Comparison of Filter and Tunable Fluorescence Detection for the HPLC Simultaneous Quantitation of Lactone and Carboxylate Forms of Topotecan in Plasma

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Abstract
The hydrolysis of the α-hydroxy-δ-lactone ring moiety in topotecan is routinely monitored using high performance liquid chromatography (HPLC) with fluorescence detection. While both tunable and filter fluorescence detectors are commercially available, only the tunable detector has been studied for clinical and in vitro applications of topotecan. In the present study we have developed a simple HPLC method for the simultaneous separation of the lactone and carboxylate forms of topotecan in plasma, which, can be utilized for both clinical and in vitro studies. Limits of detection, percent relative standard deviation, and linear range for both the lactone and carboxylate forms of the drug in plasma are presented and compared using a tunable and filter fluorescence detector. Limits of detection in plasma of 0.10 ng/mL for carboxylate and 0.26 ng/mL for lactone have been obtained using a tunable fluorescence detector. A filter fluorescence detector produced limits of detection of 0.15 ng/mL for carboxylate and 0.30 ng/mL for lactone. Reproducible quantitation using a tunable fluorescence detector from 0.25 to 250 ng/mL for carboxylate and from 0.50 to 250 ng/mL for lactone was achieved, which is an improvement over existing methods. The filter detector, which has not been previously studied, provided reproducible detection from 0.50 to 250 ng/mL for carboxylate and from 0.75 to 250 ng/mL for lactone.

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