alexa Validated liquid chromatographic determination of 5-fluorouracil in human plasma.
Chemical Engineering

Chemical Engineering

Journal of Chromatography & Separation Techniques

Author(s): Alsarra IA, Alarifi MN

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Abstract A sensitive, reproducible, selective and accurate high performance liquid chromatographic (HPLC) method for the quantitative determination of 5-flurorouracil in plasma has been developed and validated using isocratic elution and UV detection. The method provides a selective quantifications of 5-flurorouracil without any interference of the endogenous uracil. The assay is performed after a double extraction of 5-flurorouracil and thymine (internal standard) from human plasma using ethyl acetate. The drug and the internal standard were eluted from a Genesis C(18) analytical column at ambient temperature with mobile phase consisting of methanol:water (10:90, v/v) adjusted to pH 3.2 with perchloric acid at a flow rate of 1.0 ml/min. The effluent was monitored with an ultraviolet detector at 260 nm. Quantification was achieved by the measurement of the peak-height ratios and the limit of quantification for 5-flurorouracil in plasma was 30 ng/ml. The retention times for 5-flurorouracil, uracil, and thymine were 4.5, 6.0, and 9.0, respectively. The intra-day coefficient of variation (CV) ranged from 1.35 to 4.53\% at three different concentrations and the inter-day CVs varied from 1.29 to 4.98\%. The relative and absolute recoveries varied from 96 to 101\%. Stability tests showed that 5-flurorouracil is stable for at least 72 h in plasma after freezing. The simple method may permit the assessment of 5-flurorouracil plasma concentrations for pharmacokinetic studies in combination with clinical trials. This article was published in J Chromatogr B Analyt Technol Biomed Life Sci and referenced in Journal of Chromatography & Separation Techniques

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