Determination of plasma topotecan and its metabolite N-desmethyl topotecan as both lactone and total form by reversed-phase liquid chromatography with fluorescence detection

Topotecan (TPT) undergoes hepatic N-demethylation forming N-desmethyl topotecan (NDS). To evaluate the effect of drug-drug interactions on NDS disposition in children receiving TPT we developed and validated a sensitive and specific HPLC-fluorescence detection method for lactone and total (lactone plus carboxylate) TPT and NDS. Deproteinized plasma is vortexed, centrifuged, and the methanolic extract diluted with water for the lactone form of NDS and TPT or diluted with 1.5% phosphoric acid for NDS and TPT total. A 100 μL sample is injected onto a Varian ChromGuard RP column attached to an Agilent SB-C₁₈ reversed-phase analytical column held at 50°C. The mobile phase (flow-rate, 0.8 mL/min) consists of methanol-aqueous buffer (27:73, v/v) (75 mM potassium phosphate and 0.2% triethylamine, pH 6.5). TPT and NDS were detected with excitation and emission wavelengths set at 376 and 530 nm, respectively. The standard curves for both forms of TPT ranged from 0.25 to 80 ng/mL, and for NDS ranged from 0.10 to 8.0 ng/mL. Within-day and between-day precision (% RSD) was ≤4% for TPT and ≤6.2% for NDS, respectively. Within-day and between-day percentage error ranged from 1.4 to 6.3% and from 1.4 to 2.4% for TPT, and from 1.6 to 3.1% and from 0.0 to 3.7% for NDS, respectively. No significant on-column conversion from TPT or NDS lactone to carboxylate was observed. With one method we can measure lactone and total TPT and NDS with adequate sensitivity to allow for evaluation of the disposition of these compounds in children receiving TPT.