Research article Received: 24 September 2013,
Revised: 11 November 2013,
Accepted: 6 December 2013
Published online in Wiley Online Library: 15 January 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3127
Quantitative determination of enzalutamide, an anti-prostate cancer drug, in rat plasma using liquid chromatography–tandem mass spectrometry, and its application to a pharmacokinetic study Ji-Hye Songa, Tae-Heon Kima, Jong-Woo Junga, Nakjeong Kimb, Sung Hoon Ahnb, Sung-Ook Hwangc, Nam Sook Kanga, Sung-Eun Yooa and Tae-Sung Kooa* ABSTRACT: This report details a method using liquid chromatography–tandem mass spectrometry (LC-MS/MS) that allows one to determine the concentration of an atypical anticancer drug, enzalutamide, in rat plasma. Specifically, this method involves the addition of an acetonitrile and bicalutamide (internal standard) solution to plasma samples. Following centrifugation of this mixture, an aliquot of the supernatant was directly injected into the LC-MS/MS system. Separation was achieved using a column packed with octadecylsilica (5 μm, 2.1 × 50 mm) with 10 mM ammonium acetate in acetonitrile as the mobile phase; detection was accomplished using MS/MS by multiple-reaction monitoring via an electrospray ionization source. This method demonstrated a linear standard curve (r = 0.997) over a concentration range of 0.001–1 μg/mL, as well as an intra- and inter-assay precision of 2.7 and 5.1%, respectively, and an accuracy range from 100.8 to 105.6%. The lower limit of quantification was 1.0 ng/mL in 50 μL of rat plasma sample. We also demonstrated that this analytical method could be successfully applied to the pharmacokinetic study of enzalutamide in rats. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: enzalutamide; bicalutamide; pharmacokinetics; LC/MS/MS; rat plasma
Introduction
1112
Enzalutamide (EZT), formally called MDV3100, is an anticancer drug used to treat metastatic castration-resistant prostate cancer (mCRPC). Developed by Medivation and Astellas, it was approved by the US Food and Drug Administration as new drug in 2012 under the trade name of Xtandi™ (Mullard, 2013; Schrader et al., 2014). EZT has three major anticancer mechanisms – as an androgen receptor (AR) antagonist, as a translocation blocker and as a transcription inhibitor. As an AR antagonistic, EZT has a 5- to 8-fold greater affinity than bicalutamide (BCT) (Tran et al., 2009). Furthermore, unlike BCT, EZT decreases translocation to the nucleus and inhibits interactions with DNA. As a result, EZT acts to induce apoptosis to decrease tumor volume as well as the proliferation of cancer cells (Scher et al., 2010; Tran et al., 2009). EZT has been shown to be clinically effective in combating mCRPC in previous clinical trials, decreasing the level of prostate cancer specific antigens, the typical prostate cancer indicator, by more than 50% in both patients with prostate cancer and those with mCRPC after chemotherapy. Additionally, the survival of patients with mCRPC after chemotherapy was significantly prolonged through use of EZT (Cabot et al., 2012; Loriot et al., 2013; Scher et al., 2010, 2012). In modern drug discovery programs, a thorough understanding of how a reference drug functions is indispensable. Despite the
Biomed. Chromatogr. 2014; 28: 1112–1117
abundance of studies around EZT, information about the quantitative bioanalysis of EZT is not available. Also, there is very limited information about pharmacokinetics of experimental animals, especially rat, generally used as reference data. In this report, we outline a reliable analytical method of measuring EZT in rat plasma via liquid chromatography–tandem mass spectrometry (LC-MS/MS). This method was successfully used in the pharmacokinetic study of EZT in rats and may be helpful for the investigation of other similar drugs.
* Correspondence to: T.-S. Koo, Graduate School of Drug Discovery and Development, Chungnam National University, Daejeon 305-764, Korea. Email: [email protected] a
Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon, Korea
b
Division of Bio-organic Science, Korea Research Institute of Chemical Technology, Daejeon, Korea
c
Department of Obstetrics and Gynecology, Inha University Hospital, Incheon, Korea Abbreviations used: AR, androgen receptor; BCT, bicalutamide; EZT, enzalutamide; mCRPC, metastatic castration-resistant prostate cancer.
Copyright © 2014 John Wiley & Sons, Ltd.
Quantification of enzalutamide in plasma with LC-MS/MS Sample preparation
Experimental Reagents and materials EZT was obtained from the Korea Research Institute of Chemical Technology (Daejeon, Korea; Fig. 1). BCT [internal standard (IS); Fig. 1] and ammonium acetate were purchased from Sigma-Aldrich (St Louis, MO, USA). Acetonitrile and methanol [high-performance liquid chromatography (HPLC) grade] were purchased from Burdick & Jackson Inc. and the other chemicals were either of HPLC grade or the highest grade available. Rat plasma containing sodium heparin as the anticoagulant was prepared in our laboratory.
LC-MS/MS conditions HPLC was carried out using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA), which was composed of an autosampler, a binary pump, a column oven and a system controller. Compounds were separated on a Agilent Eclipse Plus C18 column (3.5 μm, 2.1 × 50 mm; Agilent Technologies) with an isocratic mobile phase comprising 10 mM ammonium acetate in 40:60 (v/v) water–methanol at a flow rate of 0.3 mL/min. The column and autosampler tray were preserved at 25 and 4 °C, respectively. The eluent was introduced directly into the tandem quadrupole mass spectrometer (API 4000 QTrap; Applied Biosystems/MDS Sciex, Foster City, CA, USA) through a turbo ion spray source with the following settings: curtain gas, 20 psi; nebulizer gas, 40 psi; turbo gas, 40 psi; ionspray voltage, 5500 V; temperature, 500 °C; declustering potentials, 96, 91 and 101 V; entrance potential, 10 V; collision energies, 47 and 51 V; collision cell exit potential, 14 and 16 V for EZT and the IS, respectively. An analytical run time of 5.0 min was used. Multiple reaction monitoring (MRM) mode was used for quantification at m/z 465 → 209 for EZT and m/z 431 → 217 for the IS. Data were analyzed using Analyst software (version 1.4.1; Applied Biosystems/AB Sciex).
Preparation of the calibration standard, quality control, and internal standard stock solutions Primary stock solutions containing a 1 mg/mL concentration of EZT and BCT were dissolved in methanol; serial dilutions of this solution were made in methanol to achieve working standard solutions at concentrations of 10, 20, 50, 200, 2000, 5000 and 10,000 ng/mL of EZT and BCT. Solutions to outline the lower limit of quantification (LLOQ) and low quality control (LQC), medium QC (MQC), high QC (HQC) and dilution QC (DQC) samples were prepared in bulk by diluting the primary stock solution to 10, 30, 1000, 9000 and 90,000 ng/mL, respectively. The IS working solution (1 μg/mL) was prepared by diluting an aliquot of stock solution (1 mg/mL) of BCT with acetonitrile. All stock solutions were stored in glass bottles at –20 °C in the dark.
To induce the precipitation of plasma proteins, 50 μL of the IS working solution and 200 μL of acetonitrile were added to 50 μL aliquots of rat plasma containing 5 μL of calibration standards. This mixture was vigorously mixed for 10 min and centrifuged at 13,500 g for 10 min, after which an aliquot (200 μL) of the supernatant was transferred to a well plate. An aliquot (5 μL) of this solution was then directly injected into the LC-MS/MS system.
Method validation The method was validated with respect to specificity, selectivity, carryover, linearity, accuracy, precision, dilution integrity, percentage recovery, matrix effects and stability (US Food and Drug Administration, 2001). To evaluate selectivity, six different rat plasma matrices (i.e. samples without EZT and the IS), zero samples (i.e. blank plasma with the IS) and LLOQ samples were used to confirm the absence of potential endogenous interfering peaks in chromatograms. The LLOQ was determined as the concentration that has a precision of
Revised: 11 November 2013,
Accepted: 6 December 2013
Published online in Wiley Online Library: 15 January 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3127
Quantitative determination of enzalutamide, an anti-prostate cancer drug, in rat plasma using liquid chromatography–tandem mass spectrometry, and its application to a pharmacokinetic study Ji-Hye Songa, Tae-Heon Kima, Jong-Woo Junga, Nakjeong Kimb, Sung Hoon Ahnb, Sung-Ook Hwangc, Nam Sook Kanga, Sung-Eun Yooa and Tae-Sung Kooa* ABSTRACT: This report details a method using liquid chromatography–tandem mass spectrometry (LC-MS/MS) that allows one to determine the concentration of an atypical anticancer drug, enzalutamide, in rat plasma. Specifically, this method involves the addition of an acetonitrile and bicalutamide (internal standard) solution to plasma samples. Following centrifugation of this mixture, an aliquot of the supernatant was directly injected into the LC-MS/MS system. Separation was achieved using a column packed with octadecylsilica (5 μm, 2.1 × 50 mm) with 10 mM ammonium acetate in acetonitrile as the mobile phase; detection was accomplished using MS/MS by multiple-reaction monitoring via an electrospray ionization source. This method demonstrated a linear standard curve (r = 0.997) over a concentration range of 0.001–1 μg/mL, as well as an intra- and inter-assay precision of 2.7 and 5.1%, respectively, and an accuracy range from 100.8 to 105.6%. The lower limit of quantification was 1.0 ng/mL in 50 μL of rat plasma sample. We also demonstrated that this analytical method could be successfully applied to the pharmacokinetic study of enzalutamide in rats. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: enzalutamide; bicalutamide; pharmacokinetics; LC/MS/MS; rat plasma
Introduction
1112
Enzalutamide (EZT), formally called MDV3100, is an anticancer drug used to treat metastatic castration-resistant prostate cancer (mCRPC). Developed by Medivation and Astellas, it was approved by the US Food and Drug Administration as new drug in 2012 under the trade name of Xtandi™ (Mullard, 2013; Schrader et al., 2014). EZT has three major anticancer mechanisms – as an androgen receptor (AR) antagonist, as a translocation blocker and as a transcription inhibitor. As an AR antagonistic, EZT has a 5- to 8-fold greater affinity than bicalutamide (BCT) (Tran et al., 2009). Furthermore, unlike BCT, EZT decreases translocation to the nucleus and inhibits interactions with DNA. As a result, EZT acts to induce apoptosis to decrease tumor volume as well as the proliferation of cancer cells (Scher et al., 2010; Tran et al., 2009). EZT has been shown to be clinically effective in combating mCRPC in previous clinical trials, decreasing the level of prostate cancer specific antigens, the typical prostate cancer indicator, by more than 50% in both patients with prostate cancer and those with mCRPC after chemotherapy. Additionally, the survival of patients with mCRPC after chemotherapy was significantly prolonged through use of EZT (Cabot et al., 2012; Loriot et al., 2013; Scher et al., 2010, 2012). In modern drug discovery programs, a thorough understanding of how a reference drug functions is indispensable. Despite the
Biomed. Chromatogr. 2014; 28: 1112–1117
abundance of studies around EZT, information about the quantitative bioanalysis of EZT is not available. Also, there is very limited information about pharmacokinetics of experimental animals, especially rat, generally used as reference data. In this report, we outline a reliable analytical method of measuring EZT in rat plasma via liquid chromatography–tandem mass spectrometry (LC-MS/MS). This method was successfully used in the pharmacokinetic study of EZT in rats and may be helpful for the investigation of other similar drugs.
* Correspondence to: T.-S. Koo, Graduate School of Drug Discovery and Development, Chungnam National University, Daejeon 305-764, Korea. Email: [email protected] a
Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon, Korea
b
Division of Bio-organic Science, Korea Research Institute of Chemical Technology, Daejeon, Korea
c
Department of Obstetrics and Gynecology, Inha University Hospital, Incheon, Korea Abbreviations used: AR, androgen receptor; BCT, bicalutamide; EZT, enzalutamide; mCRPC, metastatic castration-resistant prostate cancer.
Copyright © 2014 John Wiley & Sons, Ltd.
Quantification of enzalutamide in plasma with LC-MS/MS Sample preparation
Experimental Reagents and materials EZT was obtained from the Korea Research Institute of Chemical Technology (Daejeon, Korea; Fig. 1). BCT [internal standard (IS); Fig. 1] and ammonium acetate were purchased from Sigma-Aldrich (St Louis, MO, USA). Acetonitrile and methanol [high-performance liquid chromatography (HPLC) grade] were purchased from Burdick & Jackson Inc. and the other chemicals were either of HPLC grade or the highest grade available. Rat plasma containing sodium heparin as the anticoagulant was prepared in our laboratory.
LC-MS/MS conditions HPLC was carried out using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA), which was composed of an autosampler, a binary pump, a column oven and a system controller. Compounds were separated on a Agilent Eclipse Plus C18 column (3.5 μm, 2.1 × 50 mm; Agilent Technologies) with an isocratic mobile phase comprising 10 mM ammonium acetate in 40:60 (v/v) water–methanol at a flow rate of 0.3 mL/min. The column and autosampler tray were preserved at 25 and 4 °C, respectively. The eluent was introduced directly into the tandem quadrupole mass spectrometer (API 4000 QTrap; Applied Biosystems/MDS Sciex, Foster City, CA, USA) through a turbo ion spray source with the following settings: curtain gas, 20 psi; nebulizer gas, 40 psi; turbo gas, 40 psi; ionspray voltage, 5500 V; temperature, 500 °C; declustering potentials, 96, 91 and 101 V; entrance potential, 10 V; collision energies, 47 and 51 V; collision cell exit potential, 14 and 16 V for EZT and the IS, respectively. An analytical run time of 5.0 min was used. Multiple reaction monitoring (MRM) mode was used for quantification at m/z 465 → 209 for EZT and m/z 431 → 217 for the IS. Data were analyzed using Analyst software (version 1.4.1; Applied Biosystems/AB Sciex).
Preparation of the calibration standard, quality control, and internal standard stock solutions Primary stock solutions containing a 1 mg/mL concentration of EZT and BCT were dissolved in methanol; serial dilutions of this solution were made in methanol to achieve working standard solutions at concentrations of 10, 20, 50, 200, 2000, 5000 and 10,000 ng/mL of EZT and BCT. Solutions to outline the lower limit of quantification (LLOQ) and low quality control (LQC), medium QC (MQC), high QC (HQC) and dilution QC (DQC) samples were prepared in bulk by diluting the primary stock solution to 10, 30, 1000, 9000 and 90,000 ng/mL, respectively. The IS working solution (1 μg/mL) was prepared by diluting an aliquot of stock solution (1 mg/mL) of BCT with acetonitrile. All stock solutions were stored in glass bottles at –20 °C in the dark.
To induce the precipitation of plasma proteins, 50 μL of the IS working solution and 200 μL of acetonitrile were added to 50 μL aliquots of rat plasma containing 5 μL of calibration standards. This mixture was vigorously mixed for 10 min and centrifuged at 13,500 g for 10 min, after which an aliquot (200 μL) of the supernatant was transferred to a well plate. An aliquot (5 μL) of this solution was then directly injected into the LC-MS/MS system.
Method validation The method was validated with respect to specificity, selectivity, carryover, linearity, accuracy, precision, dilution integrity, percentage recovery, matrix effects and stability (US Food and Drug Administration, 2001). To evaluate selectivity, six different rat plasma matrices (i.e. samples without EZT and the IS), zero samples (i.e. blank plasma with the IS) and LLOQ samples were used to confirm the absence of potential endogenous interfering peaks in chromatograms. The LLOQ was determined as the concentration that has a precision of
