microl) was analyzed on a 300 x 3.9 mm Bondclone 10 C18 column coupled with a UV-Vis detector, at 449 nm. The mobile phase was 13% acetonitrile in water. Retention times of colored derivatives of hydroxyurea and methylurea were 6.5 and 12.2 min, respectively. The log-log calibration curve was linear from 0.0065 to 1.31 mM. Average accuracy was 99.9+/-4.0% and the intra- and inter-day error of assay did not exceed 11%.
Manouilov
K K
KK
UNMC/Eppley Cancer Center Core Pharmacokinetics Laboratory, Department of Pharmaceutical Sciences, College of Pharmacy, The University of Nebraska Medical Center, Omaha 69198-6025, USA.
McGuire
T R
TR
Gwilt
P R
PR
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003124:Colorimetry / Q000379:methods*; D006801:Humans; D006918:Hydroxyurea / Q000097:blood*; D015203:Reproducibility of Results
D006918:Hydroxyurea