- Email: [email protected]
MS: Application to pharmacokinetic study
Journal of Chromatography B, 1001 (2015) 22–26
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of subutinib and its active metabolite in human plasma by LC–MS/MS: Application to pharmacokinetic study Ding Li-kun, Yang Lin, Chen Su-ning., Li Jian-Kang, Wen Ai-dong ∗ Department of Pharmacy, Xijing Hospital of the Fourth Military Medical University, Chang Le West Street 127, Xi’an, Shaanxi 710032, China
a r t i c l e
i n f o
Article history: Received 27 February 2015 Received in revised form 3 July 2015 Accepted 6 July 2015 Available online 17 July 2015 Keywords: Subutinib Active metabolite LC–MS/MS Pharmacokinetics
a b s t r a c t A selective liquid chromatographic–mass spectrometric method (LC–MS/MS) has been established and validated for simultaneous determination of subutinib and its active metabolite in human plasma. Plasma samples were extracted by liquid–liquid extraction with ethyl acetate and separated on a Wondasil C18 (150 mm × 2.1 mm, 3.5 m), with methanol–0.2% formic acid solution (73:27, v/v) as mobile phase at flow rate of 0.2 ml/min. The linear range was 0.25–100 ng/mL for subutinib and 0.125–50.0 ng/mL for its active metabolite, with lower limit of quantitation of 0.25 ng/mL and 0.125 ng/mL, respectively. Intra- and interrun precision were within 7.0 and 13.1%, and the accuracies (relative errors) were < 7.0 and 8.0%, with the extraction recoveries 97.0–101.2% and 93.0–98.1% for the two analytes, respectively. The validated method was successfully applied to a pharmacokinetic study of subutinib and its active metabolite in human after oral administration of subutinib maleate capsules. © 2015 Published by Elsevier B.V.
1. Introduction
2. Experimental
Subutinib is a new type tumor inhibitor, which is smallmolecule, multi-targeted receptor tyrosine kinase (RTK) inhibitor (see Fig. 1). Through studying the structure-activity relationship of sunitinib [1], subutinib was developed that modify sunitinib molecular structure, improve the physical and chemical properties and pharmacodynamics characteristics. It could inhibit the proliferation inhibitory activity on c-Kit, PDGFR, Mo7e of KDR, VEGFR3, U87MG and A375 in vitro experiment. Compared with the positive control, subutinib is equal or slightly strong in inhibitory activity, and it has obvious selectivity to PDGFR, c-Kit, KDR and VEGFR3. The results show that it is multi-targeted tyrosine kinase inhibitors with high activity. In vivo, subutinib was metabolised to desethyl subutinib, which also has pharmacological activity. And there was no article to report the pharmacokinetics of subutinib and its active metabolite. In this study, we established a sensitive LC–MS/MS method for the simultaneous determination of subutinib and its active metabolite in human plasma, and it is successfully applied to the pharmacokinetic study of subutinib.
2.1. Chemicals and reagents
∗ Corresponding author. E-mail address: [email protected] (A.-d. Wen). http://dx.doi.org/10.1016/j.jchromb.2015.07.015 1570-0232/© 2015 Published by Elsevier B.V.
Subutinib maleate, desethyl subutinib and imatinib mesylate were purchased from JiangSu Zhengdatianqing Co., Ltd. (Jiangsu, China). The reagents used for LC–MS/MS measurement were of HPLC grade and were obtained from Sigma–Aldrich (St. Louis, Mo, USA). Double-distilled water was used throughout the study.
2.2. LC–MS/MS conditions For quantitative determination of the analytes, the LC–MS/MS instrument consisted of a Shimadzu LC-2010C HT liquid chromatograph and a QuattroMicro API mass spectrometer (Waters, Milford, MA). The separation was carried out on a Wondasil C18 column (150 mm × 2.1 mm, 3.5 m). The mobile phase consisted of methanol–0.2% formic acid solution (73:27, v/v). The flow rate was 0.2 ml/min with an injection volume of 2 L. Source conditions were optimized for quantitative analysis: electrospray capillary voltage 4.2 kV, source temperature 105 ◦ C and desolvation temperature 350 ◦ C. The cone and desolvation gas flows were 20 L/h and 550 L/h, respectively. The monitoring ions were 411.2 → 282.9, 383.2 → 282.9 and 494.1 → 393.9 for subutinib, desethyl subutinib and imatinib, respectively. The cone voltages were set at 25 V, 32 V and 30 V for subutinib, desethyl subutinib and imatinib, respectively. The collision energies were set at 18 eV, 23 eV and 28 eV for
L.-k. Ding et al. / J. Chromatogr. B 1001 (2015) 22–26
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Fig. 1. The structure of (A) Subutinib, (B) metabolite and (C) imatinib.
subutinib, desethyl subutinib and imatinib, respectively. Total data acquisition was controlled using MassLynxTM V4.0 software with QuanLynxTM program (Waters, Milford, MA).
2.3. Preparation of calibration standards and quality control Stock solutions of subutinib maleate, desethyl subutinib and imatinib were prepared by dissolving accurately weighed samples in the methanol to obtain concentrations of 1 mg/mL for analytes and 0.1 mg/L for IS. The stock solutions were then serially diluted with mobile phase to obtain reference solutions of 5.0, 10, 20, 40, 100, 200, 400, 1000, 1600, 2000, 3000 and 4000 ng/ml for subutinib; 2.5, 5.0, 10, 20, 50, 100, 200, 500, 800, 1000, 1500 and 2000 ng/ml; and 50 ng/mL for IS. All solutions were stored at 4 ◦ C and brought to room temperature before use. Calibration samples were prepared by spiking 400 L of plasma with 20 L of the solutions and 20 L of the IS solutions to obtain final concentrations of 0.2500, 0.5000, 1.000, 2.000, 5.000, 10.00, 20.00, 50.00, 80.00 and 100.0 ng/ml for subutinib maleate; 0.1250, 0.2500, 0.5000, 1.000, 2.000, 5.000, 10.00, 25.00, 40.00 and 50.00 ng/ml for desethyl subutinib. QC samples were prepared at concentrations of 1, 10, and 80 ng/mL for subutinib maleate; and 0.5, 5, and 40 ng/mL for desethyl subutinib, respectively.
2.4. Sample preparation To 400 L plasma in 2.0 ml eppendorf tube, 20 L of internal standard solution, 100 L of 0.1% ammonia water and 800 L of ethyl acetate were added. This mixture was vortex–mixed 3 min and centrifuged at 1500 × g for 10 min. The supernatant was separated out and acidified by 100 L of maleic acid. The liquid was vortex–mixed 0.5 min, then evaporated under reduced pressure. The residue was reconstituted in 200 L mobile phase and centrifuged at 6000 × g for 5 min. A 2 L aliquot sample was injected into the LC–MS/MS system for analysis.
2.5. Method 2.5.1. Selectivity and lower limit of quantification Selectivity was determined by testing plasma samples from six healthy volunteers for the presence of interfering peaks in the retention time. No interfering peaks were observed and the selectivity was considered to be acceptable. The LLOQ was the concentration at which the precision and accuracy were ≤20%, and the signal-to-noise ratio (S/N) was ≥5. Relative standard deviation (RSD, %) was used to assess precision. Relative error (RE, %) was used to estimate accuracy, calculated as ((measured conc. − nominal conc.)/Nominal conc.) × 100% [2,3]. 2.5.2. Calibration curves In the calculation of calibration curves, linear weighted leastsquares analysis and a weighting factor of 1/x2 were used. A correlation coefficient (r) > 0.99 was expected in all calibration curves. Carry over was tested for by injecting a blank sample after three injections at the highest calibration concentration; the measurement for the blank sample was required to be less than 20% of the LLOQ. 2.5.3. Precision and accuracy To determine intra-run precision and accuracy, six QC samples of each concentration were prepared and analyzed on the same run. Another two sets (six samples in each set) of QC samples were freshly prepared and analyzed over other consecutive runs. Calibration curves were freshly prepared to calculate the QC samples. RSD and RE were used to estimate the precision and accuracy, respectively. RSD values of less than 15% and RE values in the range −15–15% were required to meet our precision and accuracy requirements. 2.5.4. Recovery and matrix effects The extraction recoveries of analytes at four different QC concentrations and of IS at 50 ng/mL were measured by comparing peak areas of spiked samples of plasma, processed as described in Section 2.4 (peak area labeled as B) to those of compounds diluted
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Fig. 2. Representative MRM chromatograms of (A) blank human plasma; (B) blank human plasma spiked with 10 ng/ml of subutinib and 1 ng/ml of metabolite ans IS; (C) a plasma sample taken 4 h after administering 12.5 mg of subutinib orally to a volunteer (the measured concentration was 21.5 ng/mL of cubutinib and 0.6) ng/ml of metabolite). (I: IS; II: subutinib; III: metabolite).
Table 1 Accuracy and precision for the determination of subutinib and metabolite in plasma (n = 6). Concentration (ng/ml)
Subutinib
Concentration (ng/ml)
Accuracy
0.5 1 10 80
Precision
Desethyl subutinib
Accuracy
Intra-run RE (%)
Inter-run RE (%)
Intra-run RSD (%)
Inter-run RSD (%)
5.5 7.0 5.1 4.1
6.0 5.5 4.8 6.0
7.9 10.2 7.3 9.0
8.8 13.1 3.9 4.5
with mobile phase (peak area labeled as A). The matrix effects of human plasma were evaluated by comparing the peak area of compound mixed with plasma that remained after extraction (C) to peak area (A) at equivalent concentrations. The ratio (B/A × 100) is defined as the recovery and the ratio of C/A × 100 is defined as the matrix effect. The ratio (matrix effect of subutinib)/(matrix effect of IS) is defined as IS normalized matrix effect. An RSD value less than 15% is considered to be satisfactory. A matrix effect value of 100% indicates that the matrix components have a small impact on the quantification of the analytes. Similar recovery or matrix effect values between analytes and IS usually indicate similar impacts of the extraction procedure or matrix components on analytes and IS.
2.5.5. Stability The stability of reference solutions was tested by measuring the concentrations of analytes after 8 h at room temperature and 30 days at 4 ◦ C. The stability of the biological matrix was examined by calculating the concentration of analytes after short-term (24 h at 4 ◦ C) and long-term storage (30 days at −20 ◦ C). Freeze/thaw stability was determined by analysis of samples subjected to three freeze/thaw cycles (−20–25 ◦ C). Stability under test conditions was investigated by measuring the concentrations of analytes after storage in an autosampler rack (24 h at 25 ◦ C). Generally, a sample was considered to be stable in the biological matrix if the concentration variance was less than 15% of the freshly prepared samples.
0.25 0.5 5 40
Precision
Intra-run RE (%)
Inter-run RE (%)
Intra-run RSD (%)
Inter-run RSD (%)
8.0 7.5 5.6 4.9
7.5 6.3 4.3 6.1
5. 8.3 5.7 8.4
6.6 9.2 4.8 10.3
2.6. Pharmacokinetic study Twelve healthy Chinese volunteers (male, 19–28 years, BMI1924) were included in a pharmacokinetic study. Health status of the applicants was evaluated by reviewing medical history and tests prior to enrollment, and only eligible candidates were chosen. Prior to enrollment, the investigator have provided written information sheets and a full explanation of the purpose, methods, and possible adverse effects of the clinical research which had been reviewed and approved by the ethics committee of Xijing hospital to every healthy subject. After authorization by the ethics committee of the Xijing hospital, the pharmacokinetic study began. Each volunteer was fasted for 10 h before medication and received one subutinib maleate capsule (containing 12.5 mg subutinib maleate) with 250 ml water by oral administration. Blood samples were collected in heparin tubes before medication and 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 168 h, 240 h and 336 h after drug administration. All samples were immediately centrifuged at 1500 × g for 10 min and then stored at −20 ◦ C before analysis [4,5]. 3. Results and discussion 3.1. Method development To develop a simple and rapid method, various column and mobile phases were investigated to reduce the ion suppression
L.-k. Ding et al. / J. Chromatogr. B 1001 (2015) 22–26
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Table 2 Stability of subutinib and active metabolite in plasma (n = 3). Storage condition
Subutinib concentration(ng/ml) 0.5 Mean ± SD
Freeze-thaw Room temperature Stored at −20 ◦ C Autosampler
Cycle 3 8h 15d 30d 24h
0.5066 ± 0.038 0.4920 ± 0.021 0.4855 ± 0.052 0.4825 ± 0.060 0.5230 ± 0.046
RE (%) 1.3 −1.6 −2.9 −3.5 4.6
Metabolite concentration(ng/ml) 80 Mean ± SD
RE (%)
0.25 Mean ± SD
RE (%)
40 Mean ± SD
RE (%)
83.84 ± 3.09 82.56 ± 2.81 82.32 ± 2.34 80.08 ± 1.03 84.32 ± 4.37
4.8 3.2 2.9 0.1 5.4
0.2493 ± 0.013 0.2548 ± 0.0038 0.2453 ± 0.0066 0.2570 ± 0.016 0.2580 ± 0.021
−0.3 1.9 −1.9 2.8 3.2
41.48 ± 2.86 40.60 ± 1.91 40.92 ± 0.86 40.56 ± 2.35 41.72 ± 3.05
3.7 1.5 2.3 1.4 4.3
induced by endogenous substances and to optimize the peak shapes and retention times of subutinib, active metabolite and IS. In preliminary experiments, different columns were employed, the result showed that a Wondasil C18 (150 mm × 2.1 mm, 3.5 m) get better separation, peak shape and reproducibility, whereas the other columns showed a division of chromatographic peaks or poor sensitivity. In addition, other chromatographic variables were examined, including column temperatures at 30, 35, 40, and 45 ◦ C and flow rates at 0.2, 0.25, 0.3 and 0.4 ml/min. Eventually, the optimal conditions for chosen column, in terms of separation and peak shape were a column temperature at 40 ◦ C and a flow rate of 0.2 ml/min. The composition of the mobile phase was also optimized. The isocratic mobile phases were tested with a series of mobile phases containing methanol or acetonitrile/water compositions and different pH modifiers and found unsatisfactory due to the matrix effect and carry over effect. Finally a mobile phase consisted of water containing 0.2% formic acid and methanol was adopted due to optimal retention time, best separation of subutinib, active metabolite and IS. An efficient sample preparation was important in the method development. Although the protein precipitation was tried at initial stage with acetonitrile and methanol, the recoveries were poor. The solid-phase extraction (SPE) method also suffered low reproducibility. Then liquid–liquid extractions (LLE) were tested by several organic solvents, such as ethyl acetate, butyl acetate, methyl terti-butyl ether, hexane, and dichloromethane, with various pH from acidic to basic. All organic solvent showed poor recoveries or no peak in neutral and acidic conditions. Finally, ethyl acetate in basic condition was adopted because of its good efficiency for subutinib, active metabolite and IS.
3.2. Method validation 3.2.1. Selectivity and LLOQ In this assay, the LLOQ values reach 0.25 ng/ml of subutinib and 0.125 ng/ml of active metabolite in plasma, respectively. And no interference substance was observed at the retention times of subutinib, active metabolite and IS were completely separated using the optimized conditions described above (see Fig 2).
3.2.2. Linearity Calibration curves were prepared over the 0.250–100.0 ng/mL for subutinib and 0.125–50.0 ng/mL for its metabolite in human plasma by linear regression using a 1/x2 weighting factor. The regression equations (n = 3) were y = 0.1887 × C −0.009864 for subutinib and y = 0.1376 × C −0.002348 for its metabolite, where y is the peak area ratio of analyte to IS and C is the nominal plasma concentration. The carry over was 0.00% for subutinib and 0.00% for its active metabolite. At calibration curve concentrations, the RSD and RE values ranged from 1.35% to 6.72% and −8.4 to 13.0% for subutinib and from 3.59 to 6.03% and −9.1 to 14.9% for its active metabolite.
3.2.3. Accuracy and precision Intra- and inter-run precision and accuracy of the method are presented in Table 1. RSD values of intra- and inter-run precisions were less than 10.2 and 13.1% for subutinib, whereas the values were less than 8.4 and 10.3% for active metabolite. For subutinib and metabolite, RE values of intra- and inter-run accuracies were less than 7.0 and 6.0%, and less than 8.0 and 7.5%, respectively. All results were within the ranges of precision (%) and accuracy (%) specified by China Food and Drug Administration (CFDA) [3] for bioanalytical applications. 3.2.4. Recovery and matrix effects Recovery was found to be in the range 97.0–101.2% for subutinib, 93.8–100.8% for active metabolite and 104.8% for its IS. After the extraction procedures, the recoveries were consistent and precise between analytes and their IS. The matrix effect was assessed by comparing the signals of analytes in plasma with those of analytes dissolved in mobile phase at the same concentration. The matrix effects were 95.6%, 110.3%, 98.2% and 99.3% for subutinib; and 97.1%, 109.1%, 103.5% and 101.7% for its metabolite at four QC concentrations. The matrix effects were 92.0% for IS. IS normalized matrix effects were 95.6%, 113.2%, 100.1% and 98.6% for subutinib; and 102.6%, 108.8%, 105.3% and 104.7% for its metabolite at four QC concentrations. Negligible matrix effects were observed, indicating consistent suppression of ionization. The high precision, accuracy, recovery and low matrix effects seen in this assay can be attributed, at least in part, to the similar structures and behavior of the IS. 3.2.5. Stability The stabilities of subutinib and active metabolite in solvent and human plasma were fully evaluated under all conditions that samples might be exposed to before detection. Stability results are summarized in Table 2. Solutions of subutinib, active metabolite and IS stored at 4 ◦ C for 30 days and at room temperature (25 ◦ C) for
Fig. 3. Mean plasma concentration-time profile of subutunib and its active metabolite from 12 healthy volunteers.
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Table 3 The pharmacokinetic parameters of subutinib and metabolite in human plasma. Parameter
Subutinib
Metabolite
Cmax (ng/ml) Tmax (h) t1/2 (h) AUC(0-t) (h ng/mL) AUC(0-∞) (h ng/mL)
6.13 ± 1.66 5.0 ± 1.9 40.26 ± 3.53 278.4 ± 86.0 301.0 ± 90.8
0.90 ± 0.08 4.5 ± 1.4 68.94 ± 19.22 46.99 ± 18.80 64.33 ± 26.53
8 h showed good stabilities ranging from 102.9 to 103.6%. There was no obvious degradation of subutinib or active metabolite in plasma after short-term storage, three freeze–thaw cycles, long-term storage, or storage in the autosample rack. The expected delays in testing during normal assay operation appeared to have no effect on the detection of analytes. The method was, therefore, shown to be suitable for routine testing. 3.3. Application to the pharmacokinetic study The method was successfully used to analyze plasma samples in a subutinib pharmacokinetic study. The mean plasma concentration-time curves of subutinib and active metabolite after the oral administration to healthy Chinese volunteers and main pharmacokinetic parameters are summarized in Fig. 3 and Table 3. It indicates that this method is well suited for routine high-throughput analyses, such as in a pharmacokinetic study.
4. Conclusion In the study, a simultaneous LC–MS/MS method for subutinib and active metabolite in human plasma was developed and fully validated for clinical trials. The method met guidelines for the validation of bioanalytical methods. In addition, this method was successfully applied to a pharmacokinetic study of subutinib. The method is the first LC–MS/MS based method to be described for the analysis in human plasma and is more suitable for the routine analysis of large numbers of samples. References [1] X. Chen, Z. Wang, M. Liu, M. Liao, X. Wang, H. Du, J. Chen, M. Yao, Q. Li, Biomed. Chromatogr. (2014), http://dx.doi.org/10.1002/bmc.3331 [2] FDA/CDER. Guidance for Industry Bioanalytical Method Validation, Internet at http://www.fda.gov/cder/guidance/index.htm [3] Chinese State Food and Drug Administration, The guidance of bioavailability and bioequivalence study technique for chemistry drug in humans (no. [H] GCL2–1), 2005, Available from: http://www.sda.gov.cn/gsz05106/08pdf. [4] FDA/CDER. Guidance for industry. Bioavailability and bioequivalence studies for orally administered drug products-general considerations, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluationand Research (CDER), 2003 [online]. Available from URL: http:// www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/UCM070124pdf. [5] EMEA/CPMP. Note for guidance on the investigation of bioavailability and bioequivalence. CPMP/EWP/1401/98, European Agency for the Evaluation of Medicinal Products, Committee for Proprietary Medicinal Products (CPMP), 2001 [online]. Available from URL: http://www.emaeuropa.eu/docs/en GB/ document library/Scientific.guideline/2009/09/WC500003011.pdf.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of subutinib and its active metabolite in human plasma by LC–MS/MS: Application to pharmacokinetic study Ding Li-kun, Yang Lin, Chen Su-ning., Li Jian-Kang, Wen Ai-dong ∗ Department of Pharmacy, Xijing Hospital of the Fourth Military Medical University, Chang Le West Street 127, Xi’an, Shaanxi 710032, China
a r t i c l e
i n f o
Article history: Received 27 February 2015 Received in revised form 3 July 2015 Accepted 6 July 2015 Available online 17 July 2015 Keywords: Subutinib Active metabolite LC–MS/MS Pharmacokinetics
a b s t r a c t A selective liquid chromatographic–mass spectrometric method (LC–MS/MS) has been established and validated for simultaneous determination of subutinib and its active metabolite in human plasma. Plasma samples were extracted by liquid–liquid extraction with ethyl acetate and separated on a Wondasil C18 (150 mm × 2.1 mm, 3.5 m), with methanol–0.2% formic acid solution (73:27, v/v) as mobile phase at flow rate of 0.2 ml/min. The linear range was 0.25–100 ng/mL for subutinib and 0.125–50.0 ng/mL for its active metabolite, with lower limit of quantitation of 0.25 ng/mL and 0.125 ng/mL, respectively. Intra- and interrun precision were within 7.0 and 13.1%, and the accuracies (relative errors) were < 7.0 and 8.0%, with the extraction recoveries 97.0–101.2% and 93.0–98.1% for the two analytes, respectively. The validated method was successfully applied to a pharmacokinetic study of subutinib and its active metabolite in human after oral administration of subutinib maleate capsules. © 2015 Published by Elsevier B.V.
1. Introduction
2. Experimental
Subutinib is a new type tumor inhibitor, which is smallmolecule, multi-targeted receptor tyrosine kinase (RTK) inhibitor (see Fig. 1). Through studying the structure-activity relationship of sunitinib [1], subutinib was developed that modify sunitinib molecular structure, improve the physical and chemical properties and pharmacodynamics characteristics. It could inhibit the proliferation inhibitory activity on c-Kit, PDGFR, Mo7e of KDR, VEGFR3, U87MG and A375 in vitro experiment. Compared with the positive control, subutinib is equal or slightly strong in inhibitory activity, and it has obvious selectivity to PDGFR, c-Kit, KDR and VEGFR3. The results show that it is multi-targeted tyrosine kinase inhibitors with high activity. In vivo, subutinib was metabolised to desethyl subutinib, which also has pharmacological activity. And there was no article to report the pharmacokinetics of subutinib and its active metabolite. In this study, we established a sensitive LC–MS/MS method for the simultaneous determination of subutinib and its active metabolite in human plasma, and it is successfully applied to the pharmacokinetic study of subutinib.
2.1. Chemicals and reagents
∗ Corresponding author. E-mail address: [email protected] (A.-d. Wen). http://dx.doi.org/10.1016/j.jchromb.2015.07.015 1570-0232/© 2015 Published by Elsevier B.V.
Subutinib maleate, desethyl subutinib and imatinib mesylate were purchased from JiangSu Zhengdatianqing Co., Ltd. (Jiangsu, China). The reagents used for LC–MS/MS measurement were of HPLC grade and were obtained from Sigma–Aldrich (St. Louis, Mo, USA). Double-distilled water was used throughout the study.
2.2. LC–MS/MS conditions For quantitative determination of the analytes, the LC–MS/MS instrument consisted of a Shimadzu LC-2010C HT liquid chromatograph and a QuattroMicro API mass spectrometer (Waters, Milford, MA). The separation was carried out on a Wondasil C18 column (150 mm × 2.1 mm, 3.5 m). The mobile phase consisted of methanol–0.2% formic acid solution (73:27, v/v). The flow rate was 0.2 ml/min with an injection volume of 2 L. Source conditions were optimized for quantitative analysis: electrospray capillary voltage 4.2 kV, source temperature 105 ◦ C and desolvation temperature 350 ◦ C. The cone and desolvation gas flows were 20 L/h and 550 L/h, respectively. The monitoring ions were 411.2 → 282.9, 383.2 → 282.9 and 494.1 → 393.9 for subutinib, desethyl subutinib and imatinib, respectively. The cone voltages were set at 25 V, 32 V and 30 V for subutinib, desethyl subutinib and imatinib, respectively. The collision energies were set at 18 eV, 23 eV and 28 eV for
L.-k. Ding et al. / J. Chromatogr. B 1001 (2015) 22–26
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Fig. 1. The structure of (A) Subutinib, (B) metabolite and (C) imatinib.
subutinib, desethyl subutinib and imatinib, respectively. Total data acquisition was controlled using MassLynxTM V4.0 software with QuanLynxTM program (Waters, Milford, MA).
2.3. Preparation of calibration standards and quality control Stock solutions of subutinib maleate, desethyl subutinib and imatinib were prepared by dissolving accurately weighed samples in the methanol to obtain concentrations of 1 mg/mL for analytes and 0.1 mg/L for IS. The stock solutions were then serially diluted with mobile phase to obtain reference solutions of 5.0, 10, 20, 40, 100, 200, 400, 1000, 1600, 2000, 3000 and 4000 ng/ml for subutinib; 2.5, 5.0, 10, 20, 50, 100, 200, 500, 800, 1000, 1500 and 2000 ng/ml; and 50 ng/mL for IS. All solutions were stored at 4 ◦ C and brought to room temperature before use. Calibration samples were prepared by spiking 400 L of plasma with 20 L of the solutions and 20 L of the IS solutions to obtain final concentrations of 0.2500, 0.5000, 1.000, 2.000, 5.000, 10.00, 20.00, 50.00, 80.00 and 100.0 ng/ml for subutinib maleate; 0.1250, 0.2500, 0.5000, 1.000, 2.000, 5.000, 10.00, 25.00, 40.00 and 50.00 ng/ml for desethyl subutinib. QC samples were prepared at concentrations of 1, 10, and 80 ng/mL for subutinib maleate; and 0.5, 5, and 40 ng/mL for desethyl subutinib, respectively.
2.4. Sample preparation To 400 L plasma in 2.0 ml eppendorf tube, 20 L of internal standard solution, 100 L of 0.1% ammonia water and 800 L of ethyl acetate were added. This mixture was vortex–mixed 3 min and centrifuged at 1500 × g for 10 min. The supernatant was separated out and acidified by 100 L of maleic acid. The liquid was vortex–mixed 0.5 min, then evaporated under reduced pressure. The residue was reconstituted in 200 L mobile phase and centrifuged at 6000 × g for 5 min. A 2 L aliquot sample was injected into the LC–MS/MS system for analysis.
2.5. Method 2.5.1. Selectivity and lower limit of quantification Selectivity was determined by testing plasma samples from six healthy volunteers for the presence of interfering peaks in the retention time. No interfering peaks were observed and the selectivity was considered to be acceptable. The LLOQ was the concentration at which the precision and accuracy were ≤20%, and the signal-to-noise ratio (S/N) was ≥5. Relative standard deviation (RSD, %) was used to assess precision. Relative error (RE, %) was used to estimate accuracy, calculated as ((measured conc. − nominal conc.)/Nominal conc.) × 100% [2,3]. 2.5.2. Calibration curves In the calculation of calibration curves, linear weighted leastsquares analysis and a weighting factor of 1/x2 were used. A correlation coefficient (r) > 0.99 was expected in all calibration curves. Carry over was tested for by injecting a blank sample after three injections at the highest calibration concentration; the measurement for the blank sample was required to be less than 20% of the LLOQ. 2.5.3. Precision and accuracy To determine intra-run precision and accuracy, six QC samples of each concentration were prepared and analyzed on the same run. Another two sets (six samples in each set) of QC samples were freshly prepared and analyzed over other consecutive runs. Calibration curves were freshly prepared to calculate the QC samples. RSD and RE were used to estimate the precision and accuracy, respectively. RSD values of less than 15% and RE values in the range −15–15% were required to meet our precision and accuracy requirements. 2.5.4. Recovery and matrix effects The extraction recoveries of analytes at four different QC concentrations and of IS at 50 ng/mL were measured by comparing peak areas of spiked samples of plasma, processed as described in Section 2.4 (peak area labeled as B) to those of compounds diluted
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Fig. 2. Representative MRM chromatograms of (A) blank human plasma; (B) blank human plasma spiked with 10 ng/ml of subutinib and 1 ng/ml of metabolite ans IS; (C) a plasma sample taken 4 h after administering 12.5 mg of subutinib orally to a volunteer (the measured concentration was 21.5 ng/mL of cubutinib and 0.6) ng/ml of metabolite). (I: IS; II: subutinib; III: metabolite).
Table 1 Accuracy and precision for the determination of subutinib and metabolite in plasma (n = 6). Concentration (ng/ml)
Subutinib
Concentration (ng/ml)
Accuracy
0.5 1 10 80
Precision
Desethyl subutinib
Accuracy
Intra-run RE (%)
Inter-run RE (%)
Intra-run RSD (%)
Inter-run RSD (%)
5.5 7.0 5.1 4.1
6.0 5.5 4.8 6.0
7.9 10.2 7.3 9.0
8.8 13.1 3.9 4.5
with mobile phase (peak area labeled as A). The matrix effects of human plasma were evaluated by comparing the peak area of compound mixed with plasma that remained after extraction (C) to peak area (A) at equivalent concentrations. The ratio (B/A × 100) is defined as the recovery and the ratio of C/A × 100 is defined as the matrix effect. The ratio (matrix effect of subutinib)/(matrix effect of IS) is defined as IS normalized matrix effect. An RSD value less than 15% is considered to be satisfactory. A matrix effect value of 100% indicates that the matrix components have a small impact on the quantification of the analytes. Similar recovery or matrix effect values between analytes and IS usually indicate similar impacts of the extraction procedure or matrix components on analytes and IS.
2.5.5. Stability The stability of reference solutions was tested by measuring the concentrations of analytes after 8 h at room temperature and 30 days at 4 ◦ C. The stability of the biological matrix was examined by calculating the concentration of analytes after short-term (24 h at 4 ◦ C) and long-term storage (30 days at −20 ◦ C). Freeze/thaw stability was determined by analysis of samples subjected to three freeze/thaw cycles (−20–25 ◦ C). Stability under test conditions was investigated by measuring the concentrations of analytes after storage in an autosampler rack (24 h at 25 ◦ C). Generally, a sample was considered to be stable in the biological matrix if the concentration variance was less than 15% of the freshly prepared samples.
0.25 0.5 5 40
Precision
Intra-run RE (%)
Inter-run RE (%)
Intra-run RSD (%)
Inter-run RSD (%)
8.0 7.5 5.6 4.9
7.5 6.3 4.3 6.1
5. 8.3 5.7 8.4
6.6 9.2 4.8 10.3
2.6. Pharmacokinetic study Twelve healthy Chinese volunteers (male, 19–28 years, BMI1924) were included in a pharmacokinetic study. Health status of the applicants was evaluated by reviewing medical history and tests prior to enrollment, and only eligible candidates were chosen. Prior to enrollment, the investigator have provided written information sheets and a full explanation of the purpose, methods, and possible adverse effects of the clinical research which had been reviewed and approved by the ethics committee of Xijing hospital to every healthy subject. After authorization by the ethics committee of the Xijing hospital, the pharmacokinetic study began. Each volunteer was fasted for 10 h before medication and received one subutinib maleate capsule (containing 12.5 mg subutinib maleate) with 250 ml water by oral administration. Blood samples were collected in heparin tubes before medication and 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 168 h, 240 h and 336 h after drug administration. All samples were immediately centrifuged at 1500 × g for 10 min and then stored at −20 ◦ C before analysis [4,5]. 3. Results and discussion 3.1. Method development To develop a simple and rapid method, various column and mobile phases were investigated to reduce the ion suppression
L.-k. Ding et al. / J. Chromatogr. B 1001 (2015) 22–26
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Table 2 Stability of subutinib and active metabolite in plasma (n = 3). Storage condition
Subutinib concentration(ng/ml) 0.5 Mean ± SD
Freeze-thaw Room temperature Stored at −20 ◦ C Autosampler
Cycle 3 8h 15d 30d 24h
0.5066 ± 0.038 0.4920 ± 0.021 0.4855 ± 0.052 0.4825 ± 0.060 0.5230 ± 0.046
RE (%) 1.3 −1.6 −2.9 −3.5 4.6
Metabolite concentration(ng/ml) 80 Mean ± SD
RE (%)
0.25 Mean ± SD
RE (%)
40 Mean ± SD
RE (%)
83.84 ± 3.09 82.56 ± 2.81 82.32 ± 2.34 80.08 ± 1.03 84.32 ± 4.37
4.8 3.2 2.9 0.1 5.4
0.2493 ± 0.013 0.2548 ± 0.0038 0.2453 ± 0.0066 0.2570 ± 0.016 0.2580 ± 0.021
−0.3 1.9 −1.9 2.8 3.2
41.48 ± 2.86 40.60 ± 1.91 40.92 ± 0.86 40.56 ± 2.35 41.72 ± 3.05
3.7 1.5 2.3 1.4 4.3
induced by endogenous substances and to optimize the peak shapes and retention times of subutinib, active metabolite and IS. In preliminary experiments, different columns were employed, the result showed that a Wondasil C18 (150 mm × 2.1 mm, 3.5 m) get better separation, peak shape and reproducibility, whereas the other columns showed a division of chromatographic peaks or poor sensitivity. In addition, other chromatographic variables were examined, including column temperatures at 30, 35, 40, and 45 ◦ C and flow rates at 0.2, 0.25, 0.3 and 0.4 ml/min. Eventually, the optimal conditions for chosen column, in terms of separation and peak shape were a column temperature at 40 ◦ C and a flow rate of 0.2 ml/min. The composition of the mobile phase was also optimized. The isocratic mobile phases were tested with a series of mobile phases containing methanol or acetonitrile/water compositions and different pH modifiers and found unsatisfactory due to the matrix effect and carry over effect. Finally a mobile phase consisted of water containing 0.2% formic acid and methanol was adopted due to optimal retention time, best separation of subutinib, active metabolite and IS. An efficient sample preparation was important in the method development. Although the protein precipitation was tried at initial stage with acetonitrile and methanol, the recoveries were poor. The solid-phase extraction (SPE) method also suffered low reproducibility. Then liquid–liquid extractions (LLE) were tested by several organic solvents, such as ethyl acetate, butyl acetate, methyl terti-butyl ether, hexane, and dichloromethane, with various pH from acidic to basic. All organic solvent showed poor recoveries or no peak in neutral and acidic conditions. Finally, ethyl acetate in basic condition was adopted because of its good efficiency for subutinib, active metabolite and IS.
3.2. Method validation 3.2.1. Selectivity and LLOQ In this assay, the LLOQ values reach 0.25 ng/ml of subutinib and 0.125 ng/ml of active metabolite in plasma, respectively. And no interference substance was observed at the retention times of subutinib, active metabolite and IS were completely separated using the optimized conditions described above (see Fig 2).
3.2.2. Linearity Calibration curves were prepared over the 0.250–100.0 ng/mL for subutinib and 0.125–50.0 ng/mL for its metabolite in human plasma by linear regression using a 1/x2 weighting factor. The regression equations (n = 3) were y = 0.1887 × C −0.009864 for subutinib and y = 0.1376 × C −0.002348 for its metabolite, where y is the peak area ratio of analyte to IS and C is the nominal plasma concentration. The carry over was 0.00% for subutinib and 0.00% for its active metabolite. At calibration curve concentrations, the RSD and RE values ranged from 1.35% to 6.72% and −8.4 to 13.0% for subutinib and from 3.59 to 6.03% and −9.1 to 14.9% for its active metabolite.
3.2.3. Accuracy and precision Intra- and inter-run precision and accuracy of the method are presented in Table 1. RSD values of intra- and inter-run precisions were less than 10.2 and 13.1% for subutinib, whereas the values were less than 8.4 and 10.3% for active metabolite. For subutinib and metabolite, RE values of intra- and inter-run accuracies were less than 7.0 and 6.0%, and less than 8.0 and 7.5%, respectively. All results were within the ranges of precision (%) and accuracy (%) specified by China Food and Drug Administration (CFDA) [3] for bioanalytical applications. 3.2.4. Recovery and matrix effects Recovery was found to be in the range 97.0–101.2% for subutinib, 93.8–100.8% for active metabolite and 104.8% for its IS. After the extraction procedures, the recoveries were consistent and precise between analytes and their IS. The matrix effect was assessed by comparing the signals of analytes in plasma with those of analytes dissolved in mobile phase at the same concentration. The matrix effects were 95.6%, 110.3%, 98.2% and 99.3% for subutinib; and 97.1%, 109.1%, 103.5% and 101.7% for its metabolite at four QC concentrations. The matrix effects were 92.0% for IS. IS normalized matrix effects were 95.6%, 113.2%, 100.1% and 98.6% for subutinib; and 102.6%, 108.8%, 105.3% and 104.7% for its metabolite at four QC concentrations. Negligible matrix effects were observed, indicating consistent suppression of ionization. The high precision, accuracy, recovery and low matrix effects seen in this assay can be attributed, at least in part, to the similar structures and behavior of the IS. 3.2.5. Stability The stabilities of subutinib and active metabolite in solvent and human plasma were fully evaluated under all conditions that samples might be exposed to before detection. Stability results are summarized in Table 2. Solutions of subutinib, active metabolite and IS stored at 4 ◦ C for 30 days and at room temperature (25 ◦ C) for
Fig. 3. Mean plasma concentration-time profile of subutunib and its active metabolite from 12 healthy volunteers.
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Table 3 The pharmacokinetic parameters of subutinib and metabolite in human plasma. Parameter
Subutinib
Metabolite
Cmax (ng/ml) Tmax (h) t1/2 (h) AUC(0-t) (h ng/mL) AUC(0-∞) (h ng/mL)
6.13 ± 1.66 5.0 ± 1.9 40.26 ± 3.53 278.4 ± 86.0 301.0 ± 90.8
0.90 ± 0.08 4.5 ± 1.4 68.94 ± 19.22 46.99 ± 18.80 64.33 ± 26.53
8 h showed good stabilities ranging from 102.9 to 103.6%. There was no obvious degradation of subutinib or active metabolite in plasma after short-term storage, three freeze–thaw cycles, long-term storage, or storage in the autosample rack. The expected delays in testing during normal assay operation appeared to have no effect on the detection of analytes. The method was, therefore, shown to be suitable for routine testing. 3.3. Application to the pharmacokinetic study The method was successfully used to analyze plasma samples in a subutinib pharmacokinetic study. The mean plasma concentration-time curves of subutinib and active metabolite after the oral administration to healthy Chinese volunteers and main pharmacokinetic parameters are summarized in Fig. 3 and Table 3. It indicates that this method is well suited for routine high-throughput analyses, such as in a pharmacokinetic study.
4. Conclusion In the study, a simultaneous LC–MS/MS method for subutinib and active metabolite in human plasma was developed and fully validated for clinical trials. The method met guidelines for the validation of bioanalytical methods. In addition, this method was successfully applied to a pharmacokinetic study of subutinib. The method is the first LC–MS/MS based method to be described for the analysis in human plasma and is more suitable for the routine analysis of large numbers of samples. References [1] X. Chen, Z. Wang, M. Liu, M. Liao, X. Wang, H. Du, J. Chen, M. Yao, Q. Li, Biomed. Chromatogr. (2014), http://dx.doi.org/10.1002/bmc.3331 [2] FDA/CDER. Guidance for Industry Bioanalytical Method Validation, Internet at http://www.fda.gov/cder/guidance/index.htm [3] Chinese State Food and Drug Administration, The guidance of bioavailability and bioequivalence study technique for chemistry drug in humans (no. [H] GCL2–1), 2005, Available from: http://www.sda.gov.cn/gsz05106/08pdf. [4] FDA/CDER. Guidance for industry. Bioavailability and bioequivalence studies for orally administered drug products-general considerations, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluationand Research (CDER), 2003 [online]. Available from URL: http:// www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/UCM070124pdf. [5] EMEA/CPMP. Note for guidance on the investigation of bioavailability and bioequivalence. CPMP/EWP/1401/98, European Agency for the Evaluation of Medicinal Products, Committee for Proprietary Medicinal Products (CPMP), 2001 [online]. Available from URL: http://www.emaeuropa.eu/docs/en GB/ document library/Scientific.guideline/2009/09/WC500003011.pdf.