Determination of topotecan in human whole blood and unwashed erythrocytes by high-performance liquid chromatography

A reversed-phase HPLC method for the quantitative determination of total topotecan in human whole blood and unwashed erythrocytes has been developed and validated in terms of sensitivity, specificity, precision and accuracy. Linear calibration curves were constructed in the range of 0.20 to 50.0 ng/ml. The sample pre-treatment for whole blood involved a two-step extraction with methanol and perchloric acid. Prior to extraction, erythrocytes were separated from other blood components by centrifugation in MESED instruments. Separations were achieved on an Inertsil ODS-80A analytical column (150×4.6 mm, 5 μm particle size), eluted at 50°C and a flow-rate of 1.00 ml/min, with a mixture of 100 mM ammonium acetate (pH 6.0)–tetrahydrofuran (94.6:5.4, v/v). Fluorescence detection was performed using excitation and emission wavelengths of 381 and 525 nm, respectively. With the applied method, 80% of topotecan was extracted out of whole blood. The lower limit of quantitation in whole blood was established at 0.20 ng/ml with within-run and between-run precisions, respectively, ranging from 1.7 to 9.3% and 1.5–6.1%, while the accuracy ranged from 100 to 113%. The described method will be used in clinical studies to explore the role of erythrocytes in the overall kinetic behavior of topotecan.