Journal of Chromatography B: Biomedical Sciences and Applications
Quantification of topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces by high-performance liquid chromatographic methods
Introduction
Topotecan (Hycamtin™, Fig. 1) is a water-soluble anticancer analogue of camptothecin. The drug inhibits DNA replication and RNA transcription by stabilizing the cleavable complexes formed between the nuclear enzyme topoisomerase I and DNA [1], [2]. The lactone ring undergoes reversible hydrolysis which is pH-dependent (Fig. 1) [3]. It has been demonstrated that stabilization of the DNA–topoisomerase I complex, which is considered to mediate the antitumor activity of topotecan, requires the drug to be present in its lactone form [4]. Topotecan has been registered for the treatment of patients with metastatic carcinoma of the ovary after failure of first line chemotherapy [5]. Additionally, topotecan has shown clinical antitumor activity in the treatment of small cell lung cancer [6], [7]. Demethylation to form N-desmethyltopotecan has been identified as a metabolic pathway for topotecan in humans [8]. This metabolite was found in plasma and urine of patients treated with topotecan. Although various papers have been published on the quantification of topotecan [9], [10], [11], [12], these methodologies are not able to quantify N-desmethyltopotecan. Furthermore, no bio-analytical assays have been described for the determination of topotecan in faeces. We have developed and validated high-performance liquid chromatographic (HPLC) methods for the determination of topotecan and N-desmethyltopotecan in human plasma, urine and faeces in support of clinical studies to determine the pharmacokinetics and mass balance of topotecan.
Camptothecin derivatives are reversibly hydrolysed to their hydroxycarboxylate forms at physiologic pH. In vitro conversion is rapid and it is important that blood samples are collected in an appropriate fashion (rapid cooling, centrifuged under cooled conditions and subsequent protein precipitation) to fix the equilibrium for adequate quantitation of the lactone forms [9]. The plasma samples can then be acidified for the determination of the total levels of topotecan and N-desmethyltopotecan. The hydroxycarboxylate concentrations are determined by calculating the difference between these concentrations. Separate measurements of lactone and total levels in urine and faeces were considered irrelevant due to rapid interconversion reactions of the compounds during collection of these matrices.
Section snippets
Chemicals and control matrices
Topotecan (hydrochloride salt, SKF 104864-A, lot MM-15906-194, purity 89.2%) and N-desmethyltopotecan reference standard (hydrochloride salt, SB 209780-A, lot JW-19178-221A1, purity 86.5%) originated from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Acetonitrile and methanol (HPLC gradient grade) were obtained from Biosolve (Valkenswaard, The Netherlands). Potassium dihydrogen phosphate, disodium hydrogen phosphate di-hydrate, triethylamine, 37% (w/v) hydrochloric acid, citric acid
Optimization of the methods
The former assay for the analysis of topotecan in human plasma [10] could not be used for the quantitative determination of N-desmethyltopotecan due to insufficient resolution between both compounds. Like all known camptothecin derivatives, topotecan and N-desmethyltopotecan are reversibly hydrolysed to their carboxylate forms and it was the intention to develop a methodology enabling the quantification of both forms of the analytes in one analytical run. Using an end-capped Zorbax SB-C18
Conclusion
Accurate, precise and sensitive HPLC assays have been developed for the quantification of topotecan and N-desmethyltopotecan in several human matrices. These assays have been used to support a clinical study to determine the routes of elimination and disposition of topotecan in patients with malignant solid tumors. To minimize analyte degradation, blood samples taken from patients should be processed immediately and stored at −70°C. Under these conditions, stability data indicated that
References (14)
- et al.
J. Pharm. Biomed. Anal.
(1990) - et al.
J. Pharm. Biomed. Anal.
(1990) - et al.
J. Chromatogr. B
(1995) - et al.
J. Chromatogr. B
(1996) - et al.
J. Chromatogr. B
(1997) - et al.
J. Pharm. Biomed. Anal.
(1991) - et al.
J. Pharm. Sci.
(1992)
Cited by (37)
Topotecan exposure estimation in pediatric acute myeloid leukemia supported by LC-MS-based drug monitoring and pharmacokinetic analysis
2012, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :Noteworthy, the sample preparation time was also short (10 min) requiring only one-step liquid–liquid extraction and low volume of plasma samples (100 μl). This enabled designing the method so that it became quicker and more economical than in other previously published articles [14–20]. The method was validated, and it was fully compliant with the requirements of the bioanalytical methods.
Phase i and pharmacologic study of weekly bolus topotecan for advanced non-small-cell lung cancer
2010, Clinical Lung CancerCitation Excerpt :A urine sample was collected before the initial dose was administered and from cumulative urine collected from 0 to 24 hours and from 24 to 48 hours after the first dose during the first cycle, and 10 mL aliquots were stored at < −30°C until analyzed. The plasma concentration of the lactone form of topotecan; the total plasma concentration of topotecan (sum of the lactone form and carboxylate form); the plasma concentration of N-desmethyl topotecan, an active metabolite of topotecan20; the total urinary concentration of topotecan; and the total urinary concentration of N-desmethyl topotecan were determined by high-performance liquid chromatography (HPLC) with fluorescence detection according to the method described by Rosing et al.21 Briefly, separation was achieved on reversed-phase columns, and detection was performed fluorometrically with excitation of 380 nm and emission of 527 nm. The lower limit for quantitative determination of topotecan in plasma (lactone and total topotecan) and in urine was 0.22 nmol/L and 55 nmol/L, respectively, and the limit for N-desmethyl topotecan in plasma (lactone and total topotecan) and urine was 0.23 nmol/L and 5.6 nmol/L, respectively.
Concurrent determination of topotecan and model permeability markers (atenolol, antipyrine, propranolol and furosemide) by reversed phase liquid chromatography: Utility in Caco-2 intestinal absorption studies
2007, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesSimple and sensitive high performance liquid chromatographic method for the simultaneous quantitation of the lactone and carboxylate forms of topotecan in human plasma
2005, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesHigh-performance liquid chromatographic assay for the determination of total and free topotecan in the presence and absence of anti-topotecan antibodies in mouse plasma
2005, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesLiquid chromatography determination of 10-hydroxycamptothecin in human serum by a column-switching system containing a pre-column with restricted access media and its application to a clinical pharmacokinetic study
2003, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences