Research article Received: 13 August 2014,
Revised: 9 October 2014,
Accepted: 18 November 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3411
Study of tamoxifen urinary metabolites in rat by ultra-high-performance liquid chromatography time-of-flight mass spectrometry Juan C. Domínguez-Romeroa, Juan F. García-Reyesa*, Miriam Beneito-Cambraa, Rubén Martínez-Romerob, Esther Martinez-Larab, María L. Del Moral-Lealb and Antonio Molina-Díaza ABSTRACT: Tamoxifen (TMX) is a nonsteroidal estrogen antagonist drug used for the treatment of breast cancer. It is also included in the list of banned substances of the World Anti Doping Agency (WADA) prohibited in and out of competition. In this work, the excretion of urinary metabolites of TMX after a single therapeutic dose administration in rats has been studied using ultra-high-performance liquid chromatography electrospray time-of-flight mass spectrometry (UHPLC-TOFMS). A systematic strategy based on the search of typical biotransformations that a xenobiotic can undergo in living organisms, based on their corresponding molecular formula modification and accurate mass shifts, was applied for the identification of TMX metabolites. Prior to UHPLC-TOFMS analyses, a solid-phase extraction step with polymeric cartridges was applied to urine samples. Up to 38 TMX metabolites were detected. Additional collision induced dissociation (CID) MS/MS fragmentation was performed using UHPLC-QTOFMS. Compared with recent previous studies in human urine and plasma, new metabolites have been reported for the first time in urine. Metabolites identified in rat urine include the oxygen addition, owing to different possibilities for the hydroxylation of the rings in different positions (m/z 388.2271), the incorporation of two oxygen atoms (m/z 404.2220) (including dihydroxylated derivatives or alternatives such as epoxidation plus hydroxylation or Noxidation and hydroxylation), epoxide formation or hydroxylation and dehydrogenation [m/z 386.2114 (+O –H2)], hydroxylation of the ring accompanied by N-desmethylation (m/z 374.2115), combined hydroxylation and methoxylation (m/z 418.2377), desaturated TMX derivate (m/z 370.2165) and its N-desmethylated derivate (m/z 356.2009), the two latter modifications not previously being reported in urine. These findings confirm the usefulness of the proposed approach based on UHPLC-TOFMS. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: tamoxifen; metabolite; urine; liquid chromatography; mass spectrometry
Introduction Tamoxifen (TMX) is a nonsteroidal estrogen antagonist drug therapeutically used for the treatment of breast cancer. TMX is considered a pro-drug since its metabolites have more activity than itself. Amongst them, 4-hydroxy-tamoxifen shows from 30 to 100 times more activity than TMX (Borgna and Rochefort, 1981; Robertson et al., 1982; Jordan, 1982; Coezy et al., 1982) and its secondary metabolite 4-hydroxy-N-desmethyl-tamoxifen (endoxifen) also exhibits a similar activity (Stearns et al., 2003; Johnson et al., 2004; Young et al., 2006). From the point of view of sport drug testing, TMX is a drug included in the list of banned substances of the World Anti Doping Agency (WADA; group S4, hormone antagonist and modulators; WADA, 2014a, 2014b). Its use is prohibited at all the times (in and out of competition). Various authors have reported on the metabolism of TMX, as recently reviewed by Beijnen and co-workers (Teunissen et al., 2010). While TMX metabolism studies have been reported extensively in serum (Gjerde et al., 2005; Teunissen et al., 2009, 2011a, 2011b), plasma (Stearns et al., 2003; Murphy et al., 1987a; Stevenson et al., 1988; Kikuta and Schmid, 1989; Dahmane et al., 2010;
Biomed. Chromatogr. 2015
Jaremko et al., 2010) and other specimens such as tumoral tissues (Murphy et al., 1987b; MacCallum et al., 1997), in vitro preparations using liver microsomes (Jones et al. 1996; Lu et al., 1996; Lim et al., 1997) and synthetic gastric acids (Li et al., 2001), the urinary excretion of TMX metabolites has not been studied in such detail (De Vos et al., 1998; Mazzarino et al., 2008, 2010, 2013; Lu et al., 2014).
* Correspondence to: J. F. García-Reyes, Analytical Chemistry Research Group, Department of Physical and Analytical Chemistry, University of Jaén, 23071 Jaén, Spain. Email: [email protected] a
Analytical Chemistry Research Group, Department of Physical and Analytical Chemistry, University of Jaén, 23071, Jaén, Spain
b
Cellular Stress and Age Research Group. Department of Experimental Biology, University of Jaén, 23071, Jaén, Spain Abbreviations used: HP-921, hexakis-(1H,1H,3H-tetrafluoropropoxy) phosphazine; TMX, tamoxifen; WADA, World Anti Doping Agency; UHPLCTOFMS, ultra-high-performance liquid chromatography electrospray timeof-flight mass spectrometry.
Copyright © 2015 John Wiley & Sons, Ltd.
J. C. Domínguez-Romero et al. In the past, the identification of TMX metabolites was performed by liquid chromatography with fluorescence detection after photochemical reaction (Stearns et al., 2003; Stevenson et al., 1988; Kikuta and Schmid, 1989; De Vos et al., 1998). Nowadays, although gas chromatography (Murphy et al., 1987a, 1987b) and capillary electrophoresis (Lu et al., 1996) coupled with mass spectrometric detection have been used for TMX metabolite identification, a far more convenient technique employed for TMX metabolite identification is liquid chromatography–mass spectrometry (HPLC-MS) with different mass analyzers such as triple quadrupole (HPLC-MS/MS; Teunissen et al., 2011a, 2011b; Jaremko et al., 2010; Jones et al., 1996; Lim et al., 1997; Mazzarino et al., 2010, 2013), time-of-flight mass spectrometers (HPLCTOFMS; Li et al., 2001; Mazzarino et al., 2008, 2013; Lu et al., 2014) and orbital ion trap high-resolution mass spectrometry instruments (Dahmane et al., 2014). The main mass spectrometric strategies used so far for the identification of TMX metabolites in urine include accurate mass measurements using high resolution mass spectrometry (HRMS) (Mazzarino et al., 2008; Lu et al., 2014; Dahmane et al., 2014) or the collection of data from different (up to five) scanning modes using HPLC-MS/MS: a precursor ion scan of m/z 166, 152 and 129 and neutral loss of m/z 72 and 58; Mazzarino et al., 2010). High resolving power mass spectrometers, such as time-offlight analyzers or orbitraps, offer full-scan high sensitivity and high mass accuracy, enabling screening for a theoretically unlimited number of compounds and the analysis of unknown or nontarget compounds, which makes these instruments a powerful tool for the identification of drug metabolites, particularly when combined with ultra-high pressure liquid chromatography separations (Thevis et al., 2013). In this work, we report a study of TMX urinary metabolites in rats after a single-dose administration using solid-phase extraction followed by ultra-high-pressure liquid chromatography electrospray time-of-flight mass spectrometry (UHPLC-TOFMS). Up to 38 TMX metabolites were detected, some of the modifications detected not previously reported in urine in the literature.
Experimental Chemicals and reagents Tamoxifen was purchased from Sigma-Aldrich (Madrid, Spain). HPLCgrade acetonitrile and methanol were acquired from Sigma-Aldrich (Madrid, Spain). Formic acid was obtained from Fluka (Madrid, Spain). A Milli-Q-Plus ultra-pure water system from Millipore (Milford, MA, USA) was used throughout the study to obtain HPLC water during the analyses.
Sample collection The study was performed on adult male Wistar rats (250–300 g; Charles River Laboratories, Barcelona, Spain). The animals (n = 5) were weighed and placed in individual metabolic cages 48 h prior to treatment to acclimatize them to this environment, maintained under standard conditions of light and temperature and allowed ad libitum access to food and water to the end of the experiment. All the procedures followed the Spanish guidelines on the use of animals for research (RD 1201/2005) and were approved by the institutional Committee for Ethics. The rats were treated with TMX (10 mg/kg body weight, intraperitoneal). After drug administration, urine was collected daily in graduate cylinders for 3 days (24, 48 and 72 h). The urine collected 24 h prior to treatment was used as control.
wileyonlinelibrary.com/journal/bmc
Sample treatment A 2 mL aliquot of urine was diluted with 2 mL of formic acid/formate pH 2.6 buffer and then passed through a polymeric Bond Elut PLEXA™ SPE cartridge from Agilent Technologies (Santa Clara, CA, USA) and washed with 2 mL of H2O–methanol (95:5). The cartridges were previously conditioned with 4 mL of methanol–acetonitrile (1:1) and 4 mL of MilliQ water. The analytes were eluted from the cartridge with 4 mL of methanol–acetonitrile (1:1) and the extract was evaporated to near dryness with a TurboVap LV (Caliper LifeSciences, Hopkinton, MA, USA) and then taken up with 0.5 mL of the initial mobile phase to achieve a preconcentration factor of 4:1. Finally, the extract was filtered through a 0.22 μm PTFE syringe filter and transferred to a 2 mL glass vial prior to LC-MS analyses. Various recovery studies were conducted with TMX at different concentration levels to ensure that the compound was quantitatively recovered. The recovery rates obtained approached 100% regardless of the type of polymeric cartridge (Oasis HLB or PLEXA) used. Considering the similar structures and chromatographic properties of potential TMX phase-I metabolites, it is reasonable to expect a similar behavior.
UHPLC-TOFMS method The chromatographic separation was performed using a UHPLC system (Agilent Infinity 1290, Agilent Technologies, Santa Clara, CA, USA) equipped with a reversed-phase Zorbax high-definition (RRHD) Eclipse Plus-C18 analytical column of 2.1 × 50 mm and 1.8 μm particle size (Agilent Technologies, Santa Clara, CA, USA). Mobile phases A and B were water with 0.1% formic acid and acetonitrile. The chromatographic method held the initial mobile phase composition (10% B) constant for 5 min, followed by a linear gradient to 40% B up to 28 min, to 100% up to 33 min and finally kept for 2 min at 100%. The flow rate used was 0.5 mL/min and 20 μL of the urine extract were injected in each run. This UHPLC system was connected to a time-of-flight mass spectrometer (Agilent 6220 accurate mass TOF; Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray interface operating in positive ion mode, using the following operation parameters: capillary voltage, 3500 V; nebulizer pressure, 40 psig; drying gas flow rate, 9.0 L/min; gas temperature, 325 °C; skimmer voltage, 65 V; octapole 1 rf, 250 V; fragmentor voltage 175 and 230 V (in-source CID fragmentation); acquisition timem 1.5 spectra/s. LC-MS accurate mass spectra were recorded across the range of m/z 50–1000. The instrument performed an internal calibration using the reference mixture provided by the manufacturer over the acquired mass range, using a second sprayer with a reference solution containing the masses purine (m/z 121.0509) and hexakis-(1H,1H,3Htetrafluoropropoxy)phosphazine (HP-921; C18H18O6N3P3F24, m/z 922.0098). The instrument was operated in the 4 GHz high-resolution mode. The resolution displayed by the instrument with these conditions was ca. 14,000 at m/z 400. The full-scan data were recorded with Agilent Mass Hunter Data Acquisition software (version B.04.00) and processed with Agilent Mass Hunter Qualitative Analysis software (version B.04.00). The analytical performance of the method for TMX determination in urine was satisfactory with a limit of quantitation
Revised: 9 October 2014,
Accepted: 18 November 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3411
Study of tamoxifen urinary metabolites in rat by ultra-high-performance liquid chromatography time-of-flight mass spectrometry Juan C. Domínguez-Romeroa, Juan F. García-Reyesa*, Miriam Beneito-Cambraa, Rubén Martínez-Romerob, Esther Martinez-Larab, María L. Del Moral-Lealb and Antonio Molina-Díaza ABSTRACT: Tamoxifen (TMX) is a nonsteroidal estrogen antagonist drug used for the treatment of breast cancer. It is also included in the list of banned substances of the World Anti Doping Agency (WADA) prohibited in and out of competition. In this work, the excretion of urinary metabolites of TMX after a single therapeutic dose administration in rats has been studied using ultra-high-performance liquid chromatography electrospray time-of-flight mass spectrometry (UHPLC-TOFMS). A systematic strategy based on the search of typical biotransformations that a xenobiotic can undergo in living organisms, based on their corresponding molecular formula modification and accurate mass shifts, was applied for the identification of TMX metabolites. Prior to UHPLC-TOFMS analyses, a solid-phase extraction step with polymeric cartridges was applied to urine samples. Up to 38 TMX metabolites were detected. Additional collision induced dissociation (CID) MS/MS fragmentation was performed using UHPLC-QTOFMS. Compared with recent previous studies in human urine and plasma, new metabolites have been reported for the first time in urine. Metabolites identified in rat urine include the oxygen addition, owing to different possibilities for the hydroxylation of the rings in different positions (m/z 388.2271), the incorporation of two oxygen atoms (m/z 404.2220) (including dihydroxylated derivatives or alternatives such as epoxidation plus hydroxylation or Noxidation and hydroxylation), epoxide formation or hydroxylation and dehydrogenation [m/z 386.2114 (+O –H2)], hydroxylation of the ring accompanied by N-desmethylation (m/z 374.2115), combined hydroxylation and methoxylation (m/z 418.2377), desaturated TMX derivate (m/z 370.2165) and its N-desmethylated derivate (m/z 356.2009), the two latter modifications not previously being reported in urine. These findings confirm the usefulness of the proposed approach based on UHPLC-TOFMS. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: tamoxifen; metabolite; urine; liquid chromatography; mass spectrometry
Introduction Tamoxifen (TMX) is a nonsteroidal estrogen antagonist drug therapeutically used for the treatment of breast cancer. TMX is considered a pro-drug since its metabolites have more activity than itself. Amongst them, 4-hydroxy-tamoxifen shows from 30 to 100 times more activity than TMX (Borgna and Rochefort, 1981; Robertson et al., 1982; Jordan, 1982; Coezy et al., 1982) and its secondary metabolite 4-hydroxy-N-desmethyl-tamoxifen (endoxifen) also exhibits a similar activity (Stearns et al., 2003; Johnson et al., 2004; Young et al., 2006). From the point of view of sport drug testing, TMX is a drug included in the list of banned substances of the World Anti Doping Agency (WADA; group S4, hormone antagonist and modulators; WADA, 2014a, 2014b). Its use is prohibited at all the times (in and out of competition). Various authors have reported on the metabolism of TMX, as recently reviewed by Beijnen and co-workers (Teunissen et al., 2010). While TMX metabolism studies have been reported extensively in serum (Gjerde et al., 2005; Teunissen et al., 2009, 2011a, 2011b), plasma (Stearns et al., 2003; Murphy et al., 1987a; Stevenson et al., 1988; Kikuta and Schmid, 1989; Dahmane et al., 2010;
Biomed. Chromatogr. 2015
Jaremko et al., 2010) and other specimens such as tumoral tissues (Murphy et al., 1987b; MacCallum et al., 1997), in vitro preparations using liver microsomes (Jones et al. 1996; Lu et al., 1996; Lim et al., 1997) and synthetic gastric acids (Li et al., 2001), the urinary excretion of TMX metabolites has not been studied in such detail (De Vos et al., 1998; Mazzarino et al., 2008, 2010, 2013; Lu et al., 2014).
* Correspondence to: J. F. García-Reyes, Analytical Chemistry Research Group, Department of Physical and Analytical Chemistry, University of Jaén, 23071 Jaén, Spain. Email: [email protected] a
Analytical Chemistry Research Group, Department of Physical and Analytical Chemistry, University of Jaén, 23071, Jaén, Spain
b
Cellular Stress and Age Research Group. Department of Experimental Biology, University of Jaén, 23071, Jaén, Spain Abbreviations used: HP-921, hexakis-(1H,1H,3H-tetrafluoropropoxy) phosphazine; TMX, tamoxifen; WADA, World Anti Doping Agency; UHPLCTOFMS, ultra-high-performance liquid chromatography electrospray timeof-flight mass spectrometry.
Copyright © 2015 John Wiley & Sons, Ltd.
J. C. Domínguez-Romero et al. In the past, the identification of TMX metabolites was performed by liquid chromatography with fluorescence detection after photochemical reaction (Stearns et al., 2003; Stevenson et al., 1988; Kikuta and Schmid, 1989; De Vos et al., 1998). Nowadays, although gas chromatography (Murphy et al., 1987a, 1987b) and capillary electrophoresis (Lu et al., 1996) coupled with mass spectrometric detection have been used for TMX metabolite identification, a far more convenient technique employed for TMX metabolite identification is liquid chromatography–mass spectrometry (HPLC-MS) with different mass analyzers such as triple quadrupole (HPLC-MS/MS; Teunissen et al., 2011a, 2011b; Jaremko et al., 2010; Jones et al., 1996; Lim et al., 1997; Mazzarino et al., 2010, 2013), time-of-flight mass spectrometers (HPLCTOFMS; Li et al., 2001; Mazzarino et al., 2008, 2013; Lu et al., 2014) and orbital ion trap high-resolution mass spectrometry instruments (Dahmane et al., 2014). The main mass spectrometric strategies used so far for the identification of TMX metabolites in urine include accurate mass measurements using high resolution mass spectrometry (HRMS) (Mazzarino et al., 2008; Lu et al., 2014; Dahmane et al., 2014) or the collection of data from different (up to five) scanning modes using HPLC-MS/MS: a precursor ion scan of m/z 166, 152 and 129 and neutral loss of m/z 72 and 58; Mazzarino et al., 2010). High resolving power mass spectrometers, such as time-offlight analyzers or orbitraps, offer full-scan high sensitivity and high mass accuracy, enabling screening for a theoretically unlimited number of compounds and the analysis of unknown or nontarget compounds, which makes these instruments a powerful tool for the identification of drug metabolites, particularly when combined with ultra-high pressure liquid chromatography separations (Thevis et al., 2013). In this work, we report a study of TMX urinary metabolites in rats after a single-dose administration using solid-phase extraction followed by ultra-high-pressure liquid chromatography electrospray time-of-flight mass spectrometry (UHPLC-TOFMS). Up to 38 TMX metabolites were detected, some of the modifications detected not previously reported in urine in the literature.
Experimental Chemicals and reagents Tamoxifen was purchased from Sigma-Aldrich (Madrid, Spain). HPLCgrade acetonitrile and methanol were acquired from Sigma-Aldrich (Madrid, Spain). Formic acid was obtained from Fluka (Madrid, Spain). A Milli-Q-Plus ultra-pure water system from Millipore (Milford, MA, USA) was used throughout the study to obtain HPLC water during the analyses.
Sample collection The study was performed on adult male Wistar rats (250–300 g; Charles River Laboratories, Barcelona, Spain). The animals (n = 5) were weighed and placed in individual metabolic cages 48 h prior to treatment to acclimatize them to this environment, maintained under standard conditions of light and temperature and allowed ad libitum access to food and water to the end of the experiment. All the procedures followed the Spanish guidelines on the use of animals for research (RD 1201/2005) and were approved by the institutional Committee for Ethics. The rats were treated with TMX (10 mg/kg body weight, intraperitoneal). After drug administration, urine was collected daily in graduate cylinders for 3 days (24, 48 and 72 h). The urine collected 24 h prior to treatment was used as control.
wileyonlinelibrary.com/journal/bmc
Sample treatment A 2 mL aliquot of urine was diluted with 2 mL of formic acid/formate pH 2.6 buffer and then passed through a polymeric Bond Elut PLEXA™ SPE cartridge from Agilent Technologies (Santa Clara, CA, USA) and washed with 2 mL of H2O–methanol (95:5). The cartridges were previously conditioned with 4 mL of methanol–acetonitrile (1:1) and 4 mL of MilliQ water. The analytes were eluted from the cartridge with 4 mL of methanol–acetonitrile (1:1) and the extract was evaporated to near dryness with a TurboVap LV (Caliper LifeSciences, Hopkinton, MA, USA) and then taken up with 0.5 mL of the initial mobile phase to achieve a preconcentration factor of 4:1. Finally, the extract was filtered through a 0.22 μm PTFE syringe filter and transferred to a 2 mL glass vial prior to LC-MS analyses. Various recovery studies were conducted with TMX at different concentration levels to ensure that the compound was quantitatively recovered. The recovery rates obtained approached 100% regardless of the type of polymeric cartridge (Oasis HLB or PLEXA) used. Considering the similar structures and chromatographic properties of potential TMX phase-I metabolites, it is reasonable to expect a similar behavior.
UHPLC-TOFMS method The chromatographic separation was performed using a UHPLC system (Agilent Infinity 1290, Agilent Technologies, Santa Clara, CA, USA) equipped with a reversed-phase Zorbax high-definition (RRHD) Eclipse Plus-C18 analytical column of 2.1 × 50 mm and 1.8 μm particle size (Agilent Technologies, Santa Clara, CA, USA). Mobile phases A and B were water with 0.1% formic acid and acetonitrile. The chromatographic method held the initial mobile phase composition (10% B) constant for 5 min, followed by a linear gradient to 40% B up to 28 min, to 100% up to 33 min and finally kept for 2 min at 100%. The flow rate used was 0.5 mL/min and 20 μL of the urine extract were injected in each run. This UHPLC system was connected to a time-of-flight mass spectrometer (Agilent 6220 accurate mass TOF; Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray interface operating in positive ion mode, using the following operation parameters: capillary voltage, 3500 V; nebulizer pressure, 40 psig; drying gas flow rate, 9.0 L/min; gas temperature, 325 °C; skimmer voltage, 65 V; octapole 1 rf, 250 V; fragmentor voltage 175 and 230 V (in-source CID fragmentation); acquisition timem 1.5 spectra/s. LC-MS accurate mass spectra were recorded across the range of m/z 50–1000. The instrument performed an internal calibration using the reference mixture provided by the manufacturer over the acquired mass range, using a second sprayer with a reference solution containing the masses purine (m/z 121.0509) and hexakis-(1H,1H,3Htetrafluoropropoxy)phosphazine (HP-921; C18H18O6N3P3F24, m/z 922.0098). The instrument was operated in the 4 GHz high-resolution mode. The resolution displayed by the instrument with these conditions was ca. 14,000 at m/z 400. The full-scan data were recorded with Agilent Mass Hunter Data Acquisition software (version B.04.00) and processed with Agilent Mass Hunter Qualitative Analysis software (version B.04.00). The analytical performance of the method for TMX determination in urine was satisfactory with a limit of quantitation
