Development of A Liquid Chromatographic Method for Quantitative Determination of Triapine, A Ribonucleotide Reductase Inhibitor, by Spectrophotometric Study of Triapine Complexation Reaction

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Journal of Liquid Chromatography & Related Technologies


Triapine is a novel inhibitor of ribonucleotide reductase (RR). It can remove ferric ion from the catalytic di-iron-tyrosyl radical center of RR, inhibit de novo synthesis of deoxyribonucleotides, and impair the required ratio of DNA to cell mass during cell division and DNA repair. Recent studies indicate that triapine is particularly useful for enhancing the radiation-mediated cytotoxicity in cervical and colon cancers; hence, the interest of further clinical study of this compound is high. Due to its chelating property, quantitative measurement of triapine is an analytical challenge, which is affected by multi-factors that influence the complexation reaction between triapine and ferric ion. In this work, the effects of pH, buffer composition, and EDTA, as well as other metal ions on the spectral absorptivity and chromatographic retention of triapine have been investigated. The optimized conditions for analytical measurement of triapine have been applied to the development of an LC-UV method for quantitation of triapine, in which hydrazinecarbothioamide is used as an internal standard. Both triapine and the internal standard can be separated on a Waters Xterra RP18 column (2.1 × 150 mm, 5 µm particle size) by a mobile phase of 18.0% acetonitrile and 82.0% ammonium bicarbonate-EDTA buffer [i.e., 10.0 mM (NH4)HCO3 and 5.00 mM EDTA (v/v) at pH 8.5]. The detection of triapine and the internal standard is carried out by UV detector at wavelength of 360 nm. This method has a linear calibration range from 3.00 to 1.00 × 103 ng mL−1 with inter-assay precision (%CV) and accuracy (%RE) of 2–4% and −3–9%, respectively. This work examines the underlying factors affecting quantitative determination of triapine and provides an accurate and reliable method for quantitation of triapine.