Application of a Highly Specific and Sensitive Fluorescent HPLC Method for Topotecan Lactone in Whole Blood

Individualization of topotecan dosing reduces inter-patient variability in topotecan exposure, presumably reducing toxicity and increasing efficacy. However, logistical limitations (e.g., requirement for plasma, intensive bedside plasma processing) have prevented widespread application of this approach to dosing topotecan. Thus, the objectives of the present study were to develop and validate an HPLC with fluorescence detection method to measure topotecan lactone in whole blood samples and to evaluate its application to individualizing topotecan dosing. Plasma samples (200 μL) were prepared using methanolic precipitation, a filtration step, and then injection of 100 μL of the methanolic extract onto a Novapak® C18, 4μm, 3.9 × 150 mm column with an isocratic mobile phase. Analytes were detected with a Shimadzu Fluorescence RF-10AXL detector with an excitation and emission wavelength of 370 nm and 520 nm, respectively. This method has a lower limit of quantification of 1 ng/mL (S/N ≥5; RSD 4.9%), was validated over a linear range of 1 to 100 ng/mL, and results from a 5-day validation study demonstrated good within-day and between-day precision and accuracy. Data are presented to demonstrate that the present method can be used with whole blood samples to individualize topotecan dosing in children with cancer.