Homogeneous liquid liquid extraction using salt as mass separating agent for the Ultra High Pressure Liquid Chromatographic determination of doxorubicin in human urine

Abstract In the present report, a homogeneous liquid liquid extraction using salt as mass separating agent was developed for the determination of the anticancer drug doxorubicin in human urine. Sample preparation is based on the usage of hydrophilic acetonitrile as extraction solvent and its phase separation under high salinity conditions. The analysis of doxorubicin and its epimer, epirubicin, (used as internal standard) was carried out by Ultra High Pressure Liquid Chromatography coupled to fluorimetric detection (λex/λem = 430/580 nm). Total sample analysis was accomplished in less than 15 min including the sample preparation and LC separation steps. The parameters affecting the extraction efficiency and method sensitivity were systematically investigated and optimized using experimental design. The method was validated in-house using the accuracy profiles as a graphical decision-making tool. The β-expectation tolerance intervals did not exceed the acceptance criteria of ± 15% meaning that 95% of future results will be included in the defined bias limits. The limit of detection (LOD) of the method was adequate corresponding to 10 ng mL−1. The mean analytical bias (expressed as relative recoveries) in the spiking levels was acceptable being in the range of 94.7 – 106.3% while the relative standard deviation (RSD) was lower than 5.1%. Experimental designs were built to examine the robustness of the UHPLC method. The proposed method has been satisfactorily applied to the analysis of the selected drug in human urine samples.