Research article Received: 17 November 2013,
Revised: 23 February 2014,
Accepted: 14 April 2014
Published online in Wiley Online Library: 15 May 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3246
Development of a rapid and sensitive UPLC-MS/MS assay for the determination of TM-2 in beagle dog plasma and its application to a pharmacokinetic study Hongli Lin, Yunli Zhao†, Lei Men†, Mingjing Yang, Hui Liu, Yanjie Shao, Pei Wang, Xing Tang and Zhiguo Yu* ABSTRACT: A simple and sensitive method based on ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of TM-2, which was a novel semi-synthetic taxane derivative in beagle dog plasma. Cabazitaxel was chosen as internal standard. Following extraction by methyl tert-butyl ether, the chromatographic separation was achieved on a Thermo Syncronis C18 column (50 × 2.1 mm, 1.7 μm) by gradient elution within a runtime of 3.5 min. The mobile phase consisted of (A) acetonitrile and (B) 2 mmol/L ammonium acetate in water. The detection was accomplished using positive ion electrospray ionization in multiple reaction monitoring mode. The MS/MS ion transitions were monitored at m/z 812.39 → 551.35 for TM-2 and 836.36 → 555.26 for IS, respectively. The method was linear for TM-2 (r = 0.9924) ranging from 2.5 to 1000 ng/mL. The intra-day and inter-day precisions (relative standard deviation) were within 8.0 and 17.6%, respectively, and the accuracy (relative error) was less than 2.3%. The extraction recovery ranged from 83.1 to 97.1%. The reliable method was successfully applied to a pharmacokinetic study of TM-2 in beagle dogs after intravenous drip with different doses of 0.6, 1.2, and 2.4 mg/kg, respectively. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: TM-2; taxane; UPLC-MS/MS; pharmacokinetics; beagle dog plasma
Introduction
110
Cancer is a leading global cause of death and disability, responsible for approximately 7.6 million deaths each year (Beaglehole et al., 2011; World Health Organization, 2011). Besides surgery, ‘biological’ and radiotherapy treatment, chemotherapy is still the most effective means to prolong the survival of patients (Tamura, 2003). Taxane drugs, such as paclitaxel, docetaxel and larotaxel, are extensively used to treat mammary, prostatic, ovarian, nonsmall-cell lung carcinoma and AIDS-associated Kaposi sarcoma (Zhou and Giannakakou, 2005; Wyld and Reed, 2007). However, multidrug resistance (MDR) of neoplastic tissues is a huge challenge in cancer chemotherapy (Abal et al., 2003; Tommasi et al., 2007). To overcome the weakness of taxane, a new taxane derivative, 4,10-β-diacetoxy-2-α-benzoyloxy-5-β,20epoxy-1,13-α-dihydroxy-9-oxo-19-nor-cyclopropa[g]tax-11-ene, 13-ester with(2R,3S)-N-tert-butoxycarbonyl-3-(2-methyl propyl) isoserine dehydrate named TM-2 was developed (Fig. 1A). The molecular formula is C43H57NO14 (molecular weight 811.9 Da). There has been clear evidence that TM-2 possesses significant activity in human tumor lines in vitro and in vivo. Compared with docetaxel or larotaxel, TM-2 was more active in vitro studies involving resistant cancer cell lines KB/VCR (human cervical adenocarcinoma resistant to vincristine) and MCF-7/ADR (human breast cancer resistant to adriamycin; Li et al., 2011). In addition, the antitumor activity of TM-2 was 20% higher than that of docetaxel in nude mice bearing A549 human lung cancer xenografts. Therefore, based on the advantages of TM-2, it was selected for subsequent preclinical evaluation.
Biomed. Chromatogr. 2015; 29: 110–114
Pharmacokinetic parameters, such as the time of maximum plasma concentration (Tmax), maximum plasma concentration (Cmax), area under the plasma concentration–time curve (AUC0–t), volume of distribution (V) and clearance (CL) are of great significance for the further clinical research. For the preclinical pharmacokinetic evaluation of a new drug, two animal species are essential (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2012). The pharmacokinetic assessment in a rodent has been accomplished (unpublished data). Therefore, a preliminary pharmacokinetic study in beagle dogs (nonrodent) to obtain pharmacokinetic parameters is meaningful for the safety evaluation of TM-2. In this paper, for the first time, we developed a sensitive and selective UPLC-MS/MS method in multiple reaction monitoring mode for the quantification of TM-2 in beagle dog plasma using cabazitaxel as internal standard (IS) after liquid–liquid extraction
* Correspondence to: Z. Yu, School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, People’s Republic of China. Email: [email protected] †
The first two authors contributed equally to this work and should be regarded as co-second authors. School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, People’s Republic of China Abbreviations used: MDR, multidrug resistance.
Copyright © 2014 John Wiley & Sons, Ltd.
Determination of TM-2 in beagle dog plasma by UPLC-MS/MS
Figure 1. Chemical structure of TM-2 (A) and cabazitaxel (B).
by methyl tert-butyl ether. The total run time was 3.5 min per sample. The method was fully validated and applied to the pharmacokinetic study of TM-2 in beagle dogs after intravenous drip. This study might supply more preclinical pharmacokinetic information on TM-2 and a firm basis for evaluating the clinical efficiency.
Experimental Chemicals and reagents TM-2 (purity >98.0%) and cabazitaxel (Fig. 1B; internal standard, IS, purity >98.0%) were supplied by Fudan University (Shanghai, China). Acetonitrile and methanol of HPLC grade were obtained from Fisher Scientific (Fair Lawn, NJ, USA). HPLC-grade ammonium acetate was provided by Dikma Co. Ltd (Beijing, China). Methyl tert-butyl ether of analytical grade was purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Other chemicals were of analytical grade. Demineralized water was used throughout the study.
Preparation of TM-2 infusion TM-2 for injection, which was formulated in polysorbate 80 (40 mg/mL), was diluted with 13% ethanol (w/w) and then 5% dextrose solution to a final concentration for injection.
UPLC-MS/MS conditions
Preparation of standards and quality control samples Stock solutions of TM-2 (1 mg/mL) and IS (1 mg/mL) were prepared independently by accurately weighing the required amounts into volumetric flasks and dissolving in methanol. The working solutions of TM-2 were obtained by diluting the stock solution successively with methanol. The stock solution of IS was diluted with methanol to make a working solution of 1 μg/mL. All solutions were stored at 20°C and were brought to room temperature before use. For preparation of standard samples for calibration curve, 10 μL of the appropriate working solutions of TM-2 was added to 200 μL of blank beagle dog plasma to prepare concentrations of 2.5, 5, 10, 50, 250, 500 and 1000 ng/mL for TM-2 and 50 ng/mL for IS. Quality control (QC) samples at three concentration levels (low, 5 ng/mL; medium, 50 ng/mL; high, 800 ng/mL) were independently prepared in the same way. The standards and QC samples were freshly prepared before use.
Sample preparation A simple liquid–liquid extraction method was applied to extract the analyte and IS from beagle dog plasma. Aliquots of 200 μL beagle dog plasma sample were transferred to a 5 mL polypropylene tube followed by the addition of 10 μL IS working standard solution and 10 μL methanol. Then the mixture was extracted with 2 mL methyl tert-butyl ether by vortex-mixing for 10 min. The supernatant was transferred to another tube after centrifugation at 13000 rpm at 20°C for 10 min, and evaporated to dryness at 35°C under a gentle stream of nitrogen. Finally, the residue was reconstituted in 100 μL of the acetonitrile–water (60:40, v/v) followed by centrifugation at 13,000 rpm at 4°C for 5 min. An aliquot of 5 μL of the supernatant was injected into the UPLC-MS/MS system in the partial loop mode.
TM
Biomed. Chromatogr. 2015; 29: 110–114
Method validation The method was validated for specificity, calibration curve, accuracy, precision, recovery, matrix effect, stability and dilution effect in beagle dog plasma according to the US Food and Drug Administration guideline (US DHHS et al., 2001) and European Medicines Agency (2012) guidelines on bioanalytical method validation. Specificity. Comparing the chromatograms of blank beagle dog plasma, beagle dog plasma sample spiked with TM-2 and IS and beagle dog plasma sample after intravenous administration of TM-2, no endogenous components and metabolites of TM-2 interfered in the assay of the analyte and IS. Calibration curve. The calibration curves were constructed by plotting the peak-area ratios of each analyte to IS vs plasma concentrations 2 using a 1/x weighted least-squares linear regression model. The acceptance criterion for each back-calculated standard concentration was ±15% deviation from the nominal value, except at the LLOQ, which was within ±20%. Precision and accuracy. The intra-day precision and accuracy were determined by analyzing QC samples at three concentration levels
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
111
Assays were acquired using an Acquity ultra-performance liquid chromatography system (Waters Corp., Milford, MA, USA) with cooling autosampler and column oven enabling temperature control. A Thermo Syncronis C18 column (50 × 2.1 mm, 1.7 μm) was employed and the column temperature was maintained at 35°C. Gradient elution (delivered at 0.2 mL/min) was employed using acetonitrile (mobile phase A) and 2 mmol/L ammonium acetate in water (mobile phase B), which started at 60% A and increased linearly to 90% A at 1.5 min, then kept this proportion until 2.8 min, finally reducing the proportion to 60% A until 3.5 min to equilibrate the column. The autosampler was conditioned at TM TQD triple8°C and the injection volume was 5 μL. An Acquity quadrupole tandem mass spectrometer (Waters Corp, Milford, MA, USA) was applied to collect the mass spectrometric data. The detection was accomplished using positive ion electrospray ionization in multiple reaction monitoring mode. The MS/MS ion transitions were monitored at m/z 812.39 → 551.35 for TM-2 and 836.36 → 555.26 for IS. Detection conditions were: span, 0.2 Da; dwell time, 0.2 s. Nitrogen was used both for the cone gas (40 L/h) and the desolvation gas (550 L/h), with the source and desolvation temperatures being retained at 100 and 400°C, respectively. Argon was used as the collision gas (0.16 mL/min). The ionization source terms were as follows: capillary voltage 3.50 kV; cone voltage, 30 and 25 V; and optimized collision energy, 10 eV both for TM-2 and IS.
H. Lin et al. (low, 5 ng/mL; medium, 50 ng/mL; high, 800 ng/mL) in six replicates on the same day, while the inter-day precision and accuracy were evaluated by analyzing QC samples at three concentration levels on three continual validation days. The precision was expressed as relative standard deviation (RSD, %) and the accuracy as the relative error (RE, %). Recovery and matrix effect. The extraction recoveries of TM-2 at three QC levels with six replicates were measured by comparing the peak areas from extracted samples with those from post-extracted blank plasma samples spiked with the analytes at the same concentration. The extraction recovery of IS was evaluated in the same way. The matrix effect was measured at three QC levels by comparing the peak area from the post-extracted blank plasma spiked with TM-2
working solutions with those of corresponding standard solutions. The matrix effect of IS was evaluated using the same procedure. Stability. The stability of TM-2 in beagle dog plasma was conducted at the three QC concentration levels (n = 3) in various storage conditions. Post-preparative stability was evaluated by analyzing the processed QC samples kept in an autosampler at 8°C for 24 h. Short-term and longterm stability were studied by analyzing QC samples exposed at room temperature for 4 h and stored at 80°C for 4 months, respectively. The freeze and thaw stability was tested by analyzing QC samples undergoing three freeze–thaw ( 80°C to room temperature) cycles on three consecutive days. Dilution effect and carry-over. Dilution effect was investigated to ensure that samples could be diluted with blank matrix without affecting the final concentration. Blank beagle dog plasma samples spiked with TM-2 (1600 ng/mL) were diluted with pooled blank beagle dog plasma at dilution factors of 2 in six replicates and analyzed. The six replicates should have precision of ≤15% and accuracy within ±15%. The carry-over was determined by injecting a blank plasma sample following the injection of an upper limit of quantification sample in three independent runs. Carry-over was considered negligible if the measured peak area was 0.05), which indicated that the pharmacokinetic properties of TM-2 were linear and similar to cabazitaxel (European Medicines Agency, 2011).
Conclusion The method was successfully applied to the pharmacokinetic study of TM-2 after a single intravenous drip to beagle dogs (0.6, 1.2 and 2.4 mg/kg). This study is the first report of the pharmacokinetic parameters of TM-2 in beagle dogs, and would be useful for further characterizations of the pharmacokinetics, pharmacy and toxicity of TM-2 as a potential new drug.
References Abal M, Andreu J and Barasoain I. Taxanes: microtubule and centrosome targets, and cell cycle dependent mechanisms of action. Current Cancer Drug Targets 2003; 3(3): 193–203. Beaglehole R, Bonita R and Magnusson R. Global cancer prevention: an important pathway to global health and development. Public Health 2011; 125(12): 821–831. European Medicines Agency. Assessment report for Jevtana (Cabazitaxel): EMEA/CHMP/66633/2011. Procedure no. EMEA/H/C/002018, 2011. European Medicines Agency. Guidelines on Bioanalytical Method Validation, EMEA/CHMP/EWP/192217/2009, 1 February 2012. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2011/08/WC5001pdf (accessed 19July 2012). International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. Preclinical safety evaluation of biotechnology-derived pharmaceuticals S6 (R1), 2012. Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM194490.pdf (accessed 16 December 2012). Li YX, Geng MY, Wang JF, Liu HC, Ding N and Zhang W. Multi-drug resistant resistant derivatives and its preparation method and application of taxanes. China patent no. 201110127602.X;2011-05-17, 2011. Tamura T. Molecular-targeted therapy. Gan to kagaku ryoho/Cancer and Chemotherapy 2003; 30(2): 198. Tommasi S, Mangia A, Lacalamita R, Bellizzi A, Fedele V and Chiriatti A. Cytoskeleton and paclitaxel sensitivity in breast cancer: the role of β-tubulins. International Journal of Cancer 2007; 120(10): 2078–2085. US DHHS, FDA and CDER. Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Rat Services, Food and Drug Administration, Center for Drug Evaluation and Research and Center for Veterinary Medicine: Rockville, MD, 2001. World Health Organization. Global Status Report on Noncommunicable Diseases 2010. World Health Organization: Geneva, 2011. Wyld L and Reed M. The role of surgery in the management of older women with breast cancer. European Journal of Cancer 2007; 43(15): 2253–2263. Zhou J and Giannakakou P. Targeting microtubules for cancer chemotherapy. Current Medicinal Chemistry – Anti-Cancer Agents 2005; 5(1): 65–71.
114 wileyonlinelibrary.com/journal/bmc
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 110–114
Revised: 23 February 2014,
Accepted: 14 April 2014
Published online in Wiley Online Library: 15 May 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3246
Development of a rapid and sensitive UPLC-MS/MS assay for the determination of TM-2 in beagle dog plasma and its application to a pharmacokinetic study Hongli Lin, Yunli Zhao†, Lei Men†, Mingjing Yang, Hui Liu, Yanjie Shao, Pei Wang, Xing Tang and Zhiguo Yu* ABSTRACT: A simple and sensitive method based on ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of TM-2, which was a novel semi-synthetic taxane derivative in beagle dog plasma. Cabazitaxel was chosen as internal standard. Following extraction by methyl tert-butyl ether, the chromatographic separation was achieved on a Thermo Syncronis C18 column (50 × 2.1 mm, 1.7 μm) by gradient elution within a runtime of 3.5 min. The mobile phase consisted of (A) acetonitrile and (B) 2 mmol/L ammonium acetate in water. The detection was accomplished using positive ion electrospray ionization in multiple reaction monitoring mode. The MS/MS ion transitions were monitored at m/z 812.39 → 551.35 for TM-2 and 836.36 → 555.26 for IS, respectively. The method was linear for TM-2 (r = 0.9924) ranging from 2.5 to 1000 ng/mL. The intra-day and inter-day precisions (relative standard deviation) were within 8.0 and 17.6%, respectively, and the accuracy (relative error) was less than 2.3%. The extraction recovery ranged from 83.1 to 97.1%. The reliable method was successfully applied to a pharmacokinetic study of TM-2 in beagle dogs after intravenous drip with different doses of 0.6, 1.2, and 2.4 mg/kg, respectively. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: TM-2; taxane; UPLC-MS/MS; pharmacokinetics; beagle dog plasma
Introduction
110
Cancer is a leading global cause of death and disability, responsible for approximately 7.6 million deaths each year (Beaglehole et al., 2011; World Health Organization, 2011). Besides surgery, ‘biological’ and radiotherapy treatment, chemotherapy is still the most effective means to prolong the survival of patients (Tamura, 2003). Taxane drugs, such as paclitaxel, docetaxel and larotaxel, are extensively used to treat mammary, prostatic, ovarian, nonsmall-cell lung carcinoma and AIDS-associated Kaposi sarcoma (Zhou and Giannakakou, 2005; Wyld and Reed, 2007). However, multidrug resistance (MDR) of neoplastic tissues is a huge challenge in cancer chemotherapy (Abal et al., 2003; Tommasi et al., 2007). To overcome the weakness of taxane, a new taxane derivative, 4,10-β-diacetoxy-2-α-benzoyloxy-5-β,20epoxy-1,13-α-dihydroxy-9-oxo-19-nor-cyclopropa[g]tax-11-ene, 13-ester with(2R,3S)-N-tert-butoxycarbonyl-3-(2-methyl propyl) isoserine dehydrate named TM-2 was developed (Fig. 1A). The molecular formula is C43H57NO14 (molecular weight 811.9 Da). There has been clear evidence that TM-2 possesses significant activity in human tumor lines in vitro and in vivo. Compared with docetaxel or larotaxel, TM-2 was more active in vitro studies involving resistant cancer cell lines KB/VCR (human cervical adenocarcinoma resistant to vincristine) and MCF-7/ADR (human breast cancer resistant to adriamycin; Li et al., 2011). In addition, the antitumor activity of TM-2 was 20% higher than that of docetaxel in nude mice bearing A549 human lung cancer xenografts. Therefore, based on the advantages of TM-2, it was selected for subsequent preclinical evaluation.
Biomed. Chromatogr. 2015; 29: 110–114
Pharmacokinetic parameters, such as the time of maximum plasma concentration (Tmax), maximum plasma concentration (Cmax), area under the plasma concentration–time curve (AUC0–t), volume of distribution (V) and clearance (CL) are of great significance for the further clinical research. For the preclinical pharmacokinetic evaluation of a new drug, two animal species are essential (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2012). The pharmacokinetic assessment in a rodent has been accomplished (unpublished data). Therefore, a preliminary pharmacokinetic study in beagle dogs (nonrodent) to obtain pharmacokinetic parameters is meaningful for the safety evaluation of TM-2. In this paper, for the first time, we developed a sensitive and selective UPLC-MS/MS method in multiple reaction monitoring mode for the quantification of TM-2 in beagle dog plasma using cabazitaxel as internal standard (IS) after liquid–liquid extraction
* Correspondence to: Z. Yu, School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, People’s Republic of China. Email: [email protected] †
The first two authors contributed equally to this work and should be regarded as co-second authors. School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, People’s Republic of China Abbreviations used: MDR, multidrug resistance.
Copyright © 2014 John Wiley & Sons, Ltd.
Determination of TM-2 in beagle dog plasma by UPLC-MS/MS
Figure 1. Chemical structure of TM-2 (A) and cabazitaxel (B).
by methyl tert-butyl ether. The total run time was 3.5 min per sample. The method was fully validated and applied to the pharmacokinetic study of TM-2 in beagle dogs after intravenous drip. This study might supply more preclinical pharmacokinetic information on TM-2 and a firm basis for evaluating the clinical efficiency.
Experimental Chemicals and reagents TM-2 (purity >98.0%) and cabazitaxel (Fig. 1B; internal standard, IS, purity >98.0%) were supplied by Fudan University (Shanghai, China). Acetonitrile and methanol of HPLC grade were obtained from Fisher Scientific (Fair Lawn, NJ, USA). HPLC-grade ammonium acetate was provided by Dikma Co. Ltd (Beijing, China). Methyl tert-butyl ether of analytical grade was purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Other chemicals were of analytical grade. Demineralized water was used throughout the study.
Preparation of TM-2 infusion TM-2 for injection, which was formulated in polysorbate 80 (40 mg/mL), was diluted with 13% ethanol (w/w) and then 5% dextrose solution to a final concentration for injection.
UPLC-MS/MS conditions
Preparation of standards and quality control samples Stock solutions of TM-2 (1 mg/mL) and IS (1 mg/mL) were prepared independently by accurately weighing the required amounts into volumetric flasks and dissolving in methanol. The working solutions of TM-2 were obtained by diluting the stock solution successively with methanol. The stock solution of IS was diluted with methanol to make a working solution of 1 μg/mL. All solutions were stored at 20°C and were brought to room temperature before use. For preparation of standard samples for calibration curve, 10 μL of the appropriate working solutions of TM-2 was added to 200 μL of blank beagle dog plasma to prepare concentrations of 2.5, 5, 10, 50, 250, 500 and 1000 ng/mL for TM-2 and 50 ng/mL for IS. Quality control (QC) samples at three concentration levels (low, 5 ng/mL; medium, 50 ng/mL; high, 800 ng/mL) were independently prepared in the same way. The standards and QC samples were freshly prepared before use.
Sample preparation A simple liquid–liquid extraction method was applied to extract the analyte and IS from beagle dog plasma. Aliquots of 200 μL beagle dog plasma sample were transferred to a 5 mL polypropylene tube followed by the addition of 10 μL IS working standard solution and 10 μL methanol. Then the mixture was extracted with 2 mL methyl tert-butyl ether by vortex-mixing for 10 min. The supernatant was transferred to another tube after centrifugation at 13000 rpm at 20°C for 10 min, and evaporated to dryness at 35°C under a gentle stream of nitrogen. Finally, the residue was reconstituted in 100 μL of the acetonitrile–water (60:40, v/v) followed by centrifugation at 13,000 rpm at 4°C for 5 min. An aliquot of 5 μL of the supernatant was injected into the UPLC-MS/MS system in the partial loop mode.
TM
Biomed. Chromatogr. 2015; 29: 110–114
Method validation The method was validated for specificity, calibration curve, accuracy, precision, recovery, matrix effect, stability and dilution effect in beagle dog plasma according to the US Food and Drug Administration guideline (US DHHS et al., 2001) and European Medicines Agency (2012) guidelines on bioanalytical method validation. Specificity. Comparing the chromatograms of blank beagle dog plasma, beagle dog plasma sample spiked with TM-2 and IS and beagle dog plasma sample after intravenous administration of TM-2, no endogenous components and metabolites of TM-2 interfered in the assay of the analyte and IS. Calibration curve. The calibration curves were constructed by plotting the peak-area ratios of each analyte to IS vs plasma concentrations 2 using a 1/x weighted least-squares linear regression model. The acceptance criterion for each back-calculated standard concentration was ±15% deviation from the nominal value, except at the LLOQ, which was within ±20%. Precision and accuracy. The intra-day precision and accuracy were determined by analyzing QC samples at three concentration levels
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
111
Assays were acquired using an Acquity ultra-performance liquid chromatography system (Waters Corp., Milford, MA, USA) with cooling autosampler and column oven enabling temperature control. A Thermo Syncronis C18 column (50 × 2.1 mm, 1.7 μm) was employed and the column temperature was maintained at 35°C. Gradient elution (delivered at 0.2 mL/min) was employed using acetonitrile (mobile phase A) and 2 mmol/L ammonium acetate in water (mobile phase B), which started at 60% A and increased linearly to 90% A at 1.5 min, then kept this proportion until 2.8 min, finally reducing the proportion to 60% A until 3.5 min to equilibrate the column. The autosampler was conditioned at TM TQD triple8°C and the injection volume was 5 μL. An Acquity quadrupole tandem mass spectrometer (Waters Corp, Milford, MA, USA) was applied to collect the mass spectrometric data. The detection was accomplished using positive ion electrospray ionization in multiple reaction monitoring mode. The MS/MS ion transitions were monitored at m/z 812.39 → 551.35 for TM-2 and 836.36 → 555.26 for IS. Detection conditions were: span, 0.2 Da; dwell time, 0.2 s. Nitrogen was used both for the cone gas (40 L/h) and the desolvation gas (550 L/h), with the source and desolvation temperatures being retained at 100 and 400°C, respectively. Argon was used as the collision gas (0.16 mL/min). The ionization source terms were as follows: capillary voltage 3.50 kV; cone voltage, 30 and 25 V; and optimized collision energy, 10 eV both for TM-2 and IS.
H. Lin et al. (low, 5 ng/mL; medium, 50 ng/mL; high, 800 ng/mL) in six replicates on the same day, while the inter-day precision and accuracy were evaluated by analyzing QC samples at three concentration levels on three continual validation days. The precision was expressed as relative standard deviation (RSD, %) and the accuracy as the relative error (RE, %). Recovery and matrix effect. The extraction recoveries of TM-2 at three QC levels with six replicates were measured by comparing the peak areas from extracted samples with those from post-extracted blank plasma samples spiked with the analytes at the same concentration. The extraction recovery of IS was evaluated in the same way. The matrix effect was measured at three QC levels by comparing the peak area from the post-extracted blank plasma spiked with TM-2
working solutions with those of corresponding standard solutions. The matrix effect of IS was evaluated using the same procedure. Stability. The stability of TM-2 in beagle dog plasma was conducted at the three QC concentration levels (n = 3) in various storage conditions. Post-preparative stability was evaluated by analyzing the processed QC samples kept in an autosampler at 8°C for 24 h. Short-term and longterm stability were studied by analyzing QC samples exposed at room temperature for 4 h and stored at 80°C for 4 months, respectively. The freeze and thaw stability was tested by analyzing QC samples undergoing three freeze–thaw ( 80°C to room temperature) cycles on three consecutive days. Dilution effect and carry-over. Dilution effect was investigated to ensure that samples could be diluted with blank matrix without affecting the final concentration. Blank beagle dog plasma samples spiked with TM-2 (1600 ng/mL) were diluted with pooled blank beagle dog plasma at dilution factors of 2 in six replicates and analyzed. The six replicates should have precision of ≤15% and accuracy within ±15%. The carry-over was determined by injecting a blank plasma sample following the injection of an upper limit of quantification sample in three independent runs. Carry-over was considered negligible if the measured peak area was 0.05), which indicated that the pharmacokinetic properties of TM-2 were linear and similar to cabazitaxel (European Medicines Agency, 2011).
Conclusion The method was successfully applied to the pharmacokinetic study of TM-2 after a single intravenous drip to beagle dogs (0.6, 1.2 and 2.4 mg/kg). This study is the first report of the pharmacokinetic parameters of TM-2 in beagle dogs, and would be useful for further characterizations of the pharmacokinetics, pharmacy and toxicity of TM-2 as a potential new drug.
References Abal M, Andreu J and Barasoain I. Taxanes: microtubule and centrosome targets, and cell cycle dependent mechanisms of action. Current Cancer Drug Targets 2003; 3(3): 193–203. Beaglehole R, Bonita R and Magnusson R. Global cancer prevention: an important pathway to global health and development. Public Health 2011; 125(12): 821–831. European Medicines Agency. Assessment report for Jevtana (Cabazitaxel): EMEA/CHMP/66633/2011. Procedure no. EMEA/H/C/002018, 2011. European Medicines Agency. Guidelines on Bioanalytical Method Validation, EMEA/CHMP/EWP/192217/2009, 1 February 2012. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2011/08/WC5001pdf (accessed 19July 2012). International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. Preclinical safety evaluation of biotechnology-derived pharmaceuticals S6 (R1), 2012. Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM194490.pdf (accessed 16 December 2012). Li YX, Geng MY, Wang JF, Liu HC, Ding N and Zhang W. Multi-drug resistant resistant derivatives and its preparation method and application of taxanes. China patent no. 201110127602.X;2011-05-17, 2011. Tamura T. Molecular-targeted therapy. Gan to kagaku ryoho/Cancer and Chemotherapy 2003; 30(2): 198. Tommasi S, Mangia A, Lacalamita R, Bellizzi A, Fedele V and Chiriatti A. Cytoskeleton and paclitaxel sensitivity in breast cancer: the role of β-tubulins. International Journal of Cancer 2007; 120(10): 2078–2085. US DHHS, FDA and CDER. Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Rat Services, Food and Drug Administration, Center for Drug Evaluation and Research and Center for Veterinary Medicine: Rockville, MD, 2001. World Health Organization. Global Status Report on Noncommunicable Diseases 2010. World Health Organization: Geneva, 2011. Wyld L and Reed M. The role of surgery in the management of older women with breast cancer. European Journal of Cancer 2007; 43(15): 2253–2263. Zhou J and Giannakakou P. Targeting microtubules for cancer chemotherapy. Current Medicinal Chemistry – Anti-Cancer Agents 2005; 5(1): 65–71.
114 wileyonlinelibrary.com/journal/bmc
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 110–114
