Development and validation of a liquid chromatography–tandem mass spectrometry method for topotecan determination in beagle dog plasma and its application in a bioequivalence study

Abstract

Topotecan (TPT) is an important anti-cancer drug that inhibits topoisomerase I. A sensitive and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that potentially determines TPT in beagle dog plasma is needed for a bioequivalence study of TPT formulations. We developed and validated LC-MS/MS to evaluate TPT in beagle dog plasma in terms of specificity, linearity, precision, accuracy, stability, extraction recovery and matrix effect. Plasma samples were treated with an Ostro(TM) sorbent plate (a robust and effective tool) to eliminate phospholipids and proteins before analysis. TPT and camptothecin (internal standard) were separated on an Acquity UPLC BEH C18 column (1.7 µm, 2.1 × 50 mm) with 0.1% formic acid and methanol as the mobile phase at a flow rate of 0.25 mL/min. TPT was analyzed using positive ion electrospray ionization in multiple-reaction monitoring mode. The obtained lower limit of quantitation was 1 ng/mL (signal-to-noise ratio > 10). The standard calibration curve for TPT was linear (correlation coefficient > 0.99) at the concentration range of 1-400 ng/mL. The intra-day and inter-day precision, accuracy, stability, extraction recovery and matrix effect of TPT were within the acceptable limits. The validated method was successfully applied in a bioequivalence study of TPT in healthy beagle dogs.

Pharmacokinetic Comparison of Three Different Administration Routes for Topotecan Hydrochloride in Rats

“…The most abundant (with consistent and stable) fragment ion for multiple reaction monitoring (MRM) was m / z 422.10 → 377.15 for topotecan lactone form and m / z 453.20 → 230.30 for IS. This MRM transition of topotecan lactone form was consistent (as 422.1 → 377 in positive MRM mode) with previous reports [ 22 , 23 ]. Figure S2 shows the relevant mass spectra (including selected precursor and product ions).…”

“…The ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometer (UPLC-ESI-MS/MS) method was preferentially established in this study to quantitatively analyze the lactone or total form of topotecan from biological samples (plasma, urine, feces). The UPLC-ESI-MS/MS assay used in this study was established with reference to previously reported topotecan bioassays [ 22 , 23 ] with some modifications and improvements. In particular, mobile phase composition, gradient elution program, run time, and extraction solvent composition were optimized for our analysis system.…”

“…Quantification of total topotecan was relatively easy and simple through acidification of biosamples using 10% ( v / v ) aqueous formic acid. In previous studies, the same method was used to efficiently quantify total topotecan from plasma [ 22 , 23 ]. In this study, there was little difference when comparing the topotecan peak area in the acidified sample with the same concentration of the topotecan standard solution.…”

“…Plasma, urine, and feces concentrations of topotecan were determined using an established UPLC-ESI-MS/MS method. The analytical methods used in this study (including chromatographic conditions and sample preparation) were optimized and applied to our analytical conditions by referring to previous studies [ 22 , 23 ] that most closely matched our system. Briefly, 10 μL of IS (repaglinide, 10 ng/mL in plasma) and 500 μL of cold (−20 °C) methanol were added to 100 μL of samples (plasma, urine, and feces).…”

Mass spectrometry in the pharmacokinetic studies of anticancer natural products

“…The analytical methods for topotecan quantification in human plasma have involved liquid chromatography almost always with fluorescence detection (Rosing et al, ; Rosing et al, ; Bai et al, ; de Vries et al, ; Vali et al, ) with only two procedures based on mass spectrometry (Baczek et al, ; Li et al, ). However, mass spectrometry was successfully applied also for the determination of TPT in other matrices such as: rat serum (Shin et al, ), rat everted gut sacs (Arellano et al, ), and beagle dog plasma (Ye et al, ). To the best of our knowledge Baczeck et al (2012) was the first to apply mass spectrometry detection for the determination of topotecan in human plasma.…”