Mass spectrometric characterization of tamoxifene metabolites in human urine utilizing different scan parameters on liquid chromatography/tandem mass spectrometry

Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197 Rome, Italy.
Rapid Communications in Mass Spectrometry (Impact Factor: 2.25). 03/2010; 24(6):749-60. DOI: 10.1002/rcm.4432
Source: PubMed


Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were considered for the characterization of tamoxifene metabolites in human urine for anti-doping purpose. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 166, 152 and 129) and neutral loss scan (neutral loss of 72 Da and 58 Da) in positive ion mode were assessed to recognize common ions or common losses of tamoxifene metabolites. The applicability of these methods was checked first by infusion and then by the injection of solution of a mixture of reference standards of four tamoxifene metabolites available in our laboratory. The data obtained by the analyses of the mixture of the reference standards showed that the five methods used exhibited satisfactory results for all tamoxifene metabolites considered at a concentration level of 100 ng/mL, whereas the analysis of blank urine samples spiked with the same tamoxifene metabolites at the same concentration showed that the neutral loss scan of 58 Da lacked sufficient specificity and sensitivity. The limit of detection in urine of the compounds studied was in the concentration range 10-100 ng/mL, depending on the compound structure and on the selected product ion. The suitability of these approaches was checked by the analysis of urine samples collected after the administration of a single dose of 20 mg of tamoxifene. Six metabolites were detected: 4-hydroxytamoxifene, 3,4-dihydroxytamoxifene, 3-hydroxy-4-methoxytamoxifene, N-demethyl-4-hydroxytamoxifene, tamoxifene-N-oxide and N-demethyl-3-hydroxy-4-methoxytamoxifene, which is in conformity to our previous work using a time-of-flight (TOF) mass spectrometer in full scan acquisition mode.

Download full-text


Available from: Roberto Di Santo, Apr 18, 2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: There is increasing evidence that the clinical efficacy of tamoxifen, the first and most widely used targeted therapy for estrogen-sensitive breast cancer, depends on the formation of the active metabolites 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen (endoxifen). Large inter-individual variability in endoxifen plasma concentrations has been observed and related both to genetic and environmental (i.e. drug-induced) factors altering CYP450s metabolizing enzymes activity. In this context, we have developed an ultra performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) requiring 100 μL of plasma for the quantification of tamoxifen and three of its major metabolites in breast cancer patients. Plasma is purified by a combination of protein precipitation, evaporation at room temperature under nitrogen, and reconstitution in methanol/20 mM ammonium formate 1:1 (v/v), adjusted to pH 2.9 with formic acid. Reverse-phase chromatographic separation of tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen is performed within 13 min using elution with a gradient of 10 mM ammonium formate and acetonitrile, both containing 0.1% formic acid. Analytes quantification, using matrix-matched calibration samples spiked with their respective deuterated internal standards, is performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of relative matrix effects variability, as well as tamoxifen and metabolites short-term stability in plasma and whole blood. The method is precise (inter-day CV%: 2.5-7.8%), accurate (-1.4 to +5.8%) and sensitive (lower limits of quantification comprised between 0.4 and 2.0 ng/mL). Application of this method to patients' samples has made possible the identification of two further metabolites, 4'-hydroxy-tamoxifen and 4'-hydroxy-N-desmethyl-tamoxifen, described for the first time in breast cancer patients. This UPLC-MS/MS assay is currently applied for monitoring plasma levels of tamoxifen and its metabolites in breast cancer patients within the frame of a clinical trial aiming to assess the impact of dose increase on tamoxifen and endoxifen exposure.
    No preview · Article · Dec 2010 · Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
  • [Show abstract] [Hide abstract]
    ABSTRACT: Liquid chromatography, coupled with single-stage or tandem mass spectrometry, is a powerful tool increasingly used in analytical toxicology. However, the atmospheric pressure ionization processes involved are complex, and subject to interference from matrix components, for example. Further, the techniques used in sample preparation, chromatography and mass analysis are developing rapidly. An understanding of the advantages and limitations of LC-MS ensures appropriate analyses are performed, and that reliable results are generated. Consideration should be given to the influence of the sample preparation and chromatographic conditions on the ionization of the analyte at the mass spectrometer interface. This review aims to provide some practical guidance and examples to aid method development for commonly encountered analytes in analytical toxicology.
    No preview · Article · Feb 2011 · Biomedical Chromatography
  • [Show abstract] [Hide abstract]
    ABSTRACT: The timely update of the list of prohibited substances and methods of doping (as issued by the World Anti-Doping Agency) is an essential aspect of international anti-doping efforts and represents consensual agreement by expert panels regarding substances and the methods of performance manipulation in sports. The annual banned-substance review for human doping controls critically summarizes recent innovations in analytical approaches; its purpose is to improve the quality of doping controls by reporting emerging and advancing methods that focus on detecting known and recently outlawed substances. This review surveys new and/or enhanced procedures and techniques of doping analysis together with information relevant to doping control that has been published in the literature between October 2009 and September 2010.
    No preview · Article · Jan 2011 · Drug Testing and Analysis
Show more