Research article Received: 6 March 2014
Revised: 9 April 2014
Accepted: 10 April 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3375
Structural elucidation of new urinary tamoxifen metabolites by liquid chromatography quadrupole time-of-flight mass spectrometry Jianghai Lu,a*† Chunji He,a† Genye He,a Xiaobing Wang,a Youxuan Xu,a Yun Wu,a Ying Donga and Gangfeng Ouyangb In this study, tamoxifen metabolic profiles were investigated carefully. Tamoxifen was administered to two healthy male volunteers and one female patient suffering from breast cancer. Urinary extracts were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry using full scan and targeted MS/MS techniques with accurate mass measurement. Chromatographic peaks for potential metabolites were selected by using the theoretical [M + H]+ as precursor ion in full-scan experiment and m/z 72, 58 or 44 as characteristic product ions for N,N-dimethyl, N-desmethyl and N, N-didesmethyl metabolites in targeted MS/MS experiment, respectively. Tamoxifen and 37 metabolites were detected in extraction study samples. Chemical structures of seven unreported metabolites were elucidated particularly on the basis of fragmentation patterns observed for these metabolites. Several metabolic pathways containing mono- and di-hydroxylation, methoxylation, N-desmethylation, N,N-didesmethylation, oxidation and combinations were suggested. All the metabolites were detected in the urine samples up to 1 week. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s website. Keywords: tamoxifen; new metabolites; doping control; human urine; liquid chromatography quadruple time-of-flight mass spectrometry
Introduction
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Tamoxifen, a first-generation selective estrogen receptor modulator with a triphenylethylene structure is widely used for adjuvant breast cancer therapy.[1,2] It fights certain types of breast cancer, called hormone response or estrogen positive breast cancer, by blocking the effects of the hormone estrogen in the body. This prevents the growth of the types of breast cancer cells that require estrogen for growth and survival.[3,4] It is also used to prevent the recurrence of breast cancer in women who have been treated for the disease and treat breast cancer that has been spread to other parts of the body.[5] Since 2005, tamoxifen was included, with other similar drugs, in the Section 4 ‘agents with anti-oestrogenic activity’ of the World Anti-Doping Agency list of prohibited substances and methods.[6] Tamoxifen is extensively metabolized by the CYP3A4 and 2D6 isoforms.[7–9] The clinical goodness of tamoxifen is thought to arise from its transformation to active metabolites, mainly 4-hydroxy tamoxifen and 4-OH-N-desmethyl tamoxifen.[10–12] These metabolites bind to the estrogen receptor with up to 30-fold greater affinity than tamoxifen, which increased potency for binding translates into enhanced antiestrogen activity.[13–16] Main metabolic pathways included epoxidation, N-desmethylation, hydroxylation, dihydroxylation, reduction, methoxylation and combinations of them such as N-desmethyl--dihydroxylation (Fig. S1), and about 37 tamoxifen metabolites have been reported so far (Table S1).[17–21] The analytical methods for analysis of the drug metabolism mainly focused on the use of liquid chromatography-tandem
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mass spectrometry because of good proton affinity.[22–24] In recent years, liquid chromatography quadrupole time-of-flight (LC-QTOF) mass technique is increasingly employed in the metabolic study of the drug with accurate mass measurement in order to predict unknown metabolites.[25] Previously, we studied the metabolic pathway and metabolites of clomiphene and toemifene in human urine for doping control purpose.[26,27] The objective of this study is to identify and characterize the metabolite structures in full scan and targeted MS/MS modes using accurate mass measurements and detect the main metabolites of tamoxifen in doping control.
Materials and methods Chemicals and reagents Tamoxifen was purchased from Fuliutang pharmacy (Haidian, Beijing). All solvents were of HPLC grade obtained from Dima
* Correspondence to: Jianghai Lu, National Anti-doping Laboratory, China Anti-Doping Agency, 1st Anding Road, ChaoYang District, Beijing 100029, China. E-mail: [email protected] †
These authors contributed equally to this work.
a National Anti-doping Laboratory, China Anti-Doping Agency, 1st Anding Road, ChaoYang District, Beijing 100029, China b KLGHEI of Environment and Energy Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, 510275, China
Copyright © 2014 John Wiley & Sons, Ltd.
New urinary tamoxifen metabolites by LC-QTOF-MS Tech. Inc. (Richmond hill, CA, USA). Sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate dehydrate were purchased from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). β-Glucuronidase from E. coli, methyltesterone (internal standard) and 3-hydroxy-4-methoxy-tamoxifen were obtained from sigma-Aldrich (Shanghai, China).
subsequently centrifuged for 5 min. The organic layer was transferred to a fresh glass tube, blown to dryness at 25 ºC, and the dry residue was dissolved with 100 μl of a mixture of water (2 mM HCOONH4, 0.1% HCOOH) together with methanol (0.1% HCOOH, V/V) (50 : 50, V/V), and 20 μl of the reconstituted solution was injected for liquid chromatography-tandem mass spectrometry analysis.
Instrumentation An Agilent 1290 Series rapid-resolution LC system (Agilent Technologies, Waldbronn, Germany) was used for the chromatographic separation. The system consisted of a vacuum degasser, a high-pressure binary pump, an autosampler with a cooled sample tray and a column oven. The LC were equipped with an Agilent Zorbax XDB-C18 column (100 mm length, 2.1 mm inner diameter and 3.5 μm particle size) linked to a filter (particle size 0.2 μm), and the separation was carried out at constant temperature (40 °C). The mobile phase was composed of water (2 mM HCOONH4, 0.1% HCOOH) (eluent A) and methanol (0.1% HCOOH, V/V). A gradient was employed starting at 30% B and holding 1 min and then increasing to 90% B in 18 min, 100% B at 20 min (total run time: 26 min). The column was finally re-equilibrated at 30% B for 6 min. The flow rate was set at 0.3 ml/min. The mass spectrometer was an orthogonal acceleration QTOF MS (6538 Accurate-Mass QTOF LC/MS; Agilent Technologies, Santa Clara, CA, USA) equipped with an orthogonal electrospray ionization (ESI) source, a temperature-stabilized analog-to-digital converter operated at 4 GHz (high resolution mode) and a microchannel plate operated at 735 V. Ionization was performed in the positive mode, and nitrogen was used as the drying and nebulizing gas. The drying gas flow rate and temperature were set at 10 l/min and 330 ºC, respectively, and the nebulizer gas pressure was 35 psi. The applied capillary voltage was 5500 V. The fragmentor voltage was set at 130 V in order to avoid insource fragmentation of the protonated molecules formed in ESI from the analytes. Full-scan mass spectral data were acquired from m/z 40 to 1100 at a rate of 1.5 scan/s. The remaining QTOFMS parameters (transfer optic and ion focus voltages, quadrupole lens, TOF and detector voltages) were automatically optimized by the instrument auto-tuning procedure, performed daily. Mass calibration was performed daily before starting the analysis set by using a reference mixture of two compounds, providing [M + H]+ ions in the mass range of m/z 118.0863–2721.8950, provided by the manufacturer. Reference mass correction was used during the analysis, in order to achieve better mass accuracy, by introducing two reference compounds (hexakis (1H,1H,3H-tetrafluoropropoxy) phosphazine and purine; Agilent Technologies, Santa Clara, CA, USA) simultaneously with the samples. The reference compounds were introduced continuously into the ESI source from a second orthogonal nebulizer. Sample preparation
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One tablet containing 50 mg of tamoxifen was taken orally by two healthy male volunteers and one female patient suffered from breast cancer. The volunteers signed informed consent prior to the excretion studies. Blank urine samples were collected before administration. Samples were collected 1 week after intake.
Results and discussion Characterization of the chemical structures of tamoxifen and its metabolites The mass spectrum of tamoxifen in product ion scan mode was studied carefully (Fig. S2), which was characterized with few product ions between m/z 72 and 372. The dominant base peak ion at m/z 72.0810 obtained by QTOF was relative to the N-dimethylated side chain (C4H10N). As the parent drug was composed of a stably conjugated π–electron system, which included three benzyl functions and a double bond, the characteristic chemical structure triggered the formation of a predominant fragment ion at m/z 72 (C4H10N) in positive ion mode under ESI condition. As chemical structures of tamoxifen metabolites were similar to that of the parent drug, it was obviously deduced that the mass spectra of its metabolites in targeted MS/MS experiment were usually characterized with a dominant fragment ion corresponding to the side chain. Therefore, a reliable and convenient approach for the investigation of the type of tamoxifen metabolites could be established. On the basis of the different side chains, tamoxifen metabolites could be divided into five types of N-dimethylation, N-desmethylation, N,N-didesmethylation, N-oxidation and deamino-hydroxylation, and the corresponding dominant fragment ions in targeted MS/MS mode were m/z 72 (C4H10N), 58 (C3H8N), 44 (C2H6N), 88 (C4H10NO) and 45 (C2H5O). With the targeted MS/MS technique employed at the fixed level of collision energy (CE), the tamoxifen metabolites could be distinguished from the endogenous interfering substances according to the mass spectrometric feature of its metabolic pathways. After seeking the mass values of its metabolites out, the accurate mass values could be obtained in full-scan experiment, and the chemical structures might be deduced in targeted MS/MS experiment by means of QTOF with accurate mass measurements. In this study, 31 reported metabolites (Table S1) were detected, and seven unreported urinary metabolites of tamoxifen were identified (Fig. 1). The extraction ion chromatogram, the mass spectrum and the proposed mass spectrometry fragmentation pathway of these newly reported metabolites were presented and discussed in the succeeding text. M1a, M1b Two chromatographic peaks were detected by the extraction of the m/z 406.2378 with a mass accuracy … 72.0806
1.6 1.4 1.2 1 0.8 0.6
Figure 1. The chemical structures of tamoxifen and its new metabolites in human urine.
0.4
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unchanged tamoxifen (C26H29NO), two oxygen and two hydrogen atoms were added, and one less double bond equivalent (DBE: 12) was shown. It was proposed that the double bond underwent the epoxide reaction and then acid-catalyzed epoxide cleavage took place by backside attack of water on the protonated epoxide in a manner of SN2 reaction, which caused the hydrolysis of the epoxide intermediate to 1,2-diol and stereochemical configuration of the two adjacent diols was trans. The targeted MS/MS spectra of the protonated ion obtained by LC-QTOF-MS were shown in Fig. 2b and c. The precursor ion at m/z 406.2378 was dissociated using CE of 25 V, and targeted MS/ MS spectra of the two metabolites were very similar and characterized with a dominant fragment ion at m/z 72.0810 corresponding to an N-dimethylated side chain, which indicated the presence of two optical isomers. The m/z 167.0705 (C9H10O3, masscalc = 167.0703, error = 1.4 ppm) was suggested to be originated from the elimination of the N-containing side chain, one benzyl group and one methyl group (Fig. 3). Hence, the two metabolites were tentatively identified as 1R,2S-dihydroxyl tamoxifen (M1a) and 1S,2R-dihydroxyl tamoxifen (M1b).
(c) x103 +ESI Product Ion (7.835 min) Frag=130.0V [email protected] (406.2378[z=1] -> **) … 5
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Counts vs. Mass-to-Charge (m/z) Figure 2. The extraction ion chromatogram (a) and mass spectrum of 1R,2S-dihydroxyl tamoxifen (M1a) and 1S,2R-dihydroxyl tamoxifen (M1b) in targeted MS/MS mode by LC-QTOF-MS (b, c).
M3
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A chromatographic peak was obtained by the extraction of the m/z 434.2329 (protonated molecule’s masscalc = 434.2326, error = 0.7 ppm) (Fig. 4a), which provided evidence for the assumed elemental composition as C27H31NO4. Compared with unchanged toremifen (C26H29NO), two hydrogen, one carbon and three
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oxygen atoms were residual, and DBE was the same as tamoxifen, which was suggested that one methoxyl and two hydroxyl groups were produced and the double bond was not reduced. The precursor ions at m/z 434.2329 was dissociated using fixed CE of 20 V (Fig. 4b); the most abundant fragment ions in targeted
Copyright © 2014 John Wiley & Sons, Ltd.
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New urinary tamoxifen metabolites by LC-QTOF-MS MS/MS experiment was observed at m/z 72.0808, the origin of which has been proposed to be the N,N-dimethylated side chain. A fragment ion at m/z 225.0900 (C15H12O2, masscalc = 225.091, error = 4.4 ppm) was obtained owing to the elimination of the side
Figure 3. Proposed mass spectrometry fragmentation pathway of 1R,2Sdihydroxyl-tamoxifen.
chain and the benzyl ring A (Fig. 5). Hence, the metabolite was identified as 3′,4-dihydroxy-4′-methoxyl-tamoxifen. M4 A metabolite was generated by the accurate extraction of the m/z 434.2325 (Fig. 6a). The elemental composition of the metabolite was determined as C27H31NO4 (protonated molecule’s masscalc = 434.2326, error = 0.2 ppm). Two hydrogen, one carbon and three oxygen atoms were excess, and the DBE was the same as tamoxifen, which indicated that two hydroxyl and one methoxyl groups were given. The presence of characteristic ion at m/z 72.0809 in targeted MS/MS mode substantiated the N,Ndimethylated side chain (Fig. 6b). Neutral loss of water at m/z 416.2220 suggested that the hydroxyl group was not in phenolic ring but linked to the methyl function, and the fragment ion at m/z 371.1642 (C25H22O3, masscalc = 371.1642, error = 0 ppm) was proposed to result from the loss of the N(CH3)2 fragment
(a) x104 +ESI EIC(434.2329) Scan Frag=130.0V WXZ-PS.d 8 7 6 5 4 3 2 1 0 3.8
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Figure 4. The extraction ion chromatogram (a) and mass spectrum of 3′,4-dihydroxy-4′-methoxyl-tamoxifen in targeted MS/MS mode by LC-QTOF-MS (b).
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Figure 7. Proposed mass spectrometry fragmentation pathway of α,3,hydroxyl-4- methoxy-tamoxifen. Figure 5. Proposed mass spectrometry fragmentation pathway of 3 ,4dihydroxy-4 - methoxyl-tamoxifen.
(a) x105 +ESI EIC(434.2325) Scan Frag=130.0V WXZ-SCP-R24.d 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 4.8
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Counts vs. Acquisition Time (min)
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Figure 6. The extraction ion chromatogram (a) and mass spectrum of α,3,-hydroxyl-4- methoxy-tamoxifen in targeted MS/MS mode by LC-QTOF-MS (b).
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Copyright © 2014 John Wiley & Sons, Ltd.
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New urinary tamoxifen metabolites by LC-QTOF-MS (Fig. 7). Therefore, on the basis of our experience, the metabolite was identified as α,3-hydroxyl-4-methoxy-tamoxifen. M5 A chromatographic peak was isolated from the accurate extraction of the calculated m/z 434. 2331 (protonated molecule’s masscalc = 434.2326, error = 1.1 ppm) (Fig. 8a), which indicated the elemental composition of C27H31NO4. Three oxygen, one carbon and two hydrogen atoms were surplus by comparison with unchanged tamoxifen (C26H29NO), which demonstrated the presence of one methoxyl and two hydroxyl functions. Product ion at m/z 72.0812 in targeted MS/MS experiment indicated the presence of N,N-dimethylated side chain, an unordinary product ion at m/z 88.0761 (C4H9NO, masscalc = 88.0757, error = 4.7 ppm ) indicated that the nitrogen atom in the side chain was oxidated
(Fig. 8b). No loss of water was observed, which indicated the hydroxyl function was positioned in the aromatic rings. Hence, on the basis of the metabolism pathway of tamoxifen, the metabolite was established as 3-hydroxyl-4-methoxy-N-oxide-tamoxifen. M6 A metabolite was obtained by the accurate extraction of the m/z 390.2060 (Fig. 9a). The elemental composition of the metabolite was determined as C25H27NO3 (protonated molecule’s masscalc = 390.2064, error = 0.95 ppm). On the basis of accurate mass results, M6 had additional two oxygen atoms and a reduced carbon atom than unchanged tamoxifen, which showed the existence of two hydroxyl groups. Product ion at m/z 58.0655 (C3H7N, masscalc = 58.0651, error = 6.5 ppm) in targeted MS/MS experiment indicated the presence of N-desmethylated side chain.
(a) x105 +ESI EIC(434.2331) Scan Frag=130.0V ZLL-SCP-R24.d 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 9.5
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Figure 8. The extraction ion chromatogram (a) and mass spectrum of 3-hydroxyl-4- methoxy-N-oxide-tamoxifen in targeted MS/MS mode by LC-QTOFMS (b).
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(a) x104 +ESI EIC(390.2060) Scan Frag=130.0V ZLL-SCP-R24.d 8 7 6 5 4 3 2 1 0 2.8 2.9
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(b) x104 +ESI Product Ion (3.480 min) Frag=130.0V [email protected] (390.2064[z=1] -> **) ZLL-tam… 58.0655 4.5 4
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Counts vs. Mass-to-Charge (m/z) Figure 9. The extraction ion chromatogram (a) and mass spectrum of α,β-dihydroxyl- N-desmethlyl-tamoxifen in targeted MS/MS mode by LC-QTOF-MS (b).
The sole presence of a fragment ion at m/z 297.1281 (C22H16NO, masscalc. = 297.1274, error = 2.3 ppm) was proposed to be produced by the loss of two water, which indicated the two hydroxyl groups were linked to the ethyl function. Therefore, the metabolite was identified as α,β-dihydroxyl-N-desmethlyl-tamoxifen. M7
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The accurate m/z of M7 was measured as 390.2057 (C25H27NO3, protonated molecule’s masscalc = 390.2064, error = 1.8 ppm) (Fig. 10a). Product ion at m/z 58.0653 (C3H7N, masscalc = 58.0651, error = 3.4 ppm) in targeted MS/MS experiment supported the presence of N-desmethylated side chain (Fig. 10b). The ion at m/z 315.1360 (C22H18NO, masscalc = 315.1380, error = 6.2 ppm) was suggested to be obtained from the elimination of the N-containing side chain and a water, which supported the fact that a hydroxyl group was linked to the ethyl function. The m/z ion at 195.0801 (C14H10NO, masscalc = 195.0804, error = 1.8 ppm) was obtained owing to the elimination of a benzyl ring (C-ring) and an N-containing side chain, which demonstrated another hydroxyl
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group existed on C-ring (Fig. 11). Hence, the metabolite was identified as α,4-dihydroxyl-N-desmethyl-tamoxifen. Proposed metabolic pathway The major metabolic reactions of tamoxifen focused on oxidation in phenyl rings, and epoxidation in the double bond together with N-deaklyation in the side chain. The former included hydroxylation and methylation in para and meta position of benzene ring, and another kind of hydroxylation also took place on the ethyl function linked to the double bond. Para-hydroxylation in benzene ring was thought to be advantageous to the anti-estrogenic activity. The epoxidation took place in the double bond, which caused the unchanged tamoxifen to the trans-1,2-diols, the N-deaklyation contain N-desmethylation, N,N-didesmethylation, N-oxidation and deaminohydroxlyation. In our current study, 31 reported metabolites were also detected. Of these metabolites, 22 metabolites belonged to the type of no change in N-containing side chain, six metabolites pertained to the type of N-desmethylation and only three metabolites belonged to the type of N,N-desdimethylation.
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New urinary tamoxifen metabolites by LC-QTOF-MS
(a)
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the target metabolites can easily be fished out from the complicated matrix. In this study, seven unreported urinary metabolites of tamoxifen were identified after oral administration using LC-QTOF-MS. It is notable that product ion analysis of tamoxifen and its metabolites gave the same fragmentation patterns and were characteristic of the dominant base peak ion relative to N-containing side chain and few fragment ions, which could be due to the existence of the steady conjugated system between three benzene rings. The seven newly found metabolites could be detected in all urine samples during 1 week after intake, which suggested that they are potential biomarkers for monitoring the oral administration of tamoxifen in doping control.
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Acknowledgements
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This research was supported by the Foundation of General Administration of Sport of China (2013B006) and the National Science Found for Distinguished Young Scholars (21225731).
References +ESI Product Ion (6.758min) Frag=130.0V [email protected] (390.2064[z=1] -> **)… 58.065
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Counts vs. Mass-to-Charge Figure 10. The extraction ion chromatogram (a) and mass spectrum of α,β-dihydroxyl- N-desmethlyl-tamoxifen in targeted MS/MS mode by LC-QTOF-MS (b).
Figure 11. Proposed mass spectrometry fragmentation pathway of α,4-dihydroxyl-N-desmethyl-tamoxifen.
Moreover, trace of unchanged tamoxifen could be detected in all urine samples up to 1 week, the abundance of 3-hydroxyl-4methoxy-tamoxifen was much higher than that of others, which had been reported to be the main metabolite in human urine.
Conclusions
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It has been shown that LC-QTOF-MS is a powerful tool in the elucidation of tamoxifen metabolism. By the accurate m/z extraction,
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J. Lu et al. [15] V. Stearns, M. D. Johnson, J. M. Rae. Active tamoxifen metabolite plasma concentrations after Coadministration of tamoxifen and the selective serotonin reuptake inhibitor paroxetine. J. Natl. Cancer Inst. 2003, 95, 1758. [16] M. D. Johnson, H. Zuo, K. H. Lee. Pharmacological characterization of 4-hydroxy-N-desmethyl tamoxifen, a novel active metabolite of tamoxifen. Breast Cancer Res. Treat. 2004, 85, 151. [17] X. F. Li, S. Carter, N. J. Dovichi, J. Y. Zhao, P. Kovarik, T. Sakuma. Analysis of tamoxifen and its metabolites in synthetic gastric fluid digests and urine samples using high-performance liquid chromatography with electrospray mass spectrometry. J. Chromatogr. A 2001, 914, 5. [18] E. R. Kisanga, G. Mellgren, E. A. Lien. Excretion of hydroxylated metabolites of tamoxifen in human bile and urine. Anticancer Res 2005, 25, 4487. [19] C. K. Lim, Z. X. Yuan, R. M. Jones, I. N. H. White, L. L. Smith. Identification and mechanism of formation of potentially genotoxic metabolites of tamoxifen: study by LC-MS/MS. J. Pharmaceut. Biomed. Anal. 1997, 15, 1335. [20] K. P. Grace, M. F. Graham, M. Prkah. Electrospray ionization mass spectrometry for analysis of low-molecular-weight anticancer drugs and their analogues. J. Am. Soc. Mass Spectrom. 1993, 4, 588. [21] J. R. Flores, J. J. Nevado, G. C. Peñalvo, N. M. Diez. Development of a Micellar electrokinetic capillary chromatography method for the determination of three drugs employed in the erectile dysfunction therapy. J. Chromatogr. B 2004, 811, 231. [22] S. F. Teunissen, H. Rosing, M. D. Seoane, L. Brunsveld, J. H. M. Schellens, A. H. Schinkel, J. H. Beijinen. Investigational study of tamoxifen phase I metabolites using chromatographic and spectroscopic analytical techniques. J. Pharm. Biomed. Anal. 2011, 55, 518.
[23] S. P. Singh, Wahajuddin, M. M. Ali, K. Kohli, G. K. Jain. Liquid chromatography-mass spectrometry method for the quantification of tamoxifen and its metabolite 4-hydroxytamoxifen in rat plasma: application to interaction study with biochanin a (an isoflavone). J. Chromatogr. B 2011, 879, 2845. [24] S. F. Teunissen, N. G. Jager, H. Rosing, A. H. Schinkel, J. H. Schellens, J. H. Beijnen. Development and validation of a quantitative assay for the determination of tamoxifen and its five main phase I metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry. J. Chromatogr. B 2011, 879, 1677. [25] M. Thevis, T. Kuuranne, H. Geyer, W. Schänzer. Annual bannedsubstance review: analytical approaches in human sports drug testing. Drug Test. Anal. 2013, 5, 1. [26] J. Lu, G. He, X. Wang, Y. Xu, Y. Wu, Y. Dong, Z. He, X. Liu, T. Bo, G. Ouyang. Mass spectrometric identification and characterization of new clomiphene metabolites in human urine by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry. J. Chromatogr. A 2012, 1243, 23. [27] J. Lu, G. He, X. Wang, Y. Xu, Y. Wu, Y. Dong, M. Wu. Mass spectrometric characterization of toremifene metabolites in human urine by liquid chromatography-tandem mass spectrometry with different scan modes. Analyst 2011, 136, 467.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s website.
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Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 570–578
Revised: 9 April 2014
Accepted: 10 April 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3375
Structural elucidation of new urinary tamoxifen metabolites by liquid chromatography quadrupole time-of-flight mass spectrometry Jianghai Lu,a*† Chunji He,a† Genye He,a Xiaobing Wang,a Youxuan Xu,a Yun Wu,a Ying Donga and Gangfeng Ouyangb In this study, tamoxifen metabolic profiles were investigated carefully. Tamoxifen was administered to two healthy male volunteers and one female patient suffering from breast cancer. Urinary extracts were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry using full scan and targeted MS/MS techniques with accurate mass measurement. Chromatographic peaks for potential metabolites were selected by using the theoretical [M + H]+ as precursor ion in full-scan experiment and m/z 72, 58 or 44 as characteristic product ions for N,N-dimethyl, N-desmethyl and N, N-didesmethyl metabolites in targeted MS/MS experiment, respectively. Tamoxifen and 37 metabolites were detected in extraction study samples. Chemical structures of seven unreported metabolites were elucidated particularly on the basis of fragmentation patterns observed for these metabolites. Several metabolic pathways containing mono- and di-hydroxylation, methoxylation, N-desmethylation, N,N-didesmethylation, oxidation and combinations were suggested. All the metabolites were detected in the urine samples up to 1 week. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s website. Keywords: tamoxifen; new metabolites; doping control; human urine; liquid chromatography quadruple time-of-flight mass spectrometry
Introduction
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Tamoxifen, a first-generation selective estrogen receptor modulator with a triphenylethylene structure is widely used for adjuvant breast cancer therapy.[1,2] It fights certain types of breast cancer, called hormone response or estrogen positive breast cancer, by blocking the effects of the hormone estrogen in the body. This prevents the growth of the types of breast cancer cells that require estrogen for growth and survival.[3,4] It is also used to prevent the recurrence of breast cancer in women who have been treated for the disease and treat breast cancer that has been spread to other parts of the body.[5] Since 2005, tamoxifen was included, with other similar drugs, in the Section 4 ‘agents with anti-oestrogenic activity’ of the World Anti-Doping Agency list of prohibited substances and methods.[6] Tamoxifen is extensively metabolized by the CYP3A4 and 2D6 isoforms.[7–9] The clinical goodness of tamoxifen is thought to arise from its transformation to active metabolites, mainly 4-hydroxy tamoxifen and 4-OH-N-desmethyl tamoxifen.[10–12] These metabolites bind to the estrogen receptor with up to 30-fold greater affinity than tamoxifen, which increased potency for binding translates into enhanced antiestrogen activity.[13–16] Main metabolic pathways included epoxidation, N-desmethylation, hydroxylation, dihydroxylation, reduction, methoxylation and combinations of them such as N-desmethyl--dihydroxylation (Fig. S1), and about 37 tamoxifen metabolites have been reported so far (Table S1).[17–21] The analytical methods for analysis of the drug metabolism mainly focused on the use of liquid chromatography-tandem
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mass spectrometry because of good proton affinity.[22–24] In recent years, liquid chromatography quadrupole time-of-flight (LC-QTOF) mass technique is increasingly employed in the metabolic study of the drug with accurate mass measurement in order to predict unknown metabolites.[25] Previously, we studied the metabolic pathway and metabolites of clomiphene and toemifene in human urine for doping control purpose.[26,27] The objective of this study is to identify and characterize the metabolite structures in full scan and targeted MS/MS modes using accurate mass measurements and detect the main metabolites of tamoxifen in doping control.
Materials and methods Chemicals and reagents Tamoxifen was purchased from Fuliutang pharmacy (Haidian, Beijing). All solvents were of HPLC grade obtained from Dima
* Correspondence to: Jianghai Lu, National Anti-doping Laboratory, China Anti-Doping Agency, 1st Anding Road, ChaoYang District, Beijing 100029, China. E-mail: [email protected] †
These authors contributed equally to this work.
a National Anti-doping Laboratory, China Anti-Doping Agency, 1st Anding Road, ChaoYang District, Beijing 100029, China b KLGHEI of Environment and Energy Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, 510275, China
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New urinary tamoxifen metabolites by LC-QTOF-MS Tech. Inc. (Richmond hill, CA, USA). Sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate dehydrate were purchased from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). β-Glucuronidase from E. coli, methyltesterone (internal standard) and 3-hydroxy-4-methoxy-tamoxifen were obtained from sigma-Aldrich (Shanghai, China).
subsequently centrifuged for 5 min. The organic layer was transferred to a fresh glass tube, blown to dryness at 25 ºC, and the dry residue was dissolved with 100 μl of a mixture of water (2 mM HCOONH4, 0.1% HCOOH) together with methanol (0.1% HCOOH, V/V) (50 : 50, V/V), and 20 μl of the reconstituted solution was injected for liquid chromatography-tandem mass spectrometry analysis.
Instrumentation An Agilent 1290 Series rapid-resolution LC system (Agilent Technologies, Waldbronn, Germany) was used for the chromatographic separation. The system consisted of a vacuum degasser, a high-pressure binary pump, an autosampler with a cooled sample tray and a column oven. The LC were equipped with an Agilent Zorbax XDB-C18 column (100 mm length, 2.1 mm inner diameter and 3.5 μm particle size) linked to a filter (particle size 0.2 μm), and the separation was carried out at constant temperature (40 °C). The mobile phase was composed of water (2 mM HCOONH4, 0.1% HCOOH) (eluent A) and methanol (0.1% HCOOH, V/V). A gradient was employed starting at 30% B and holding 1 min and then increasing to 90% B in 18 min, 100% B at 20 min (total run time: 26 min). The column was finally re-equilibrated at 30% B for 6 min. The flow rate was set at 0.3 ml/min. The mass spectrometer was an orthogonal acceleration QTOF MS (6538 Accurate-Mass QTOF LC/MS; Agilent Technologies, Santa Clara, CA, USA) equipped with an orthogonal electrospray ionization (ESI) source, a temperature-stabilized analog-to-digital converter operated at 4 GHz (high resolution mode) and a microchannel plate operated at 735 V. Ionization was performed in the positive mode, and nitrogen was used as the drying and nebulizing gas. The drying gas flow rate and temperature were set at 10 l/min and 330 ºC, respectively, and the nebulizer gas pressure was 35 psi. The applied capillary voltage was 5500 V. The fragmentor voltage was set at 130 V in order to avoid insource fragmentation of the protonated molecules formed in ESI from the analytes. Full-scan mass spectral data were acquired from m/z 40 to 1100 at a rate of 1.5 scan/s. The remaining QTOFMS parameters (transfer optic and ion focus voltages, quadrupole lens, TOF and detector voltages) were automatically optimized by the instrument auto-tuning procedure, performed daily. Mass calibration was performed daily before starting the analysis set by using a reference mixture of two compounds, providing [M + H]+ ions in the mass range of m/z 118.0863–2721.8950, provided by the manufacturer. Reference mass correction was used during the analysis, in order to achieve better mass accuracy, by introducing two reference compounds (hexakis (1H,1H,3H-tetrafluoropropoxy) phosphazine and purine; Agilent Technologies, Santa Clara, CA, USA) simultaneously with the samples. The reference compounds were introduced continuously into the ESI source from a second orthogonal nebulizer. Sample preparation
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One tablet containing 50 mg of tamoxifen was taken orally by two healthy male volunteers and one female patient suffered from breast cancer. The volunteers signed informed consent prior to the excretion studies. Blank urine samples were collected before administration. Samples were collected 1 week after intake.
Results and discussion Characterization of the chemical structures of tamoxifen and its metabolites The mass spectrum of tamoxifen in product ion scan mode was studied carefully (Fig. S2), which was characterized with few product ions between m/z 72 and 372. The dominant base peak ion at m/z 72.0810 obtained by QTOF was relative to the N-dimethylated side chain (C4H10N). As the parent drug was composed of a stably conjugated π–electron system, which included three benzyl functions and a double bond, the characteristic chemical structure triggered the formation of a predominant fragment ion at m/z 72 (C4H10N) in positive ion mode under ESI condition. As chemical structures of tamoxifen metabolites were similar to that of the parent drug, it was obviously deduced that the mass spectra of its metabolites in targeted MS/MS experiment were usually characterized with a dominant fragment ion corresponding to the side chain. Therefore, a reliable and convenient approach for the investigation of the type of tamoxifen metabolites could be established. On the basis of the different side chains, tamoxifen metabolites could be divided into five types of N-dimethylation, N-desmethylation, N,N-didesmethylation, N-oxidation and deamino-hydroxylation, and the corresponding dominant fragment ions in targeted MS/MS mode were m/z 72 (C4H10N), 58 (C3H8N), 44 (C2H6N), 88 (C4H10NO) and 45 (C2H5O). With the targeted MS/MS technique employed at the fixed level of collision energy (CE), the tamoxifen metabolites could be distinguished from the endogenous interfering substances according to the mass spectrometric feature of its metabolic pathways. After seeking the mass values of its metabolites out, the accurate mass values could be obtained in full-scan experiment, and the chemical structures might be deduced in targeted MS/MS experiment by means of QTOF with accurate mass measurements. In this study, 31 reported metabolites (Table S1) were detected, and seven unreported urinary metabolites of tamoxifen were identified (Fig. 1). The extraction ion chromatogram, the mass spectrum and the proposed mass spectrometry fragmentation pathway of these newly reported metabolites were presented and discussed in the succeeding text. M1a, M1b Two chromatographic peaks were detected by the extraction of the m/z 406.2378 with a mass accuracy … 72.0806
1.6 1.4 1.2 1 0.8 0.6
Figure 1. The chemical structures of tamoxifen and its new metabolites in human urine.
0.4
167.0705
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unchanged tamoxifen (C26H29NO), two oxygen and two hydrogen atoms were added, and one less double bond equivalent (DBE: 12) was shown. It was proposed that the double bond underwent the epoxide reaction and then acid-catalyzed epoxide cleavage took place by backside attack of water on the protonated epoxide in a manner of SN2 reaction, which caused the hydrolysis of the epoxide intermediate to 1,2-diol and stereochemical configuration of the two adjacent diols was trans. The targeted MS/MS spectra of the protonated ion obtained by LC-QTOF-MS were shown in Fig. 2b and c. The precursor ion at m/z 406.2378 was dissociated using CE of 25 V, and targeted MS/ MS spectra of the two metabolites were very similar and characterized with a dominant fragment ion at m/z 72.0810 corresponding to an N-dimethylated side chain, which indicated the presence of two optical isomers. The m/z 167.0705 (C9H10O3, masscalc = 167.0703, error = 1.4 ppm) was suggested to be originated from the elimination of the N-containing side chain, one benzyl group and one methyl group (Fig. 3). Hence, the two metabolites were tentatively identified as 1R,2S-dihydroxyl tamoxifen (M1a) and 1S,2R-dihydroxyl tamoxifen (M1b).
(c) x103 +ESI Product Ion (7.835 min) Frag=130.0V [email protected] (406.2378[z=1] -> **) … 5
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Counts vs. Mass-to-Charge (m/z) Figure 2. The extraction ion chromatogram (a) and mass spectrum of 1R,2S-dihydroxyl tamoxifen (M1a) and 1S,2R-dihydroxyl tamoxifen (M1b) in targeted MS/MS mode by LC-QTOF-MS (b, c).
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A chromatographic peak was obtained by the extraction of the m/z 434.2329 (protonated molecule’s masscalc = 434.2326, error = 0.7 ppm) (Fig. 4a), which provided evidence for the assumed elemental composition as C27H31NO4. Compared with unchanged toremifen (C26H29NO), two hydrogen, one carbon and three
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oxygen atoms were residual, and DBE was the same as tamoxifen, which was suggested that one methoxyl and two hydroxyl groups were produced and the double bond was not reduced. The precursor ions at m/z 434.2329 was dissociated using fixed CE of 20 V (Fig. 4b); the most abundant fragment ions in targeted
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J. Mass Spectrom. 2014, 49, 570–578
New urinary tamoxifen metabolites by LC-QTOF-MS MS/MS experiment was observed at m/z 72.0808, the origin of which has been proposed to be the N,N-dimethylated side chain. A fragment ion at m/z 225.0900 (C15H12O2, masscalc = 225.091, error = 4.4 ppm) was obtained owing to the elimination of the side
Figure 3. Proposed mass spectrometry fragmentation pathway of 1R,2Sdihydroxyl-tamoxifen.
chain and the benzyl ring A (Fig. 5). Hence, the metabolite was identified as 3′,4-dihydroxy-4′-methoxyl-tamoxifen. M4 A metabolite was generated by the accurate extraction of the m/z 434.2325 (Fig. 6a). The elemental composition of the metabolite was determined as C27H31NO4 (protonated molecule’s masscalc = 434.2326, error = 0.2 ppm). Two hydrogen, one carbon and three oxygen atoms were excess, and the DBE was the same as tamoxifen, which indicated that two hydroxyl and one methoxyl groups were given. The presence of characteristic ion at m/z 72.0809 in targeted MS/MS mode substantiated the N,Ndimethylated side chain (Fig. 6b). Neutral loss of water at m/z 416.2220 suggested that the hydroxyl group was not in phenolic ring but linked to the methyl function, and the fragment ion at m/z 371.1642 (C25H22O3, masscalc = 371.1642, error = 0 ppm) was proposed to result from the loss of the N(CH3)2 fragment
(a) x104 +ESI EIC(434.2329) Scan Frag=130.0V WXZ-PS.d 8 7 6 5 4 3 2 1 0 3.8
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Figure 4. The extraction ion chromatogram (a) and mass spectrum of 3′,4-dihydroxy-4′-methoxyl-tamoxifen in targeted MS/MS mode by LC-QTOF-MS (b).
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Figure 7. Proposed mass spectrometry fragmentation pathway of α,3,hydroxyl-4- methoxy-tamoxifen. Figure 5. Proposed mass spectrometry fragmentation pathway of 3 ,4dihydroxy-4 - methoxyl-tamoxifen.
(a) x105 +ESI EIC(434.2325) Scan Frag=130.0V WXZ-SCP-R24.d 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 4.8
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Figure 6. The extraction ion chromatogram (a) and mass spectrum of α,3,-hydroxyl-4- methoxy-tamoxifen in targeted MS/MS mode by LC-QTOF-MS (b).
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Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 570–578
New urinary tamoxifen metabolites by LC-QTOF-MS (Fig. 7). Therefore, on the basis of our experience, the metabolite was identified as α,3-hydroxyl-4-methoxy-tamoxifen. M5 A chromatographic peak was isolated from the accurate extraction of the calculated m/z 434. 2331 (protonated molecule’s masscalc = 434.2326, error = 1.1 ppm) (Fig. 8a), which indicated the elemental composition of C27H31NO4. Three oxygen, one carbon and two hydrogen atoms were surplus by comparison with unchanged tamoxifen (C26H29NO), which demonstrated the presence of one methoxyl and two hydroxyl functions. Product ion at m/z 72.0812 in targeted MS/MS experiment indicated the presence of N,N-dimethylated side chain, an unordinary product ion at m/z 88.0761 (C4H9NO, masscalc = 88.0757, error = 4.7 ppm ) indicated that the nitrogen atom in the side chain was oxidated
(Fig. 8b). No loss of water was observed, which indicated the hydroxyl function was positioned in the aromatic rings. Hence, on the basis of the metabolism pathway of tamoxifen, the metabolite was established as 3-hydroxyl-4-methoxy-N-oxide-tamoxifen. M6 A metabolite was obtained by the accurate extraction of the m/z 390.2060 (Fig. 9a). The elemental composition of the metabolite was determined as C25H27NO3 (protonated molecule’s masscalc = 390.2064, error = 0.95 ppm). On the basis of accurate mass results, M6 had additional two oxygen atoms and a reduced carbon atom than unchanged tamoxifen, which showed the existence of two hydroxyl groups. Product ion at m/z 58.0655 (C3H7N, masscalc = 58.0651, error = 6.5 ppm) in targeted MS/MS experiment indicated the presence of N-desmethylated side chain.
(a) x105 +ESI EIC(434.2331) Scan Frag=130.0V ZLL-SCP-R24.d 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 9.5
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Figure 8. The extraction ion chromatogram (a) and mass spectrum of 3-hydroxyl-4- methoxy-N-oxide-tamoxifen in targeted MS/MS mode by LC-QTOFMS (b).
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(a) x104 +ESI EIC(390.2060) Scan Frag=130.0V ZLL-SCP-R24.d 8 7 6 5 4 3 2 1 0 2.8 2.9
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(b) x104 +ESI Product Ion (3.480 min) Frag=130.0V [email protected] (390.2064[z=1] -> **) ZLL-tam… 58.0655 4.5 4
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Counts vs. Mass-to-Charge (m/z) Figure 9. The extraction ion chromatogram (a) and mass spectrum of α,β-dihydroxyl- N-desmethlyl-tamoxifen in targeted MS/MS mode by LC-QTOF-MS (b).
The sole presence of a fragment ion at m/z 297.1281 (C22H16NO, masscalc. = 297.1274, error = 2.3 ppm) was proposed to be produced by the loss of two water, which indicated the two hydroxyl groups were linked to the ethyl function. Therefore, the metabolite was identified as α,β-dihydroxyl-N-desmethlyl-tamoxifen. M7
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The accurate m/z of M7 was measured as 390.2057 (C25H27NO3, protonated molecule’s masscalc = 390.2064, error = 1.8 ppm) (Fig. 10a). Product ion at m/z 58.0653 (C3H7N, masscalc = 58.0651, error = 3.4 ppm) in targeted MS/MS experiment supported the presence of N-desmethylated side chain (Fig. 10b). The ion at m/z 315.1360 (C22H18NO, masscalc = 315.1380, error = 6.2 ppm) was suggested to be obtained from the elimination of the N-containing side chain and a water, which supported the fact that a hydroxyl group was linked to the ethyl function. The m/z ion at 195.0801 (C14H10NO, masscalc = 195.0804, error = 1.8 ppm) was obtained owing to the elimination of a benzyl ring (C-ring) and an N-containing side chain, which demonstrated another hydroxyl
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group existed on C-ring (Fig. 11). Hence, the metabolite was identified as α,4-dihydroxyl-N-desmethyl-tamoxifen. Proposed metabolic pathway The major metabolic reactions of tamoxifen focused on oxidation in phenyl rings, and epoxidation in the double bond together with N-deaklyation in the side chain. The former included hydroxylation and methylation in para and meta position of benzene ring, and another kind of hydroxylation also took place on the ethyl function linked to the double bond. Para-hydroxylation in benzene ring was thought to be advantageous to the anti-estrogenic activity. The epoxidation took place in the double bond, which caused the unchanged tamoxifen to the trans-1,2-diols, the N-deaklyation contain N-desmethylation, N,N-didesmethylation, N-oxidation and deaminohydroxlyation. In our current study, 31 reported metabolites were also detected. Of these metabolites, 22 metabolites belonged to the type of no change in N-containing side chain, six metabolites pertained to the type of N-desmethylation and only three metabolites belonged to the type of N,N-desdimethylation.
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J. Mass Spectrom. 2014, 49, 570–578
New urinary tamoxifen metabolites by LC-QTOF-MS
(a)
0.4
the target metabolites can easily be fished out from the complicated matrix. In this study, seven unreported urinary metabolites of tamoxifen were identified after oral administration using LC-QTOF-MS. It is notable that product ion analysis of tamoxifen and its metabolites gave the same fragmentation patterns and were characteristic of the dominant base peak ion relative to N-containing side chain and few fragment ions, which could be due to the existence of the steady conjugated system between three benzene rings. The seven newly found metabolites could be detected in all urine samples during 1 week after intake, which suggested that they are potential biomarkers for monitoring the oral administration of tamoxifen in doping control.
0.2
Acknowledgements
x105
+ESI EIC(390.2057) Scan Frag=130.0V ZLL-SCP-R24.d
1.8 1.6 1.4 1.2 1 0.8 0.6
0 6
6.2
6.4
6.6
6.8
7
7.2
7.4
7.6
Counts vs. Acquisition Time (min)
(b) x104 2.7
This research was supported by the Foundation of General Administration of Sport of China (2013B006) and the National Science Found for Distinguished Young Scholars (21225731).
References +ESI Product Ion (6.758min) Frag=130.0V [email protected] (390.2064[z=1] -> **)… 58.065
2. 2.2 2 1.7 1. 1.2 1 0.7
390.205
0. 195.080
0.2
315.136
0 5
7 10 12 15 17 20 22 25 27 30 32 35 37 40 42
Counts vs. Mass-to-Charge Figure 10. The extraction ion chromatogram (a) and mass spectrum of α,β-dihydroxyl- N-desmethlyl-tamoxifen in targeted MS/MS mode by LC-QTOF-MS (b).
Figure 11. Proposed mass spectrometry fragmentation pathway of α,4-dihydroxyl-N-desmethyl-tamoxifen.
Moreover, trace of unchanged tamoxifen could be detected in all urine samples up to 1 week, the abundance of 3-hydroxyl-4methoxy-tamoxifen was much higher than that of others, which had been reported to be the main metabolite in human urine.
Conclusions
J. Mass Spectrom. 2014, 49, 570–578
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It has been shown that LC-QTOF-MS is a powerful tool in the elucidation of tamoxifen metabolism. By the accurate m/z extraction,
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