Quantification of tamoxifen and three metabolites in plasma by high-performance liquid chromatography with fluorescence detection: application to a clinical trial

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Abstract

A sensitive and reproducible assay employing liquid–liquid extraction and high-performance liquid chromatography with fluorescence detection for the quantification of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen, and Z-4-hydroxy-N-desmethyltamoxifen in human plasma is described. The compounds and internal standard, propranolol, were separated with a cyano column and a mobile phase of acetonitrile–20 mM potassium phosphate buffer (pH 3; 35:65, v/v) then detected with fluorescence using a modified version of a method originally described by Fried and Wainer [J. Chromatogr. B 655 (1994) 261]. The coefficients of variation for the midpoint of the standard curve for each compound were less than 10%. This method was applied to a pharmacokinetic study of tamoxifen disposition in breast cancer patients.

Introduction

Tamoxifen [trans-1-(4-β-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene], a non-steroidal triphenylethylene, is currently the endocrine therapeutic agent of choice for all stages of breast cancer and has also been approved in the United States for use as a chemopreventive agent in women at high risk for the disease. The metabolism of tamoxifen could play an important role in modulating the biological activity of the drug because active metabolites may interact with the parent drug at the estrogen receptors. It has been known for many years that tamoxifen is extensively metabolized by hepatic cytochrome P450 (CYP) in humans [2], [3], [4], [5], [6], [7], [8], [9]. The structures of tamoxifen and its major metabolites are illustrated in Fig. 1 [10].

The widespread use of tamoxifen has stimulated efforts to develop routine assays for this drug and its metabolites in human plasma. Procedures based on gas chromatography with mass spectrometry are highly specific, but require derivatization of sample and involve equipment not generally available [11]. Thin-layer and high-performance liquid chromatographic (HPLC) methods [10], [12], [13] involve photochemical conversion of tamoxifen and its metabolites to fluorescent phenanthrene derivatives [14]. In 1994, Fried and Wainer [1] reported a HPLC assay that incorporates post column fluorescent activation and that avoids problems related to the variable photochemical degradation of the phenanthrenes. This method applies solvent to deproteinate the sample, an expeditious procedure that prevents degradation of parent compound and metabolites, that is mixed and spun prior to injection into the HPLC [1]. The method we report here represents an evolution of the Fried and Wainer [1] method with three major differences; the incorporation of an internal standard in the assay, the use of a liquid–liquid extraction procedure to further purify and allow concentration of the sample, and the quantification of 4-hydroxy-N-desmethyltamoxifen, a metabolite that may have anti-cancer activity. Thus, a sensitive and reproducible HPLC assay with fluorescence detection using propranolol as the internal standard for the quantification of tamoxifen and three metabolites in human plasma is described in this paper.

Section snippets

Chemicals and reagents

Tamoxifen, 4-hydroxytamoxifen, and propranolol were purchased from Sigma (St. Louis, MO, USA). N-Desmethyltamoxifen was a gift from Dr Irving W. Wainer (Department of Pharmacology, Georgetown University, Washington, DC, USA). 4-Hydroxy-N-desmethyltamoxifen (approximately 20 mg) was synthesized in our laboratory according to a previously published method with slight modifications using Z-4-hydroxytamoxifen (purity >98%) as a starting material [15]. The Z-isomer of 4-hydroxy-N-desmethyltamoxifen,

Chromatography

Z-4-hydroxy-N-desmethyltamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, and tamoxifen exhibited good chromatography with baseline resolution of each compound (Fig. 2, Fig. 3). The retention times for Z-4-hydroxy-N-desmethyltamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, and tamoxifen were 29.9, 32.3, 53.3, and 58.5 min, respectively.

Many compounds were tested for possible use as an internal standard, e.g. nelfinavir, dextromethorphan, imipramine, and propranolol. Propranolol exhibited

Conclusions

The method presented here describes a specific, sensitive and reproducible human plasma assay using HPLC with internal standard and fluorescence detection. The major evolutionary changes of this assay relative to its predecessor [1] are the incorporation of an internal standard in the assay, the use of a liquid–liquid extraction procedure to further purify and allow concentration of the sample and the quantification of 4-hydroxy-N-desmethyltamoxifen. This method should make it possible to

Acknowledgments

This work was supported in part by a Pharmacogenetic Network Grant from the NIGMS, Bethesda, MD, USA (U-01-GM61373) and by a Clinical Pharmacology training grant to the Division of Clinical Pharmacology at Indiana University School of Medicine (T-32-GM08425). The contribution of Kyung-Hoon Lee, MD, PhD has been made possible by the postdoctoral fellowship program of the Korean Science and Engineering Foundation (KOSEF). Data from these studies have been deposited at the Pharmacogenetics Network

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