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 combin...

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Bibliographic Details
Published in:Journal of chromatography. B, Biomedical sciences and applications Biomedical sciences and applications, 1998-04, Vol.708 (1), p.321-324
Main Authors: Manouilov, Konstantine K, McGuire, Timothy R, Gwilt, Peter R
Format: Article
Language:English
Subjects:
Analysis
Biological and medical sciences
Chromatography, High Pressure Liquid - methods
Colorimetry - methods
General pharmacology
Humans
Hydroxyurea
Hydroxyurea - blood
Medical sciences
Pharmacology. Drug treatments
Reproducibility of Results
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Summary: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 μl) was analyzed on a 300×3.9 mm Bondclone 10 C 18 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 m M. Average accuracy was 99.9±4.0% and the intra- and inter-day error of assay did not exceed 11%.
ISSN:0378-4347
1387-2273
DOI:10.1016/S0378-4347(97)00634-8