Journal of Pharmaceutical and Biomedical Analysis 107 (2015) 223–228
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Development and validation of a liquid chromatography–tandem mass spectroscopy method for simultaneous determination of (+)-(13aS)-deoxytylophorinine and its pharmacologically active 3-O-desmethyl metabolite in rat plasma Feifei Yu, Haining Lv, Wujun Dong ∗ , Jun Ye, Huazhen Hao, Shuanggang Ma, Shishan Yu, Yuling Liu ∗ Department of Pharmaceutics, Beijing Key Laboratory of Drug Delivery Technology and Novel Formulation, State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
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Article history: Received 21 September 2014 Received in revised form 24 December 2014 Accepted 26 December 2014 Available online 3 January 2015 Keywords: (+)-(13aS)-Deoxytylophorinine Metabolite LC–MS/MS Pharmacokinetics Rat plasma
a b s t r a c t CAT ((+)-(13aS)-deoxytylophorinine) is a novel anticancer drug belonging to phenanthroindolizidine alkaloids. A sensitive and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of CAT and its pharmacologically active 3-O-desmethyl metabolite (S-4) was developed and validated in rat plasma using rotundine as the internal standard (IS). CAT, S-4 and IS were extracted by acetonitrile protein precipitation and separated on an Eclipse XDB-C18 column (1.8 m, 4.6 mm × 50 mm) with acetonitrile–water (27:73, v/v) mobile phase containing 0.1% formic acid at a 0.4 mL/min flow rate. Positive ion electrospray ionization in multiple reaction monitoring mode was employed to measure CAT, S-4 and IS by monitoring the transitions m/z 364.2 → 70.1 for CAT, 350.1 → 70.1 for S-4 and 356.2 → 192.2 for IS. Good linear correlation (r2 > 0.991) was achieved for CAT and S-4 over the range of 0.214–128.16 and 0.044–11.00 ng/mL, respectively. The lower limit of quantification was 0.214 ng/mL for CAT and 0.044 ng/mL for S-4, using 50 L rat plasma samples. The intraand inter-day precisions were not exceed 15% and the accuracy ranged between 94.80% and 108.22%. The average extraction recoveries of both analytes were greater than 94.62%. The method was successfully applied to the pharmacokinetic study of CAT and S-4 in rats after oral administration. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Phenanthroindolizidine alkaloids are found mainly in the plants of Cynanchum, Pergularia, Tylophora, and some other genera of Asclepiadaceae family [1–4]. These compounds are among the many alkaloids reported to possess bioactive and pharmacological activities, including anti-arthritis [5], anti-inflammatory [6], and anti-tumor properties [7,8]. (+)-(13aS)-Deoxytylophorinine (CAT, Fig. 1A) is a new anticancer drug belonging to phenanthroindolizidine alkaloids initially isolated from the roots of Tylophora atrofolliculata and Tylophora ovata [9]. Previous studies [10] had found that CAT has significant anti-tumor activity on H22 mouse hepatoma xenografts after oral administration. The anti-tumor activity of CAT is primarily attributed to its ability to intercalate into AT-repeated base pairs in double-helical DNA sequences. Further
∗ Corresponding authors. Tel.: +86 10 63159373; fax: +86 10 63159373. E-mail addresses: [email protected] (W. Dong), [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.jpba.2014.12.042 0731-7085/© 2015 Elsevier B.V. All rights reserved.
studies have shown that CAT potently suppresses the proliferation of common cancer cell lines such as HepG2 and A549 cells, with an IC50 of approximately 10−7 mol/L [11,12]. CAT was susceptible to metabolism by rat liver microsomes and its three major monodesmethyl metabolites were synthesized recently [13], among which 3-O-desmethyldeoxytylophorinine (S-4, Fig. 1B) exhibited highest cytotoxic potency and the cytotoxicity significantly higher than parent compound. Hence, it was imperative to determine CAT and its pharmacologically active metabolite, S-4 simultaneously in order to fully understand the contribution of S-4 to the efficacy profile of CAT. Bioanalytical assays for CAT qualification by LC–MS/MS in mouse plasma or rat tissues were reported [14,15]. However, in the assays, neither report entirely validated LC–MS/MS method nor did they support the determination of S-4. Recently, the use of methods combining LC–MS/MS and nuclear magnetic resonance (NMR) was reported in the identification of CAT and its metabolites in rat urine [16], but no information was provided on the measurement of CAT and its metabolites. So far, little is known about
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Fig. 1. Mass spectroscopy spectra of (A) CAT, (B) S-4 and (C) internal standard (IS).
the oral plasma pharmacokinetic characteristics of CAT and S-4 in rats. In this study, we developed and validated a sensitive LC–MS/MS method for simultaneous determination of CAT and S-4 levels in rat plasma. To the best of our knowledge, this is the first report of a fully validated LC–MS/MS method for quantification of CAT and S-4 in a biological matrix. The LC–MS/MS method was successfully applied in pharmacokinetic study of CAT and its pharmacologically active 3O-desmethyl metabolite, S-4 in rats following oral administration. 2. Materials and methods 2.1. Chemicals and reagents CAT (purity > 99.0%) and S-4 (purity > 99.0%) was kindly provided by Prof. Shishan Yu (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China) and rotundine (Fig. 1C, internal standard, IS) was obtained from National institutes for food and drug control (Beijing, China). HPLC-grade acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ, USA), while MS-grade formic acid was purchased from Sigma (St. Louis, MO, USA). Pure water (Wahaha, Hangzhou, China) was obtained from a local market. Other chemicals were all of analytical grade.
Agilent 1200 RRLC system (Agilent Co.) coupled to a triple quadrupole MS analyzer with an electrospray ionization (ESI) source. Chromatographic separations were achieved using an Eclipse XDB-C18 column (1.8 m, 4.6 mm × 50 mm, Agilent Co.). The mobile phase, consisting of acetonitrile–deionized water (27:63, v/v) with 0.1% formic acid, was used in isocratic mode at a flow rate of 0.4 mL/min. Aliquots (10 L) of the sample solution were injected for each analysis. The separation run was finished within 6 min of sample injection at 35 ◦ C. Quantification was performed in a multiple reaction monitoring (MRM) mode using ESI in the positive ion mode: for CAT, m/z 364.2 → 70.1 (fragmentor, 120 V; collision energy, 30 V); for S-4, m/z 350.1 → 70.1 (fragmentor, 130 V; collision energy, 35 V); for rotundine (IS), 356.2 → 192.2 (fragmentor, 140 V; collision energy, 25 V) were used as the quantitative ion-pairs, respectively. The optimum operating parameters were as follows: drying gas temperature, 350 ◦ C; drying gas flow, 10 L/min; nebulizer pressure, 50 psi; and capillary voltage, 4000 V. Data acquisition and processing were performed using MassHunter workstation (Agilent Co.). 2.3. Stock solutions, calibration curve, and quality control (QC) samples
2.2. Instrumentation and chromatographic conditions The analysis was performed on a 6410B triple quadrupole LC–MS system (Agilent Corporation, MA, USA) consisting of an
A stock solution of CAT and S-4 was prepared in ethanol at a concentration of 50 g/mL and 11 g/mL, respectively. The stock solution was diluted in mobile phase to obtain working
F. Yu et al. / Journal of Pharmaceutical and Biomedical Analysis 107 (2015) 223–228
solutions with concentrations of 1.068/0.22, 2.136/0.55, 6.408/2.20, 16.02/5.50, 64.08/11.00, 160.2/22.00, 534.00/44.00, and 640.8/55.00 ng/mL. A solution of IS at a final concentration of 12.04 ng/mL was prepared in ethanol. All solutions were stored at 4 ◦ C until LC–MS/MS analysis. Calibrations standards of CAT and S-4 (0.214/0.044, 0.427/0.11, 1.282/0.44, 3.204/1.10, 12.82/2.20, 32.04/4.40, 106.80/8.80, and 128.16/11.00 ng/mL) were prepared by spiking the blank plasma with the corresponding working solution. The quality control (QC) samples of CAT and S-4 with four concentrations at 0.214/0.044, 0.427/0.11, 12.82/2.20, and 106.80/8.80 ng/mL were similarly prepared. 2.4. Sample preparation All plasma samples were thawed to room temperature before analysis. Aliquots of 50 L plasma and 20 L of IS working solution (12.04 ng/mL) were added into 1.5-mL centrifuge tubes. After the mixture was vortexed for 30 s, 600 L of acetonitrile was added. The mixture was vortex-mixed for 2 min and centrifuged at 14,000 rpm for 5 min at room temperature. The upper organic layer was transferred into another tube and evaporated to dryness under a gentle stream of nitrogen gas. The residue was reconstituted with 50 L mobile phase by vortexing for 2 min and centrifuged at 14,000 rpm for 10 min. Finally, 10 L of supernatant was injected onto the LC–MS/MS system. 2.5. Method validation A full validation of the method was performed in accordance with the FDA Guidance for Industry Bioanalytical Method Validation [17]. 2.5.1. Specificity The specificity of the method was evaluated by analyzing blank plasma samples from six individual rats to test for potential interference by endogenous compounds. The analyses of the blank samples were compared with the corresponding plasma samples spiked the LLOQ level and samples taken from treated animals. 2.5.2. Calibration curve Five calibration curves were freshly prepared at eight concentrations covering the range of 0.214–128.16 ng/mL for CAT and 0.044–11.00 ng/mL for S-4. The linearity was investigated by plotting the peak area ratios of analytes to IS versus the concentrations of analytes and applying the least squares linear regression. A correlation coefficient (r2 ) calculated in the calibration curve should be no less than 0.99. 2.5.3. Accuracy and precision Intra-day accuracy and precision were estimated by analyzing the QC samples of plasma at four concentrations (n = 5 at each concentration) on a single day. Inter-day accuracy and precision were determined on three consecutive days. Precision was expressed as the relative standard deviation (RSD%), while accuracy was presented as percentage calculated using the following equation: accuracy = (measured concentration/nominal spiked concentration) × 100. 2.5.4. Carry-over Carry-over was evaluated by sequentially injecting an extracted sample at the upper limit of quantification (ULOQ) concentration and two extracted blank samples. 2.5.5. Matrix effect and extraction recovery The matrix effect was determined at four QC concentrations by comparing peak responses of post-extraction blank plasma (from six different individuals) spiked QC samples with those of neat
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standard solutions. The extraction recovery was measured by comparing the responses of analytes obtained from extracted samples with blank plasma extracts spiked with QC samples. The matrix effect and extraction recovery of IS were also calculated at a single concentration. 2.5.6. Stability The stability of CAT and S-4 was assessed in plasma under different conditions at four QC concentrations with five replicates for each concentration. The conditions included the freeze-thaw stability (three cycles at −20 ◦ C), post-preparation stability (sample left in autosampler vials at ambient temperature for 24 h), shortterm stability (kept at room temperature for 8 h), and long-term stability (stored at −20 ◦ C for 30 days). 2.6. Pharmacokinetic study The validated method was applied to study the pharmacokinetic profile of CAT and S-4 in rats after oral administration. Animal experiments were carried out according to the protocol approved by the Institute Animal Care and Welfare Committee. Sprague-Dawley male rats (weight, 200–220 g) were purchased from Beijing Vital River Laboratories (Beijing, China). Animals were kept under temperature-controlled conditions with a 12 h light/dark cycle and had free access to food and water prior to the study. Rats were fasted for 12 h before the experiments, and no food was allowed until 4 h following the dose, but they were given free access to water at all times. Blood samples (approximately 200 L) were collected from the orbital vein by capillary tube and transferred to heparinized tubes at baseline (pre-dose), 0.083, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12 and 24 h after a single oral dose of CAT (5 mg/kg), which was prepared by mixing CAT with 0.5% aqueous carboxymethyl cellulose sodium [10]. Tubes were immediately centrifuged at 4500 rpm for 10 min. Plasma was separated and maintained at −20 ◦ C until analysis. The measured values were expressed as means ± SD for six rats at each time point and the mean concentration–time curves were plotted. The pharmacokinetic parameters of CAT and S-4 were calculated using DAS software package (version 2.0, Chinese Pharmacological Association, China) with the non-compartmental model. 3. Results and discussion 3.1. Method development To develop ESI conditions for CAT, S-4 and IS, the full scan precursor was used in both positive and negative ion mode, with the results showing analytes and IS to be detectable with greater sensitivity in positive ion mode. In the infusing experiment, CAT, S-4 and IS yielded [M+H]+ ion peaks at m/z 364.2, 350.1 and 356.2, respectively. After selecting the precursor ion, the fragmentor voltage was then optimized in the range of 50–170 V, and 120 V gave best fragmentation for CAT, 130 V for S-4, and 140 V for IS, respectively. In product ion mode, both CAT and S-4 showed predominant ions at m/z 70.1 with collision energy voltage of 30 and 35 V, respectively. While for IS, the prominent product ion at 192.2 was observed with CE of 25 V, as shown in Fig. 1. The other parameters were set according to the values recommended for the instrument by the manufacturer. Rotundine was selected as the IS due to the similarities of its molecular structure, polarity, ionization condition, and retention time to those of analytes, as well as its m/z value and remarkable extraction recovery efficiency. Additionally, the conditions of the chromatographic analysis were optimized to obtain good
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Table 1 Intra-day and inter-day precision and accuracy of CAT and S-4 in rat plasma by LC–MS/MS. Spiked conc. (ng/mL)
Intra-day (n = 5) Measured conc. (mean ± SD, ng/mL)
Inter-day (n = 15) Precision (RSD%)
Accuracy (%)
Measured conc. (mean ± SD, ng/mL)
Precision (RSD%)
Accuracy (%)
CAT 0.214 0.427 12.82 106.80
0.202 0.444 13.87 107.04
± ± ± ±
0.019 0.022 0.21 4.45
9.2 4.9 1.5 4.2
94.80 103.93 108.22 100.22
0.215 0.455 13.77 101.43
± ± ± ±
0.022 0.017 0.80 2.50
10.4 3.6 5.8 2.5
100.76 106.45 107.48 94.97
S-4 0.044 0.11 2.20 8.80
0.043 0.109 2.27 8.61
± ± ± ±
0.002 0.005 0.16 0.47
4.8 4.8 7.2 5.5
97.80 99.17 103.20 97.87
0.044 0.110 2.31 9.35
± ± ± ±
0.006 0.012 0.14 0.76
13.4 10.7 5.8 8.1
99.43 99.67 105.00 106.25
resolution and high sensitivity for CAT, S-4 and IS. Following comparisons and testing of different categories of columns and mobile phases, an Eclipse XDB-C18 column (1.8 m, 4.6 mm × 50 mm) with acetonitrile–water mobile phase was chosen. The flow rate in the range of 0.2–0.4 mL/min was evaluated to obtain faster elution of all compounds. With mobile flow rate of 0.4 mL/min, CAT, S-4 and IS eluted with good separation at retention times of 3.6, 2.2 and 1.8 min, respectively. Finally, optimal peak shapes were achieved by adding 0.1% acetic acid to the mobile phase and increasing the temperature to 35 ◦ C. Considering the solubility of analytes and IS, a simple protein precipitation method was used to prepare the plasma samples, using acetonitrile as a precipitation agent. Previous work has shown acetonitrile to produce a leading peak when used as an analyte solvent (data not shown). To obtain a fine peak, an evaporation process was carried out to remove acetonitrile first and then samples were reconstituted by mobile phase. Almost no matrix effect was observed in LC–MS/MS analysis after the precipitation procedure, and the extraction recovery of analytes exceeded 90% with excellent reproducibility when assessed at LLOQ, low, medium, and high concentrations.
3.2. Method validation 3.2.1. Specificity No significant endogenous interference was observed at the retention times of CAT, S-4 and IS in blank plasma or plasma samples. Typical MRM chromatograms of blank rat plasma, blank plasma sample spiked with analytes (at LLOQ), and IS, as well as a plasma sample from a CAT-treated rat, are shown in Fig. 2. 3.2.2. Calibration curve and LLOQ The calibration curves of analytes were linear over the concentration range of 0.214–128.16 ng/mL for CAT and 0.044–11.00 ng/mL for S-4, respectively. The typical linear regression equation of the calibration curves was y = 0.1698x + 0.0118 (r2 = 0.9959) using weight factor 1/x2 for CAT and y = 0.0526x + 0.00016135 (r2 = 0.9996) using weight factor 1/x for S-4, where y is the peak area ratio of analyte to IS, and x represents concentrations of analyte. The correlation coefficients (r2 ) were >0.99 for all calibration curves. The LLOQ of CAT and S-4 were calculated as 0.214 ng/mL and 0.044 ng/mL, respectively, which were sufficient for the pharmacokinetics study in rats.
Fig. 2. Typical MRM chromatograms for (A) blank plasma, (B) blank plasma spiked with CAT and S-4 at lower limit of quantitation (LLOQ) and internal standard (IS), (C) plasma sample 45 min after oral administration of CAT (5 mg/kg). Peak at 2.49 and 2.64 min were 6-O-desmethyl and 7-O-desmethyl metabolites of CAT, respectively.
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3.2.3. Accuracy and precision The inter- and intra-day precision and accuracy of CAT level measurement are presented in Table 1. The inter- and intra-day precision at four QC concentrations was less than 15% and accuracy was between 94.80% and 108.22%. 3.2.4. Carry-over To eliminate the auto-sampler memory effect, a standard needle wash procedure using mobile phase as wash solvent after aspiration was conducted in our experiment. The results showed that there was no carry-over. 3.2.5. Matrix effect and extraction recovery The matrix effect values were 94.81%, 91.26%, 87.47% and 93.03% for CAT, 91.41%, 89.91%, 88.63% and 87.27% for S-4 at LLOQ, low, medium and high concentration levels. Matrix effect for IS was 93.67 ± 1.5% at a concentration of 12.06 ng/mL. The results indicate that ionization of analytes is not notably influenced by the co-eluting endogenous substances. The recoveries of analytes in rat plasma at four concentration levels were 95.09–99.90% with RSD% values below 5.4% for CAT and 94.62–101.86% with RSD% values below 8.4% for S-4, respectively. The recovery of IS was 96.28% with RSD% of 3.2%. 3.2.6. Stability Results of stability assessments in rat plasma are presented in Table 2. Our results show CAT and S-4 to remain stable after three freeze-thaw cycles, post-preparation placement in auto-sample vial, short-term storage at room temperature, and long-term storage at −20 ◦ C. The precision and accuracy of the assay were found to be well within the general assay acceptability criteria. 3.3. Pharmacokinetic study The validated LC–MS/MS method was successfully applied in a study evaluating the pharmacokinetics of CAT and its pharmacologically active 3-O-desmethyl metabolite, S-4 in rat plasma after oral administration. The mean plasma concentrations versus time curves of CAT and S-4 are presented in Fig. 3 and the related pharmacokinetic parameters are summarized as means ± SD (Table 3). As shown in Fig. 2C, due to the same precursor ions mass with S-4, the other two monodesmethyl metabolites (peak at 2.49
Fig. 3. Mean plasma concentration–time profile of CAT and S-4 in rats following oral administration of CAT (5 mg/kg, mean ± SD, n = 6).
Table 2 Stability of CAT and S-4 in rat plasma (n = 5). Conditions
CAT
S-4 Accuracy (%)
Spiked conc. (ng/mL)
Measured conc. (mean ± SD, ng/mL)
0.009 0.035 1.02 12.50
96.63 105.21 110.36 99.13
0.044 0.11 2.20 8.80
0.043 0.104 2.35 8.86
± ± ± ±
0.006 0.009 0.15 0.17
98.47 94.60 106.88 100.64
± ± ± ±
0.012 0.036 0.29 1.63
95.21 98.58 91.08 101.86
0.044 0.11 2.20 8.80
0.042 0.102 2.01 8.28
± ± ± ±
0.004 0.007 0.11 0.38
96.40 92.51 91.49 94.13
0.195 0.417 13.22 98.16
± ± ± ±
0.020 0.021 0.73 3.26
91.16 97.68 103.13 91.91
0.044 0.11 2.20 8.80
0.044 0.105 2.30 8.93
± ± ± ±
0.005 0.009 0.10 0.22
100.22 95.23 104.76 101.48
0.194 0.423 14.12 99.44
± ± ± ±
0.018 0.020 0.18 1.36
91.02 98.98 110.15 93.11
0.044 0.11 2.20 8.80
0.041 0.112 2.31 7.88
± ± ± ±
0.003 0.010 0.17 0.29
92.79 101.59 105.21 89.49
Spiked conc. (ng/mL)
Measured conc. (mean ± SD, ng/mL)
Three freeze-thaw cycles
0.214 0.427 12.82 106.80
0.206 0.449 14.14 105.87
± ± ± ±
Post-preparation (in autosampler vial for 24 h)
0.214 0.427 12.82 106.80
0.203 0.421 11.67 108.79
Short-term (room temperature for 12 h)
0.214 0.427 12.82 106.80
Long-term (−20 ◦ C for 30 days)
0.214 0.427 12.82 106.80
Accuracy (%)
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Table 3 Mean pharmacokinetic parameters of CAT and S-4 after oral administration (mean ± SD, n = 6). Parameter
Unit
Mean ± SD CAT
AUC0–t AUC0–∞ MRT0–t MRT0–∞ Tmax t1/2 z CLz/F Vz/F Cmax
g/L h g/L h h h h h L/h/kg L/kg g/L
62.16 65.98 3.13 3.91 0.63 3.06 77.13 340.56 23.88
S-4 ± ± ± ± ± ± ± ± ±
9.04 9.63 0.31 0.48 0.26 0.48 11.17 71.20 5.91
3.97 ± 4.36 ± 7.59 ± 9.98 ± 0.71 ± 6.82 ± – – 0.46 ±
0.75 0.76 0.42 0.68 0.10 0.66
0.11
and 2.64 min) of CAT were also observed in the plasma sample chromatograms. Considering their lower cytotoxic activities and concentration levels than parent drug [13], we did not quantify them in this study. In the present study, CAT was rapidly absorbed in rats and its pharmacologically active 3-O-desmethyl metabolite was promptly measured in the plasma. The exposures of S-4, relative to CAT, were 6.61% calculating by ratio of individual AUC0–∞ . Although the results indicated that S-4 are minor metabolites of CAT, considering its potent cytotoxicity in vitro, the existing of S-4 might contribute to the efficacy of CAT to some extent. However, further studies need to clarify the therapeutic index and safe profile of S-4 in vivo. 4. Conclusions A reliable and sensitive LC–MS/MS method was developed and fully validated for simultaneous quantification of CAT and its pharmacologically active 3-O-desmethyl metabolite, S-4 in rat plasma for the first time. The established LC–MS/MS method had low LLOQ (0.214 ng/mL for CAT and 0.044 ng/mL for S-4) with small sample volumes (50 L) and a relatively short chromatographic run time. Following validation, the method was successfully applied in a study evaluating pharmacokinetic properties of CAT and S-4 in rats after oral administration. Acknowledgements This work was supported by the Ph.D. Innovation Founding of the Peking Union Medical College, China (2012-1007-018). We thank Professor Shishan Yu for providing CAT and S-4 compound.
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Development and validation of a liquid chromatography–tandem mass spectroscopy method for simultaneous determination of (+)-(13aS)-deoxytylophorinine and its pharmacologically active 3-O-desmethyl metabolite in rat plasma Feifei Yu, Haining Lv, Wujun Dong ∗ , Jun Ye, Huazhen Hao, Shuanggang Ma, Shishan Yu, Yuling Liu ∗ Department of Pharmaceutics, Beijing Key Laboratory of Drug Delivery Technology and Novel Formulation, State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
a r t i c l e
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Article history: Received 21 September 2014 Received in revised form 24 December 2014 Accepted 26 December 2014 Available online 3 January 2015 Keywords: (+)-(13aS)-Deoxytylophorinine Metabolite LC–MS/MS Pharmacokinetics Rat plasma
a b s t r a c t CAT ((+)-(13aS)-deoxytylophorinine) is a novel anticancer drug belonging to phenanthroindolizidine alkaloids. A sensitive and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of CAT and its pharmacologically active 3-O-desmethyl metabolite (S-4) was developed and validated in rat plasma using rotundine as the internal standard (IS). CAT, S-4 and IS were extracted by acetonitrile protein precipitation and separated on an Eclipse XDB-C18 column (1.8 m, 4.6 mm × 50 mm) with acetonitrile–water (27:73, v/v) mobile phase containing 0.1% formic acid at a 0.4 mL/min flow rate. Positive ion electrospray ionization in multiple reaction monitoring mode was employed to measure CAT, S-4 and IS by monitoring the transitions m/z 364.2 → 70.1 for CAT, 350.1 → 70.1 for S-4 and 356.2 → 192.2 for IS. Good linear correlation (r2 > 0.991) was achieved for CAT and S-4 over the range of 0.214–128.16 and 0.044–11.00 ng/mL, respectively. The lower limit of quantification was 0.214 ng/mL for CAT and 0.044 ng/mL for S-4, using 50 L rat plasma samples. The intraand inter-day precisions were not exceed 15% and the accuracy ranged between 94.80% and 108.22%. The average extraction recoveries of both analytes were greater than 94.62%. The method was successfully applied to the pharmacokinetic study of CAT and S-4 in rats after oral administration. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Phenanthroindolizidine alkaloids are found mainly in the plants of Cynanchum, Pergularia, Tylophora, and some other genera of Asclepiadaceae family [1–4]. These compounds are among the many alkaloids reported to possess bioactive and pharmacological activities, including anti-arthritis [5], anti-inflammatory [6], and anti-tumor properties [7,8]. (+)-(13aS)-Deoxytylophorinine (CAT, Fig. 1A) is a new anticancer drug belonging to phenanthroindolizidine alkaloids initially isolated from the roots of Tylophora atrofolliculata and Tylophora ovata [9]. Previous studies [10] had found that CAT has significant anti-tumor activity on H22 mouse hepatoma xenografts after oral administration. The anti-tumor activity of CAT is primarily attributed to its ability to intercalate into AT-repeated base pairs in double-helical DNA sequences. Further
∗ Corresponding authors. Tel.: +86 10 63159373; fax: +86 10 63159373. E-mail addresses: [email protected] (W. Dong), [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.jpba.2014.12.042 0731-7085/© 2015 Elsevier B.V. All rights reserved.
studies have shown that CAT potently suppresses the proliferation of common cancer cell lines such as HepG2 and A549 cells, with an IC50 of approximately 10−7 mol/L [11,12]. CAT was susceptible to metabolism by rat liver microsomes and its three major monodesmethyl metabolites were synthesized recently [13], among which 3-O-desmethyldeoxytylophorinine (S-4, Fig. 1B) exhibited highest cytotoxic potency and the cytotoxicity significantly higher than parent compound. Hence, it was imperative to determine CAT and its pharmacologically active metabolite, S-4 simultaneously in order to fully understand the contribution of S-4 to the efficacy profile of CAT. Bioanalytical assays for CAT qualification by LC–MS/MS in mouse plasma or rat tissues were reported [14,15]. However, in the assays, neither report entirely validated LC–MS/MS method nor did they support the determination of S-4. Recently, the use of methods combining LC–MS/MS and nuclear magnetic resonance (NMR) was reported in the identification of CAT and its metabolites in rat urine [16], but no information was provided on the measurement of CAT and its metabolites. So far, little is known about
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Fig. 1. Mass spectroscopy spectra of (A) CAT, (B) S-4 and (C) internal standard (IS).
the oral plasma pharmacokinetic characteristics of CAT and S-4 in rats. In this study, we developed and validated a sensitive LC–MS/MS method for simultaneous determination of CAT and S-4 levels in rat plasma. To the best of our knowledge, this is the first report of a fully validated LC–MS/MS method for quantification of CAT and S-4 in a biological matrix. The LC–MS/MS method was successfully applied in pharmacokinetic study of CAT and its pharmacologically active 3O-desmethyl metabolite, S-4 in rats following oral administration. 2. Materials and methods 2.1. Chemicals and reagents CAT (purity > 99.0%) and S-4 (purity > 99.0%) was kindly provided by Prof. Shishan Yu (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China) and rotundine (Fig. 1C, internal standard, IS) was obtained from National institutes for food and drug control (Beijing, China). HPLC-grade acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ, USA), while MS-grade formic acid was purchased from Sigma (St. Louis, MO, USA). Pure water (Wahaha, Hangzhou, China) was obtained from a local market. Other chemicals were all of analytical grade.
Agilent 1200 RRLC system (Agilent Co.) coupled to a triple quadrupole MS analyzer with an electrospray ionization (ESI) source. Chromatographic separations were achieved using an Eclipse XDB-C18 column (1.8 m, 4.6 mm × 50 mm, Agilent Co.). The mobile phase, consisting of acetonitrile–deionized water (27:63, v/v) with 0.1% formic acid, was used in isocratic mode at a flow rate of 0.4 mL/min. Aliquots (10 L) of the sample solution were injected for each analysis. The separation run was finished within 6 min of sample injection at 35 ◦ C. Quantification was performed in a multiple reaction monitoring (MRM) mode using ESI in the positive ion mode: for CAT, m/z 364.2 → 70.1 (fragmentor, 120 V; collision energy, 30 V); for S-4, m/z 350.1 → 70.1 (fragmentor, 130 V; collision energy, 35 V); for rotundine (IS), 356.2 → 192.2 (fragmentor, 140 V; collision energy, 25 V) were used as the quantitative ion-pairs, respectively. The optimum operating parameters were as follows: drying gas temperature, 350 ◦ C; drying gas flow, 10 L/min; nebulizer pressure, 50 psi; and capillary voltage, 4000 V. Data acquisition and processing were performed using MassHunter workstation (Agilent Co.). 2.3. Stock solutions, calibration curve, and quality control (QC) samples
2.2. Instrumentation and chromatographic conditions The analysis was performed on a 6410B triple quadrupole LC–MS system (Agilent Corporation, MA, USA) consisting of an
A stock solution of CAT and S-4 was prepared in ethanol at a concentration of 50 g/mL and 11 g/mL, respectively. The stock solution was diluted in mobile phase to obtain working
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solutions with concentrations of 1.068/0.22, 2.136/0.55, 6.408/2.20, 16.02/5.50, 64.08/11.00, 160.2/22.00, 534.00/44.00, and 640.8/55.00 ng/mL. A solution of IS at a final concentration of 12.04 ng/mL was prepared in ethanol. All solutions were stored at 4 ◦ C until LC–MS/MS analysis. Calibrations standards of CAT and S-4 (0.214/0.044, 0.427/0.11, 1.282/0.44, 3.204/1.10, 12.82/2.20, 32.04/4.40, 106.80/8.80, and 128.16/11.00 ng/mL) were prepared by spiking the blank plasma with the corresponding working solution. The quality control (QC) samples of CAT and S-4 with four concentrations at 0.214/0.044, 0.427/0.11, 12.82/2.20, and 106.80/8.80 ng/mL were similarly prepared. 2.4. Sample preparation All plasma samples were thawed to room temperature before analysis. Aliquots of 50 L plasma and 20 L of IS working solution (12.04 ng/mL) were added into 1.5-mL centrifuge tubes. After the mixture was vortexed for 30 s, 600 L of acetonitrile was added. The mixture was vortex-mixed for 2 min and centrifuged at 14,000 rpm for 5 min at room temperature. The upper organic layer was transferred into another tube and evaporated to dryness under a gentle stream of nitrogen gas. The residue was reconstituted with 50 L mobile phase by vortexing for 2 min and centrifuged at 14,000 rpm for 10 min. Finally, 10 L of supernatant was injected onto the LC–MS/MS system. 2.5. Method validation A full validation of the method was performed in accordance with the FDA Guidance for Industry Bioanalytical Method Validation [17]. 2.5.1. Specificity The specificity of the method was evaluated by analyzing blank plasma samples from six individual rats to test for potential interference by endogenous compounds. The analyses of the blank samples were compared with the corresponding plasma samples spiked the LLOQ level and samples taken from treated animals. 2.5.2. Calibration curve Five calibration curves were freshly prepared at eight concentrations covering the range of 0.214–128.16 ng/mL for CAT and 0.044–11.00 ng/mL for S-4. The linearity was investigated by plotting the peak area ratios of analytes to IS versus the concentrations of analytes and applying the least squares linear regression. A correlation coefficient (r2 ) calculated in the calibration curve should be no less than 0.99. 2.5.3. Accuracy and precision Intra-day accuracy and precision were estimated by analyzing the QC samples of plasma at four concentrations (n = 5 at each concentration) on a single day. Inter-day accuracy and precision were determined on three consecutive days. Precision was expressed as the relative standard deviation (RSD%), while accuracy was presented as percentage calculated using the following equation: accuracy = (measured concentration/nominal spiked concentration) × 100. 2.5.4. Carry-over Carry-over was evaluated by sequentially injecting an extracted sample at the upper limit of quantification (ULOQ) concentration and two extracted blank samples. 2.5.5. Matrix effect and extraction recovery The matrix effect was determined at four QC concentrations by comparing peak responses of post-extraction blank plasma (from six different individuals) spiked QC samples with those of neat
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standard solutions. The extraction recovery was measured by comparing the responses of analytes obtained from extracted samples with blank plasma extracts spiked with QC samples. The matrix effect and extraction recovery of IS were also calculated at a single concentration. 2.5.6. Stability The stability of CAT and S-4 was assessed in plasma under different conditions at four QC concentrations with five replicates for each concentration. The conditions included the freeze-thaw stability (three cycles at −20 ◦ C), post-preparation stability (sample left in autosampler vials at ambient temperature for 24 h), shortterm stability (kept at room temperature for 8 h), and long-term stability (stored at −20 ◦ C for 30 days). 2.6. Pharmacokinetic study The validated method was applied to study the pharmacokinetic profile of CAT and S-4 in rats after oral administration. Animal experiments were carried out according to the protocol approved by the Institute Animal Care and Welfare Committee. Sprague-Dawley male rats (weight, 200–220 g) were purchased from Beijing Vital River Laboratories (Beijing, China). Animals were kept under temperature-controlled conditions with a 12 h light/dark cycle and had free access to food and water prior to the study. Rats were fasted for 12 h before the experiments, and no food was allowed until 4 h following the dose, but they were given free access to water at all times. Blood samples (approximately 200 L) were collected from the orbital vein by capillary tube and transferred to heparinized tubes at baseline (pre-dose), 0.083, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12 and 24 h after a single oral dose of CAT (5 mg/kg), which was prepared by mixing CAT with 0.5% aqueous carboxymethyl cellulose sodium [10]. Tubes were immediately centrifuged at 4500 rpm for 10 min. Plasma was separated and maintained at −20 ◦ C until analysis. The measured values were expressed as means ± SD for six rats at each time point and the mean concentration–time curves were plotted. The pharmacokinetic parameters of CAT and S-4 were calculated using DAS software package (version 2.0, Chinese Pharmacological Association, China) with the non-compartmental model. 3. Results and discussion 3.1. Method development To develop ESI conditions for CAT, S-4 and IS, the full scan precursor was used in both positive and negative ion mode, with the results showing analytes and IS to be detectable with greater sensitivity in positive ion mode. In the infusing experiment, CAT, S-4 and IS yielded [M+H]+ ion peaks at m/z 364.2, 350.1 and 356.2, respectively. After selecting the precursor ion, the fragmentor voltage was then optimized in the range of 50–170 V, and 120 V gave best fragmentation for CAT, 130 V for S-4, and 140 V for IS, respectively. In product ion mode, both CAT and S-4 showed predominant ions at m/z 70.1 with collision energy voltage of 30 and 35 V, respectively. While for IS, the prominent product ion at 192.2 was observed with CE of 25 V, as shown in Fig. 1. The other parameters were set according to the values recommended for the instrument by the manufacturer. Rotundine was selected as the IS due to the similarities of its molecular structure, polarity, ionization condition, and retention time to those of analytes, as well as its m/z value and remarkable extraction recovery efficiency. Additionally, the conditions of the chromatographic analysis were optimized to obtain good
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Table 1 Intra-day and inter-day precision and accuracy of CAT and S-4 in rat plasma by LC–MS/MS. Spiked conc. (ng/mL)
Intra-day (n = 5) Measured conc. (mean ± SD, ng/mL)
Inter-day (n = 15) Precision (RSD%)
Accuracy (%)
Measured conc. (mean ± SD, ng/mL)
Precision (RSD%)
Accuracy (%)
CAT 0.214 0.427 12.82 106.80
0.202 0.444 13.87 107.04
± ± ± ±
0.019 0.022 0.21 4.45
9.2 4.9 1.5 4.2
94.80 103.93 108.22 100.22
0.215 0.455 13.77 101.43
± ± ± ±
0.022 0.017 0.80 2.50
10.4 3.6 5.8 2.5
100.76 106.45 107.48 94.97
S-4 0.044 0.11 2.20 8.80
0.043 0.109 2.27 8.61
± ± ± ±
0.002 0.005 0.16 0.47
4.8 4.8 7.2 5.5
97.80 99.17 103.20 97.87
0.044 0.110 2.31 9.35
± ± ± ±
0.006 0.012 0.14 0.76
13.4 10.7 5.8 8.1
99.43 99.67 105.00 106.25
resolution and high sensitivity for CAT, S-4 and IS. Following comparisons and testing of different categories of columns and mobile phases, an Eclipse XDB-C18 column (1.8 m, 4.6 mm × 50 mm) with acetonitrile–water mobile phase was chosen. The flow rate in the range of 0.2–0.4 mL/min was evaluated to obtain faster elution of all compounds. With mobile flow rate of 0.4 mL/min, CAT, S-4 and IS eluted with good separation at retention times of 3.6, 2.2 and 1.8 min, respectively. Finally, optimal peak shapes were achieved by adding 0.1% acetic acid to the mobile phase and increasing the temperature to 35 ◦ C. Considering the solubility of analytes and IS, a simple protein precipitation method was used to prepare the plasma samples, using acetonitrile as a precipitation agent. Previous work has shown acetonitrile to produce a leading peak when used as an analyte solvent (data not shown). To obtain a fine peak, an evaporation process was carried out to remove acetonitrile first and then samples were reconstituted by mobile phase. Almost no matrix effect was observed in LC–MS/MS analysis after the precipitation procedure, and the extraction recovery of analytes exceeded 90% with excellent reproducibility when assessed at LLOQ, low, medium, and high concentrations.
3.2. Method validation 3.2.1. Specificity No significant endogenous interference was observed at the retention times of CAT, S-4 and IS in blank plasma or plasma samples. Typical MRM chromatograms of blank rat plasma, blank plasma sample spiked with analytes (at LLOQ), and IS, as well as a plasma sample from a CAT-treated rat, are shown in Fig. 2. 3.2.2. Calibration curve and LLOQ The calibration curves of analytes were linear over the concentration range of 0.214–128.16 ng/mL for CAT and 0.044–11.00 ng/mL for S-4, respectively. The typical linear regression equation of the calibration curves was y = 0.1698x + 0.0118 (r2 = 0.9959) using weight factor 1/x2 for CAT and y = 0.0526x + 0.00016135 (r2 = 0.9996) using weight factor 1/x for S-4, where y is the peak area ratio of analyte to IS, and x represents concentrations of analyte. The correlation coefficients (r2 ) were >0.99 for all calibration curves. The LLOQ of CAT and S-4 were calculated as 0.214 ng/mL and 0.044 ng/mL, respectively, which were sufficient for the pharmacokinetics study in rats.
Fig. 2. Typical MRM chromatograms for (A) blank plasma, (B) blank plasma spiked with CAT and S-4 at lower limit of quantitation (LLOQ) and internal standard (IS), (C) plasma sample 45 min after oral administration of CAT (5 mg/kg). Peak at 2.49 and 2.64 min were 6-O-desmethyl and 7-O-desmethyl metabolites of CAT, respectively.
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3.2.3. Accuracy and precision The inter- and intra-day precision and accuracy of CAT level measurement are presented in Table 1. The inter- and intra-day precision at four QC concentrations was less than 15% and accuracy was between 94.80% and 108.22%. 3.2.4. Carry-over To eliminate the auto-sampler memory effect, a standard needle wash procedure using mobile phase as wash solvent after aspiration was conducted in our experiment. The results showed that there was no carry-over. 3.2.5. Matrix effect and extraction recovery The matrix effect values were 94.81%, 91.26%, 87.47% and 93.03% for CAT, 91.41%, 89.91%, 88.63% and 87.27% for S-4 at LLOQ, low, medium and high concentration levels. Matrix effect for IS was 93.67 ± 1.5% at a concentration of 12.06 ng/mL. The results indicate that ionization of analytes is not notably influenced by the co-eluting endogenous substances. The recoveries of analytes in rat plasma at four concentration levels were 95.09–99.90% with RSD% values below 5.4% for CAT and 94.62–101.86% with RSD% values below 8.4% for S-4, respectively. The recovery of IS was 96.28% with RSD% of 3.2%. 3.2.6. Stability Results of stability assessments in rat plasma are presented in Table 2. Our results show CAT and S-4 to remain stable after three freeze-thaw cycles, post-preparation placement in auto-sample vial, short-term storage at room temperature, and long-term storage at −20 ◦ C. The precision and accuracy of the assay were found to be well within the general assay acceptability criteria. 3.3. Pharmacokinetic study The validated LC–MS/MS method was successfully applied in a study evaluating the pharmacokinetics of CAT and its pharmacologically active 3-O-desmethyl metabolite, S-4 in rat plasma after oral administration. The mean plasma concentrations versus time curves of CAT and S-4 are presented in Fig. 3 and the related pharmacokinetic parameters are summarized as means ± SD (Table 3). As shown in Fig. 2C, due to the same precursor ions mass with S-4, the other two monodesmethyl metabolites (peak at 2.49
Fig. 3. Mean plasma concentration–time profile of CAT and S-4 in rats following oral administration of CAT (5 mg/kg, mean ± SD, n = 6).
Table 2 Stability of CAT and S-4 in rat plasma (n = 5). Conditions
CAT
S-4 Accuracy (%)
Spiked conc. (ng/mL)
Measured conc. (mean ± SD, ng/mL)
0.009 0.035 1.02 12.50
96.63 105.21 110.36 99.13
0.044 0.11 2.20 8.80
0.043 0.104 2.35 8.86
± ± ± ±
0.006 0.009 0.15 0.17
98.47 94.60 106.88 100.64
± ± ± ±
0.012 0.036 0.29 1.63
95.21 98.58 91.08 101.86
0.044 0.11 2.20 8.80
0.042 0.102 2.01 8.28
± ± ± ±
0.004 0.007 0.11 0.38
96.40 92.51 91.49 94.13
0.195 0.417 13.22 98.16
± ± ± ±
0.020 0.021 0.73 3.26
91.16 97.68 103.13 91.91
0.044 0.11 2.20 8.80
0.044 0.105 2.30 8.93
± ± ± ±
0.005 0.009 0.10 0.22
100.22 95.23 104.76 101.48
0.194 0.423 14.12 99.44
± ± ± ±
0.018 0.020 0.18 1.36
91.02 98.98 110.15 93.11
0.044 0.11 2.20 8.80
0.041 0.112 2.31 7.88
± ± ± ±
0.003 0.010 0.17 0.29
92.79 101.59 105.21 89.49
Spiked conc. (ng/mL)
Measured conc. (mean ± SD, ng/mL)
Three freeze-thaw cycles
0.214 0.427 12.82 106.80
0.206 0.449 14.14 105.87
± ± ± ±
Post-preparation (in autosampler vial for 24 h)
0.214 0.427 12.82 106.80
0.203 0.421 11.67 108.79
Short-term (room temperature for 12 h)
0.214 0.427 12.82 106.80
Long-term (−20 ◦ C for 30 days)
0.214 0.427 12.82 106.80
Accuracy (%)
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Table 3 Mean pharmacokinetic parameters of CAT and S-4 after oral administration (mean ± SD, n = 6). Parameter
Unit
Mean ± SD CAT
AUC0–t AUC0–∞ MRT0–t MRT0–∞ Tmax t1/2 z CLz/F Vz/F Cmax
g/L h g/L h h h h h L/h/kg L/kg g/L
62.16 65.98 3.13 3.91 0.63 3.06 77.13 340.56 23.88
S-4 ± ± ± ± ± ± ± ± ±
9.04 9.63 0.31 0.48 0.26 0.48 11.17 71.20 5.91
3.97 ± 4.36 ± 7.59 ± 9.98 ± 0.71 ± 6.82 ± – – 0.46 ±
0.75 0.76 0.42 0.68 0.10 0.66
0.11
and 2.64 min) of CAT were also observed in the plasma sample chromatograms. Considering their lower cytotoxic activities and concentration levels than parent drug [13], we did not quantify them in this study. In the present study, CAT was rapidly absorbed in rats and its pharmacologically active 3-O-desmethyl metabolite was promptly measured in the plasma. The exposures of S-4, relative to CAT, were 6.61% calculating by ratio of individual AUC0–∞ . Although the results indicated that S-4 are minor metabolites of CAT, considering its potent cytotoxicity in vitro, the existing of S-4 might contribute to the efficacy of CAT to some extent. However, further studies need to clarify the therapeutic index and safe profile of S-4 in vivo. 4. Conclusions A reliable and sensitive LC–MS/MS method was developed and fully validated for simultaneous quantification of CAT and its pharmacologically active 3-O-desmethyl metabolite, S-4 in rat plasma for the first time. The established LC–MS/MS method had low LLOQ (0.214 ng/mL for CAT and 0.044 ng/mL for S-4) with small sample volumes (50 L) and a relatively short chromatographic run time. Following validation, the method was successfully applied in a study evaluating pharmacokinetic properties of CAT and S-4 in rats after oral administration. Acknowledgements This work was supported by the Ph.D. Innovation Founding of the Peking Union Medical College, China (2012-1007-018). We thank Professor Shishan Yu for providing CAT and S-4 compound.
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