A simple and sensitive preconcentration strategy using sequential electrokinetic and hydrodynamic injection modes in micellar electrokinetic chromatography with diode array detection was developed and applied for the separation and determination of anticancer agent, 5‐fluorouracil and its metabolite, 5‐fluoro‐2′‐deoxyuridine, in human plasma. Sequential injection modes with increased analyte loading capacity using the anionic pseudo‐stationary phase facilitated collection of the dispersed neutral and charged analytes into narrow zones and improved sensitivity. Several important parameters affecting sample enrichment performance were evaluated and optimized in this study. Under the optimized experimental conditions, 614‐ and 643‐fold and 782‐ and 803‐fold sensitivity improvement were obtained for 5‐fluorouracil and its metabolite when compared with normal hydrodynamic and electrokinetic injection, respectively. The method has good linearity (1–1,000 ng/ml) with acceptable coefficient of determination (r2 > 0.993), low limits of detection (0.11–0.14 ng/ml) and satisfactory analyte relative recovery (97.4–99.7%) with relative standard deviations of 4.6–9.3% (n = 6). Validation results as well as the application to analysis of human plasma samples from cancer patients demonstrate the applicability of the proposed method to clinical studies.
5-fluorouracil (5-FU) refers to a fluorinated pyrimidine analogue that has been widely used as an anticancer agent for colon, head, and neck cancers. Detection of 5-FU and its metabolites; 5-fluorouridine and 5-fluoro-2-deoxyuridine in biological samples allows optimization of pharmacotherapy and encourages fundamental investigations of this medication. The development of accurate and reliable sample preparation, as well as analytical methods, is critical to isolate targeted analytes from complex matrices, apart from increasing detection sensitivity of analytes. With that, this paper presents a review of prior studies pertaining to chromatographic and electrophoretic methods that focused on the analysis of 5-FU and its metabolites in biological matrices such as plasma and urine. This paper concentrates on HPLC, GC and CE systems, which are the most commonly used strategies for analytical separation of 5-FU and its metabolites from samples. Detection of these antineoplastic agents at trace level demands highly sensitive and selective analytical methodologies. Application of these analytical techniques to biological matrices is reviewed with a focus on method development strategies, including types of mobile phases and background electrolytes employed in LC and CE systems.
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