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

Abstract

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.

Mass spectrometric characterization of urinary toremifene metabolites for doping control analyses

“…Metabolic pathways detected for toremifene include N-desmethylation, hydroxylation, dihydroxylation, reduction, methylation and dehalogenation and combinations of them. Some of proposed metabolic pathways (N,N-didesmethylation, N-desmethylation + hydroxylation + hydroxymethylation, hydroxy-quinine and hydroxylation + oxidation) were not detected (Table 1) despite the fact that they were previously reported for tamoxifen, clomifene and toremifene [3,4,14,[18][19][20][21]25,[28][29][30]. As it can be observed in Table 1, N-desmethylated and dehalogenated metabolites were detected mainly in free form.…”

“…Metabolic pathways studied were based on those metabolites reported in previous studies for toremifene [3,13,14,20,21,24,26,[28][29][30] and other related compounds, such as tamoxifen and clomifene [3,4,17,[31][32][33][34][35][36][37][38][39][40][41]. All previously reported metabolic pathways and their combination were included in SRM method, with the main exception of those proposed after metabolic removal of the amine function because of no ionization in positive ion mode was expected for these compounds [13][14][15][18][19][20][21][22][23][24][25]27,42] ( Table 1).…”

“…There are no well-established clinical indications for anti-estrogens in men [1,2]. In athletes the use of antiestrogenic compounds may compensate an extensive abuse of anabolic androgenic steroids [3,4]. For these reasons, the use of agents with anti-estrogenic activity has been banned in sports by the World Anti-doping Agency (WADA) [5], and anti-doping control laboratories have to be able to detect the administration of the drug.…”

“…Few analytical methods for the urinary detection of toremifene administration have been developed [3,14,[26][27][28][29]. Different metabolic pathways have been described as the most characteristic for toremifene and related compounds such as tamoxifen and clomifene including, hydroxylation, N-desmethylation, N,Ndidesmethylation, deaminohydroxylation, deaminocarboxylation, N-oxide formation, among others [3,4,17,[30][31][32][33][34][35][36][37][38][39][40][41][42]. However, no systematic study on toremifene metabolites appearing in urine after drug administration has been performed.…”

An ultra performance liquid chromatography–tandem MS assay for tamoxifen metabolites profiling in plasma: First evidence of 4′-hydroxylated metabolites in breast cancer patients

“…In that context, several analytical methods have been published for the monitoring of tamoxifen and its metabolites in human biological fluids, including GC-MS [35], CE-MS [36], conventional and micellar liquid chromatography methods coupled to fluorescence detection [37][38][39][40] and LC-MS/MS methods [41][42][43][44][45][46]. Reports have also been published describing liquid chromatography method coupled to mass spectrometry or fluorescence detection for the study of tamoxifen metabolism in vitro and in vivo [47][48][49][50][51][52][53][54]. For mass spectrometry techniques, conventional HPLC [42,45,46] and fast liquid chromatography coupled to tandem MS methods using monolithic [41] or small particles (3 m) packed columns [43,44] have been proposed for the quantification of tamoxifen and/or its metabolites.…”