Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 483–488
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Quantitative determination of Lx2-32c, a novel taxane derivative, in rat plasma by liquid chromatography–tandem mass spectrometry Jinping Hu, Feng You, Shu Yang, Yan Li ∗ State Key Laboratory of Bioactive Substance and Function of Natural Medicines and Department of Drug Metabolism, Institute of Materia Medica, Chinese Academy of Medical Sciences & Perking Union Medical College, Beijing 100050, PR China
a r t i c l e
i n f o
Article history: Received 1 July 2013 Received in revised form 3 September 2013 Accepted 11 September 2013 Available online 5 October 2013 Keywords: Lx2-32c Taxane derivative LC–MS/MS Pharmacokinetic Bioavailability
a b s t r a c t A sensitive and reliable LC–MS/MS method for the determination of Lx2-32c, a novel taxane derived from cephalomannine, has been developed and validated. Plasma samples containing Lx2-32c and paclitaxel (internal standard) were prepared based on a simple protein precipitation by the addition of two volumes of acetonitrile. The analyte and internal standard were separated on a Zorbax SB-C18 column (3.5 m, 2.1 mm × 100 mm) with the mobile phase of acetonitrile/water containing 0.1% formic acid (v/v) with gradient elution at a flow rate of 0.2 ml/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with atmospheric pressure chemical ionization (APCI) by multiple reactions monitoring (MRM) of the transitions at m/z 887.5 → 264.3 for Lx2-32c and 854.5 → 286.2 for IS. Linear detection responses were obtained for Lx2-32c ranging from 1 to 1000 ng/ml. Inter- and intra-day precision (R.S.D.%) were all within 15% and the accuracy (R.E.%) was equal or lower than 8%. The lower limit of quantitation (LLOQ) was 1 ng/ml and the average recovery was greater than 91.5%. The method was successfully applied to the pharmacokinetic study of Lx2-32c in rat plasma. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Taxanes, a class anti-tumor drug isolated from the bark of Pacific Yew tree, have been widely employed for the cancer chemotherapy [1,2]. However, these agents suffer from poor drug solubility, toxicity and, in particular, drug resistance [3–5]. To overcome the above clinical problems, the structural of taxanes has been extensively modified to obtain the new molecules with a better therapeutic index, and the ability to overcome drug resistance [6]. Lx2-32c, a novel taxane derivative, is a semisynthetic analog from cephalomannine [7]. Our previous studies had shown that Lx2-32c had significant antitumor activity on BGC-823 (human gastric carcinoma) and A549 (human non-small cell lung carcinoma) xenograft in nude mice [8]. The further study found that Lx2-32c has the potent antiproliferative effect on several drug-resistant tumor cell lines such as Paclitaxel-resistant MX-1/T, A549/T, MCF7/T, BGC-823/T, vincristin-resistant KB/V and fluorouracil-resistant Bel-7402/5-Fu cells. Moreover, Lx2-32c also displayed robust anti-paclitaxel-resistance activity in nude mice bearing paclitaxelresistant MX-1/T tumors. Hence Lx2-32c is expected to overcome, at least in part, drug resistance in the clinical treatment [9]. Despite
∗ Corresponding author. Tel.: +86 10 6316 5172; fax: +86 10 6316 5172. E-mail addresses: [email protected], [email protected] (Y. Li). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.09.018
extensive researches in the pharmacological activities of Lx2-32c, little is known about its pharmacokinetics. Therefore, it is necessary to develop a reliable method for the pharmacokinetic study of Lx2-32c. To our knowledge there have been several LC–MS methods reported for the quantitative bioanalysis of paclitaxel. But most of them employed time-consuming solid-phase extraction (SPE) and liquid–liquid extraction (LLE) as a means of sample pretreatment to achieve low quantitation limits [10–16]. In the present study, a simple, sensitive, accurate and reproducible LC–MS/MS method was developed and validated for the determination Lx2-32c in rats following intravenous and intraperitoneal administration at 3 and 30 mg/kg.
2. Experimental 2.1. Chemicals and reagents Lx2-32c (purity >99.5%) were synthesized at Laboratory of Chemistry of Natural Products (Chinese Academy of Medical Sciences). Paclitaxel, 100% ethanol and polyoxyl 35 castor oil were kindly obtained from Beijing Union Pharmaceutical Factory. Acetonitrile was of HPLC grade (Fisher, USA). All other chemicals were of analytical reagent grade. Ultrapure water, prepared by a Milli-Q Reagent water system (Millipore, MA, USA), was used throughout the study.
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2.2. Preparation of stocks, calibration standards and quality control samples Standard stock solutions of Lx2-32c and paclitaxel (internal standard) were prepared in methanol at 1 mg/ml. Then the stock solutions were diluted with methanol to obtain fresh standard working solution. Calibrations standards were prepared by adding corresponding working solutions in drug-free plasma. The final concentrations of Lx2-32c in plasma were 1, 2.5, 10, 50, 200, 500, 750, 1000 ng/ml, and IS was 1000 ng/ml, respectively. Low-, mid- and high-level quality control samples containing 2.5, 200 and 800 ng/ml of Lx2-32c, were prepared in a manner similar to that used for the preparation of calibration samples. All stock solutions, working solutions, calibration standards and quality controls were stored at −20 ◦ C and were brought to room temperature before analysis. 2.3. Sample processing A 20 l of IS working solution (1 g/ml) and 180 l acetonitrile were added to 100 l of plasma sample. The mixture was vortexed for 30 s followed by centrifugation at 14,000 rpm for 5 min at room temperature. A 5 l aliquot of each supernatant was injected into the LC/MS/MS system for the analysis. 2.4. Instrumental analysis Chromatographic separations were carried out on an Agilent 1260 HPLC system using a Zorbax SB-C18 column (3.5 m, 2.1 mm × 100 mm, Agilent, USA). The column temperature was set to 25 ◦ C. The mobile phase consisted of deionized water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B). Separation was performed at a flow-rate of 0.2 ml/min with the following gradient elution: from 0 to 1 min, 35% B; linear increase to 95% B in 0.2 min; 95% B during 3 min, decrease to 35% B in 0.2 min; and stabilization at initial conditions for 8 min. The total run time was 12 min. The sample volume injected was 5 l. The HPLC instrument was coupled to API 4000 triple quadruple mass spectrometer from Applied Biosystems Sciex (Toronto, Canada) with atmospheric pressure chemical ionization (APCI) ion source in the positive mode. The detection was operated in the multiple reaction monitoring (MRM) mode with a dwell time of 200 ms for each transition. The MRM transitions of Lx2-32c and its internal standard (IS) were 887.5 → 264.3 and 854.5 → 286.2, respectively. The mass spectrometric condition was optimized as follows: collision gas, 6 psi; curtain gas, 15 psi; ion source gas (GS1), 30 psi; nebulizer current, 3.0 A; temperature, 300 ◦ C. Declustering potential (DP), collision energy (CE) and collision cell exit potential (CXP) was 61, 21 and 10 V for Lx2-32c, and 80, 27 and 8 V for IS, respectively. Data acquisition and processing were performed using Analyst software (version 1.5.2). 2.5. Method validation 2.5.1. Selectivity Six different blank rat plasma samples were analyzed to detect the potential interferences co-eluting with the analyte and IS. Chromatographic peaks of analyte and IS were identified on the basis of their retention times and MRM responses. 2.5.2. Linearity, precision and accuracy The linearity of LC/MS/MS method for the determination of Lx2-32c was evaluated by a calibration curve in the range of 1–1000 ng/ml. The calibration curve was obtained by plotting the
ratio of chromatographic peaks area (Lx2-32c/IS) versus the concentration of Lx2-32c. Least squares linear regression analysis was used to determine the slope, intercept and correlation coefficient. The calibration curve requires a correlation coefficient (r2 ) of 0.99 or better. To evaluate the precision, at least five QC samples of three different concentrations of Lx2-32c were processed and injected on a single day (intra-day) and at different days (inter-day). The variability of Lx2-32c determination was expressed as the relative standard deviation (R.S.D.%) which should not exceed 15% at all concentrations. Accuracy is expressed as relative error (R.E.%) which should be within the limits of ±15% at all concentrations of Lx2-32c. 2.5.3. Matrix effect and recovery The matrix effect was defined as the ion suppression/enhancement on the ionization of analyte, which was evaluated by comparing the responses of deproteinized samples of blank plasma from six rats spiked Lx2-32c samples (n = 5) with those of standard samples at equivalent concentrations. The extraction recovery was determined at three Lx2-32c levels and calculated by comparing the analyte standard peak areas obtained from extracted samples with post-extracted samples spiked with the analyte. 2.5.4. Stability studies Three freeze–thaws, long-term, short-term and post-extracted stabilities of Lx2-32c in plasma was tested using high-, midand low-quality control samples. The freeze–thaw stability of the analyte was determined over three freeze–thaw cycles. In each freeze–thaw cycle, the samples were frozen and stored at −20 ◦ C for 24 h, then thawed at ambient temperature. To evaluate longterm stability of Lx2-32c, the plasma samples were stored at −20 ◦ C for 30 days. For the short-term stability, fresh plasma samples were kept at room temperature for 24 h before treatment. The stability of treated plasma in auto-sampler was tested after keeping the samples at 15 ◦ C for 24 h. Dilution stability was assessed using a spiked sample the concentration of which was higher than the upper limit of quantification samples. The result was obtained by comparing the back-calculated value of the sample after being diluted 5 times with the nominal value. 2.6. Pharmacokinetic experiments in rats The validated assay was used to determine the plasma pharmacokinetic of Lx2-32c in rats after a single intraperitoneal (i.p.) or intravenous (i.v.) injection. All animal protocols were approved by Institute Animal Care and Welfare Committee. Sprague–Dawley rats (adult male), weighing 200–220 g, were obtained from Beijing Vital River Experimental Animal Co., Ltd. The rats were quarantined for 1 week prior to the study and maintained on a 12 h light/12 h dark cycle at 22 ± ◦ C and at 60% relative humidity. Before the experiment animals were of free access to water and food. The dosing solutions used for the animal studies were prepared by dissolving the required amounts of Lx2-32c in 100% ethanol, followed by mixing with polyoxyl 35 castor oil to yield a 12.5 mg/ml stock (50% ethanol:50% polyoxyl 35 castor oil) that were diluted in saline immediately before administration. After i.p. or i.v. injection of Lx2-32c at a dose of 30 or 3 mg/kg to rats, approximately 0.2 ml blood samples were collected in heparinized 1.5 ml polythene tubes by orbital bleeding via capillary tubes at 0.03, 0.08, 0.17, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h postdose. The blood samples were immediately centrifuged at 5000 rpm for 10 min. The plasma was separated and frozen at −20 ◦ C until analysis. The plasma concentrations of Lx2-32c were expressed as mean ± S.D. and the mean concentration–time curves were plotted. Data fitting and
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Fig. 1. Product ion mass spectra of [M+H]+ of (A) Lx2-32c, (B) paclitaxel (IS).
Table 1 Intra- and inter-day accuracy and precision of Lx2-32c in rat plasma. Concentrations (ng/ml)
Intra-day
Inter-day
Mean ± S.D. (n = 5) 1.00 2.50 200.00 800.00
1.04 2.55 197.61 809.02
± ± ± ±
0.09 0.21 3.22 24.53
Accuracy (R.E.%)
Precision (R.S.D.%)
Mean ± S.D. (n = 5)
4.0 2.0 -1.2 1.1
8.7 8.2 1.6 3.0
1.06 2.58 196.73 815.54
± ± ± ±
0.12 0.28 9.85 46.48
Accuracy (R.E.%)
Precision (R.S.D.%)
6.0 3.2 -1.6 1.9
11.3 10.9 5.0 5.7
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Fig. 2. Typical MRM chromatograms of Lx2-32c and IS: (A) blank rat plasma; (B) blank plasma spiked with Lx2-32c (LLOQ of 1 ng/ml) and IS; and (C) rat plasma sample at 24 h after i.p. administration of Lx2-32c at 30 mg/kg spiked with IS. The retention time of Lx2-32c and IS was 8.17 and 7.19 min, respectively.
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Table 2 Stability of Lx2-32c in rat plasma (n = 5). Stability tests
Theoretical cone (ng/ml)
Found cone (ng/ml)
R.E.%
R.S.D%
2.50 200.00 800.00
2.65 ± 0.13 200.72 ± 2.92 834.75 ± 15.02
6.0 0.4 4.3
4.9 1.5 1.8
2.50 200.00 800.00
2.38 ± 0.23 195.75 ± 1.58 794.08 ± 9.15
−4.8 −2.1 −0.7
9.7 0.8 1.2
2.50 200.00 800.00
2.59 ± 0.24 205.76 ± 13.54 847.08 ± 45.36
3.6 2.9 5.9
9.3 6.6 5.4
2.50 200.00 800.00
2.69 ± 0.29 208.36 ± 7.16 839.89 ± 27.83
7.6 4.2 5.0
10.8 3.4 3.3
−6.1
4.9
Room temperature (24 h)
Post-treated (15 ◦ C for 24 h)
Three freeze–thaw cycles
Stored at −20 ◦ C (30 day)
Dilution stability (dilution factor: 5) 4000.00
pharmacokinetic parameter estimates were carried out using WinNonLin Software (Version 6.1, Pharsight Corporation). 3. Results and discussion 3.1. Method development To get appropriate retention time, better resolution and sensitivity, the different HPLC parameters including mobile phase, category of column and flow rate of mobile phase were tested and compared. Finally, the mobile phase of acetonitrile/water containing 0.1% formic acid was found to be suitable for the formation of molecular ions of Lx2-32c and IS. Zorbax SB-C18 column (3.5 m, 2.1 mm × 100 mm) was selected for the chromatographic separation. The system provided high resolution, great baseline stability and high ionization efficiency. By investigating the full-scan mass spectra of Lx2-32c and IS, we found that the signal intensity in the positive ion mode was much higher than that in the negative ion mode. During a direct infusion experiment, the mass spectra for Lx2-32c and IS revealed peaks at m/z 887.5 and 854.5, respectively, as protonated molecular ion [M+H]+ . As shown in Fig. 1, the most abundant and stable productions were at m/z 264.3 for Lx2-32c, and at m/z 286.2 for IS, after fragmentation in the collision cell. The most suitable mass spectrometric conditions were determined by optimizing all the parameters of mass spectrometer such as collision gas, curtain gas, ion source gas (GS1), nebulizer current, temperature, declustering potential (DP), collision energy (CE) and collision cell exit potential (CXP) to obtain high and stable signal. 3.2. Method validation 3.2.1. Selectivity Representative MRM chromatograms of blank plasma samples, plasma sample spiked with drugs and a real rat plasma sample were obtained under the selected analytical conditions. The result showed the absence of any interference at the retention time of the analyte and IS. The retention times of Lx2-32c and IS are 8.17 and 7.19 min, respectively (Fig. 2). 3.2.2. Calibration curve and lower limit of quantification The calibration curves generated from the detection of rat plasma containing known amounts of Lx2-32c were linear over the concentration range tested (1–1000 ng/ml). The
3756.72 ± 182.45
regression equations obtained by least squared regression were y = 0.0193x + 0.00634 (n = 5), where y is the peak area ratios of analyte to IS, and x is the concentrations of analyte. The correlation coefficients (r2 ) were ≥0.99 for all calibration curves. The lower limit of quantification of Lx2-32c in rat plasma was found to be 1 ng/ml, which are sufficient for the pharmacokinetics study. 3.2.3. Precision and accuracy The intra- and inter-assay variations were found to be within the accepted limits. The intra-and inter-day precision of Lx2-32c at three QC concentrations, as presented in Table 1 were less than 11.3%. Assay accuracy was found to be within ±8.0%. The results indicated that the present method was reliable and reproducible for the quantitative determination of Lx2-32c. 3.2.4. Matrix effect and recovery The matrix effect values were 107.2%, 103.6% and 101.0% for Lx232c at low, medium and high concentration levels, respectively. These results suggested that the matrix effect on the ionization of Lx2-32c was not obvious under our experimental conditions. The recoveries in rat plasma were 91.5–101.1% for Lx2-32c at three concentration levels. 3.2.5. Stability The stability of Lx2-32c in rat plasma is summarized in Table 2. Lx2-32c in rat plasma was found to be stable after being placed at room temperature for 24 h, stored at −20 ◦ C for 30 days or through three freeze–thaw cycles. Furthermore, samples after treatment were stable at 15 ◦ C in auto-sampler for a period of 24 h, which indicated that a large number of samples could be determined in each analytical run. 3.3. Pharmacokinetic study The present method was successfully applied to the pharmacokinetic studies of Lx2-32c in rats after i.p. or i.v. injection. A plot of plasma concentration versus time for Lx2-32c in rats is presented in Fig. 3. The corresponding pharmacokinetic parameters calculated using non-compartmental analysis are listed as mean ± SD in Table 3. The plasma peak of Lx2-32c reached at 2 h after a single i.p. dose at 30 mg/kg, and the values of Cmax and AUC0−t were 0.6 g/ml and 5.4(g/ml) h, respectively. The AUC0−t of Lx2-32c after an i.v. dosing of 3 mg/kg was 1.6(g/ml) h. Lx2-32c displayed much smaller volume of distribution and lower clearance after i.v.
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Table 3 Mean pharmacokinetic parameters of Lx2-32c in rats following a single i.p. dose of 30 mg/kg and i.v. dose of 3 mg/kg of Lx2-32c (n = 5). Parameter
AUC(0−t) (mg/l h) MRT(0−t) (h) tl/2z (h) Tmax (h) CLz/F (l/h kg) Vz/F (l/kg) Cmax (mg/l) F (%)
Mean ± SD i.p. 30 mg/kg
i.v. 3 mg/kg
4.5 ± 2.4 7.3 ± 2.0 5.9 ± 2.5 2.6 ± 2.4 9.4 ± 8.9 85.5 ± 94.3 0.6 ± 0.4 28.1
1.6 5.1 10.9 – 1.7 24.8 3.3
± 0.2 ± 0.3 ± 6.0 ± 0.4 ± 10.4 ± 0.4
Fig. 3. Plasma concentration–time profile of Lx2-32c in rats following a single i.p. dose of 30 mg/kg and i.v. dose of 3 mg/kg of Lx2-32c (n = 5). Data are expressed as mean ± SD.
dosing than that of i.p. administration. The bioavailability of Lx232c after i.p. dosing was 28.1%. Acknowledgments This work was supported by the National Science & Technology Major Project, China (2012ZX09103101-054, 2012ZX09301002001-007, and 2012ZX09301002-006). We thank Professor Weishuo Fang for providing Lx2-32c compound.
References [1] C. Ferlini, D. Gallo, G. Scambia, New taxanes in development, Expert Opin. Investig. Drugs 17 (2008) 335–347. [2] B.T. McGrogan, B. Gilmartin, D.N. Carney, A. McCann., Taxanes, microtubules and chemoresistant breast cancer, Biochim. Biophys. Acta 1785 (2008) 96–132. [3] A. Moreno-Aspitia, E.A. Perez, Anthracycline- and/or taxane-resistant breast cancer: results of a literature review to determine the clinical challenges and current treatment trends, Clin. Ther. 31 (2009) 1619–1640. [4] E. Galletti, M. Magnani, M.L. Renzulli, M. Botta, Paclitaxel and docetaxel resistance: molecular mechanisms and development of new generation taxanes, Chem. Med. Chem. 2 (2007) 920–942. [5] G.A. Orr, P. Verdier-Pinard, H. McDaid, S.B. Horwitz, Mechanisms of Taxol resistance related to microtubules, Oncogene 22 (2003) 7280–7295. [6] W.S. Fang, X.T. Liang, Recent progress in structure activity relationship and mechanistic studies of taxol analogues, Mini Rev. Med. Chem. 5 (2005) 1–12. [7] X. Xiao, J. Wu, C. Trigili, H. Chen, J.W. Chu, Y. Zhao, P. Lu, L. Sheng, Y. Li, F.J. Sharom, I. Barasoain, J.F. Diaz, W.S. Fang, Effects of C7 substitutions in a high affinity microtubule-binding taxane on antitumor activity and drug transport, Bioorg. Med. Chem. Lett. 21 (2011) 4852–4856. [8] H. Wang, H. Li, M. Zuo, Y. Zhang, H. Liu, W. Fang, X. Chen, Lx2-32c a novel taxane and its antitumor activities in vitro and in vivo, Cancer Lett. 268 (2008) 89–97. [9] Q. Zhou, Y. Li, J. Jin, L. Lang, Z. Zhu, W. Fang, X. Chen., Lx2-32c, a novel taxane derivative, exerts anti-resistance activity by initiating intrinsic apoptosis pathway in vitro and inhibits the growth of resistant tumor in vivo, Biol. Pharm. Bull. 35 (2012) 2170–2179. [10] P. Guo, J. Ma, S. Li, J.M. Gallo, Determination of paclitaxel in mouse plasma and brain tissue by liquid chromatography–mass spectrometry, J. Chromatogr. B 798 (2003) 79–86. [11] R.A. Parise, R.K. Ramanathan, W.C. Zamboni, M.J. Egorin, Sensitive liquidchromatography–mass spectrometry assay for quantitation of docetaxel and paclitaxel in human plasma, J. Chromatogr. B 783 (2003) 231–236. [12] A. Schellen, B. Ooms, M. van Gils, O. Halmingh, E. van der Vlies, D. van de Lagemaat, E. Verheij, High throughput on-line solid phase extraction/tandem mass spectrometric determination of paclitaxel in human serum, Rapid Commun. Mass Spectrom. 14 (2000) 230–233. [13] M.S. Alexander, M.M. Kiser, T. Culley, J.R. Kern, J.W. Dolan, J.D. McChesney, J. Zygmunt, S.J. Bannister, Measurement of paclitaxel in biological matrices: high-throughput liquid chromatographic–tandem mass spectrometric quantification of paclitaxel and metabolites in human and dog plasma, J. Chromatogr. B 785 (2003) 253–261. [14] G. Basileo, M. Breda, G. Fonte, R. Pisano, C.A. James, Quantitative determination of paclitaxel in human plasma using semi-automated liquid extraction in conjunction with liquid chromatography/tandem mass spectrometry, J. Pharm. Biomed. Anal. 32 (2003) 591–600. [15] E. Stokvis, M. Ouwehand, L.G. Nan, E.M. Kemper, O. van Tellingen, H. Rosing, J.H. Beijnen, Liquid chromatography–mass spectrometry for the quantitative bioanalysis of anticancer drugs, J. Mass Spectrom. 39 (2004) 1506–1512. [16] W. Zhang, G.E. Dutschman, X. Li, Y.C. Cheng, Quantitation of paclitaxel and its two major metabolites using a liquid chromatography–electrospray ionization tandem mass spectrometry, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 879 (2011) 2018–2022.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Quantitative determination of Lx2-32c, a novel taxane derivative, in rat plasma by liquid chromatography–tandem mass spectrometry Jinping Hu, Feng You, Shu Yang, Yan Li ∗ State Key Laboratory of Bioactive Substance and Function of Natural Medicines and Department of Drug Metabolism, Institute of Materia Medica, Chinese Academy of Medical Sciences & Perking Union Medical College, Beijing 100050, PR China
a r t i c l e
i n f o
Article history: Received 1 July 2013 Received in revised form 3 September 2013 Accepted 11 September 2013 Available online 5 October 2013 Keywords: Lx2-32c Taxane derivative LC–MS/MS Pharmacokinetic Bioavailability
a b s t r a c t A sensitive and reliable LC–MS/MS method for the determination of Lx2-32c, a novel taxane derived from cephalomannine, has been developed and validated. Plasma samples containing Lx2-32c and paclitaxel (internal standard) were prepared based on a simple protein precipitation by the addition of two volumes of acetonitrile. The analyte and internal standard were separated on a Zorbax SB-C18 column (3.5 m, 2.1 mm × 100 mm) with the mobile phase of acetonitrile/water containing 0.1% formic acid (v/v) with gradient elution at a flow rate of 0.2 ml/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with atmospheric pressure chemical ionization (APCI) by multiple reactions monitoring (MRM) of the transitions at m/z 887.5 → 264.3 for Lx2-32c and 854.5 → 286.2 for IS. Linear detection responses were obtained for Lx2-32c ranging from 1 to 1000 ng/ml. Inter- and intra-day precision (R.S.D.%) were all within 15% and the accuracy (R.E.%) was equal or lower than 8%. The lower limit of quantitation (LLOQ) was 1 ng/ml and the average recovery was greater than 91.5%. The method was successfully applied to the pharmacokinetic study of Lx2-32c in rat plasma. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Taxanes, a class anti-tumor drug isolated from the bark of Pacific Yew tree, have been widely employed for the cancer chemotherapy [1,2]. However, these agents suffer from poor drug solubility, toxicity and, in particular, drug resistance [3–5]. To overcome the above clinical problems, the structural of taxanes has been extensively modified to obtain the new molecules with a better therapeutic index, and the ability to overcome drug resistance [6]. Lx2-32c, a novel taxane derivative, is a semisynthetic analog from cephalomannine [7]. Our previous studies had shown that Lx2-32c had significant antitumor activity on BGC-823 (human gastric carcinoma) and A549 (human non-small cell lung carcinoma) xenograft in nude mice [8]. The further study found that Lx2-32c has the potent antiproliferative effect on several drug-resistant tumor cell lines such as Paclitaxel-resistant MX-1/T, A549/T, MCF7/T, BGC-823/T, vincristin-resistant KB/V and fluorouracil-resistant Bel-7402/5-Fu cells. Moreover, Lx2-32c also displayed robust anti-paclitaxel-resistance activity in nude mice bearing paclitaxelresistant MX-1/T tumors. Hence Lx2-32c is expected to overcome, at least in part, drug resistance in the clinical treatment [9]. Despite
∗ Corresponding author. Tel.: +86 10 6316 5172; fax: +86 10 6316 5172. E-mail addresses: [email protected], [email protected] (Y. Li). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.09.018
extensive researches in the pharmacological activities of Lx2-32c, little is known about its pharmacokinetics. Therefore, it is necessary to develop a reliable method for the pharmacokinetic study of Lx2-32c. To our knowledge there have been several LC–MS methods reported for the quantitative bioanalysis of paclitaxel. But most of them employed time-consuming solid-phase extraction (SPE) and liquid–liquid extraction (LLE) as a means of sample pretreatment to achieve low quantitation limits [10–16]. In the present study, a simple, sensitive, accurate and reproducible LC–MS/MS method was developed and validated for the determination Lx2-32c in rats following intravenous and intraperitoneal administration at 3 and 30 mg/kg.
2. Experimental 2.1. Chemicals and reagents Lx2-32c (purity >99.5%) were synthesized at Laboratory of Chemistry of Natural Products (Chinese Academy of Medical Sciences). Paclitaxel, 100% ethanol and polyoxyl 35 castor oil were kindly obtained from Beijing Union Pharmaceutical Factory. Acetonitrile was of HPLC grade (Fisher, USA). All other chemicals were of analytical reagent grade. Ultrapure water, prepared by a Milli-Q Reagent water system (Millipore, MA, USA), was used throughout the study.
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2.2. Preparation of stocks, calibration standards and quality control samples Standard stock solutions of Lx2-32c and paclitaxel (internal standard) were prepared in methanol at 1 mg/ml. Then the stock solutions were diluted with methanol to obtain fresh standard working solution. Calibrations standards were prepared by adding corresponding working solutions in drug-free plasma. The final concentrations of Lx2-32c in plasma were 1, 2.5, 10, 50, 200, 500, 750, 1000 ng/ml, and IS was 1000 ng/ml, respectively. Low-, mid- and high-level quality control samples containing 2.5, 200 and 800 ng/ml of Lx2-32c, were prepared in a manner similar to that used for the preparation of calibration samples. All stock solutions, working solutions, calibration standards and quality controls were stored at −20 ◦ C and were brought to room temperature before analysis. 2.3. Sample processing A 20 l of IS working solution (1 g/ml) and 180 l acetonitrile were added to 100 l of plasma sample. The mixture was vortexed for 30 s followed by centrifugation at 14,000 rpm for 5 min at room temperature. A 5 l aliquot of each supernatant was injected into the LC/MS/MS system for the analysis. 2.4. Instrumental analysis Chromatographic separations were carried out on an Agilent 1260 HPLC system using a Zorbax SB-C18 column (3.5 m, 2.1 mm × 100 mm, Agilent, USA). The column temperature was set to 25 ◦ C. The mobile phase consisted of deionized water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B). Separation was performed at a flow-rate of 0.2 ml/min with the following gradient elution: from 0 to 1 min, 35% B; linear increase to 95% B in 0.2 min; 95% B during 3 min, decrease to 35% B in 0.2 min; and stabilization at initial conditions for 8 min. The total run time was 12 min. The sample volume injected was 5 l. The HPLC instrument was coupled to API 4000 triple quadruple mass spectrometer from Applied Biosystems Sciex (Toronto, Canada) with atmospheric pressure chemical ionization (APCI) ion source in the positive mode. The detection was operated in the multiple reaction monitoring (MRM) mode with a dwell time of 200 ms for each transition. The MRM transitions of Lx2-32c and its internal standard (IS) were 887.5 → 264.3 and 854.5 → 286.2, respectively. The mass spectrometric condition was optimized as follows: collision gas, 6 psi; curtain gas, 15 psi; ion source gas (GS1), 30 psi; nebulizer current, 3.0 A; temperature, 300 ◦ C. Declustering potential (DP), collision energy (CE) and collision cell exit potential (CXP) was 61, 21 and 10 V for Lx2-32c, and 80, 27 and 8 V for IS, respectively. Data acquisition and processing were performed using Analyst software (version 1.5.2). 2.5. Method validation 2.5.1. Selectivity Six different blank rat plasma samples were analyzed to detect the potential interferences co-eluting with the analyte and IS. Chromatographic peaks of analyte and IS were identified on the basis of their retention times and MRM responses. 2.5.2. Linearity, precision and accuracy The linearity of LC/MS/MS method for the determination of Lx2-32c was evaluated by a calibration curve in the range of 1–1000 ng/ml. The calibration curve was obtained by plotting the
ratio of chromatographic peaks area (Lx2-32c/IS) versus the concentration of Lx2-32c. Least squares linear regression analysis was used to determine the slope, intercept and correlation coefficient. The calibration curve requires a correlation coefficient (r2 ) of 0.99 or better. To evaluate the precision, at least five QC samples of three different concentrations of Lx2-32c were processed and injected on a single day (intra-day) and at different days (inter-day). The variability of Lx2-32c determination was expressed as the relative standard deviation (R.S.D.%) which should not exceed 15% at all concentrations. Accuracy is expressed as relative error (R.E.%) which should be within the limits of ±15% at all concentrations of Lx2-32c. 2.5.3. Matrix effect and recovery The matrix effect was defined as the ion suppression/enhancement on the ionization of analyte, which was evaluated by comparing the responses of deproteinized samples of blank plasma from six rats spiked Lx2-32c samples (n = 5) with those of standard samples at equivalent concentrations. The extraction recovery was determined at three Lx2-32c levels and calculated by comparing the analyte standard peak areas obtained from extracted samples with post-extracted samples spiked with the analyte. 2.5.4. Stability studies Three freeze–thaws, long-term, short-term and post-extracted stabilities of Lx2-32c in plasma was tested using high-, midand low-quality control samples. The freeze–thaw stability of the analyte was determined over three freeze–thaw cycles. In each freeze–thaw cycle, the samples were frozen and stored at −20 ◦ C for 24 h, then thawed at ambient temperature. To evaluate longterm stability of Lx2-32c, the plasma samples were stored at −20 ◦ C for 30 days. For the short-term stability, fresh plasma samples were kept at room temperature for 24 h before treatment. The stability of treated plasma in auto-sampler was tested after keeping the samples at 15 ◦ C for 24 h. Dilution stability was assessed using a spiked sample the concentration of which was higher than the upper limit of quantification samples. The result was obtained by comparing the back-calculated value of the sample after being diluted 5 times with the nominal value. 2.6. Pharmacokinetic experiments in rats The validated assay was used to determine the plasma pharmacokinetic of Lx2-32c in rats after a single intraperitoneal (i.p.) or intravenous (i.v.) injection. All animal protocols were approved by Institute Animal Care and Welfare Committee. Sprague–Dawley rats (adult male), weighing 200–220 g, were obtained from Beijing Vital River Experimental Animal Co., Ltd. The rats were quarantined for 1 week prior to the study and maintained on a 12 h light/12 h dark cycle at 22 ± ◦ C and at 60% relative humidity. Before the experiment animals were of free access to water and food. The dosing solutions used for the animal studies were prepared by dissolving the required amounts of Lx2-32c in 100% ethanol, followed by mixing with polyoxyl 35 castor oil to yield a 12.5 mg/ml stock (50% ethanol:50% polyoxyl 35 castor oil) that were diluted in saline immediately before administration. After i.p. or i.v. injection of Lx2-32c at a dose of 30 or 3 mg/kg to rats, approximately 0.2 ml blood samples were collected in heparinized 1.5 ml polythene tubes by orbital bleeding via capillary tubes at 0.03, 0.08, 0.17, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h postdose. The blood samples were immediately centrifuged at 5000 rpm for 10 min. The plasma was separated and frozen at −20 ◦ C until analysis. The plasma concentrations of Lx2-32c were expressed as mean ± S.D. and the mean concentration–time curves were plotted. Data fitting and
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Fig. 1. Product ion mass spectra of [M+H]+ of (A) Lx2-32c, (B) paclitaxel (IS).
Table 1 Intra- and inter-day accuracy and precision of Lx2-32c in rat plasma. Concentrations (ng/ml)
Intra-day
Inter-day
Mean ± S.D. (n = 5) 1.00 2.50 200.00 800.00
1.04 2.55 197.61 809.02
± ± ± ±
0.09 0.21 3.22 24.53
Accuracy (R.E.%)
Precision (R.S.D.%)
Mean ± S.D. (n = 5)
4.0 2.0 -1.2 1.1
8.7 8.2 1.6 3.0
1.06 2.58 196.73 815.54
± ± ± ±
0.12 0.28 9.85 46.48
Accuracy (R.E.%)
Precision (R.S.D.%)
6.0 3.2 -1.6 1.9
11.3 10.9 5.0 5.7
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Fig. 2. Typical MRM chromatograms of Lx2-32c and IS: (A) blank rat plasma; (B) blank plasma spiked with Lx2-32c (LLOQ of 1 ng/ml) and IS; and (C) rat plasma sample at 24 h after i.p. administration of Lx2-32c at 30 mg/kg spiked with IS. The retention time of Lx2-32c and IS was 8.17 and 7.19 min, respectively.
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Table 2 Stability of Lx2-32c in rat plasma (n = 5). Stability tests
Theoretical cone (ng/ml)
Found cone (ng/ml)
R.E.%
R.S.D%
2.50 200.00 800.00
2.65 ± 0.13 200.72 ± 2.92 834.75 ± 15.02
6.0 0.4 4.3
4.9 1.5 1.8
2.50 200.00 800.00
2.38 ± 0.23 195.75 ± 1.58 794.08 ± 9.15
−4.8 −2.1 −0.7
9.7 0.8 1.2
2.50 200.00 800.00
2.59 ± 0.24 205.76 ± 13.54 847.08 ± 45.36
3.6 2.9 5.9
9.3 6.6 5.4
2.50 200.00 800.00
2.69 ± 0.29 208.36 ± 7.16 839.89 ± 27.83
7.6 4.2 5.0
10.8 3.4 3.3
−6.1
4.9
Room temperature (24 h)
Post-treated (15 ◦ C for 24 h)
Three freeze–thaw cycles
Stored at −20 ◦ C (30 day)
Dilution stability (dilution factor: 5) 4000.00
pharmacokinetic parameter estimates were carried out using WinNonLin Software (Version 6.1, Pharsight Corporation). 3. Results and discussion 3.1. Method development To get appropriate retention time, better resolution and sensitivity, the different HPLC parameters including mobile phase, category of column and flow rate of mobile phase were tested and compared. Finally, the mobile phase of acetonitrile/water containing 0.1% formic acid was found to be suitable for the formation of molecular ions of Lx2-32c and IS. Zorbax SB-C18 column (3.5 m, 2.1 mm × 100 mm) was selected for the chromatographic separation. The system provided high resolution, great baseline stability and high ionization efficiency. By investigating the full-scan mass spectra of Lx2-32c and IS, we found that the signal intensity in the positive ion mode was much higher than that in the negative ion mode. During a direct infusion experiment, the mass spectra for Lx2-32c and IS revealed peaks at m/z 887.5 and 854.5, respectively, as protonated molecular ion [M+H]+ . As shown in Fig. 1, the most abundant and stable productions were at m/z 264.3 for Lx2-32c, and at m/z 286.2 for IS, after fragmentation in the collision cell. The most suitable mass spectrometric conditions were determined by optimizing all the parameters of mass spectrometer such as collision gas, curtain gas, ion source gas (GS1), nebulizer current, temperature, declustering potential (DP), collision energy (CE) and collision cell exit potential (CXP) to obtain high and stable signal. 3.2. Method validation 3.2.1. Selectivity Representative MRM chromatograms of blank plasma samples, plasma sample spiked with drugs and a real rat plasma sample were obtained under the selected analytical conditions. The result showed the absence of any interference at the retention time of the analyte and IS. The retention times of Lx2-32c and IS are 8.17 and 7.19 min, respectively (Fig. 2). 3.2.2. Calibration curve and lower limit of quantification The calibration curves generated from the detection of rat plasma containing known amounts of Lx2-32c were linear over the concentration range tested (1–1000 ng/ml). The
3756.72 ± 182.45
regression equations obtained by least squared regression were y = 0.0193x + 0.00634 (n = 5), where y is the peak area ratios of analyte to IS, and x is the concentrations of analyte. The correlation coefficients (r2 ) were ≥0.99 for all calibration curves. The lower limit of quantification of Lx2-32c in rat plasma was found to be 1 ng/ml, which are sufficient for the pharmacokinetics study. 3.2.3. Precision and accuracy The intra- and inter-assay variations were found to be within the accepted limits. The intra-and inter-day precision of Lx2-32c at three QC concentrations, as presented in Table 1 were less than 11.3%. Assay accuracy was found to be within ±8.0%. The results indicated that the present method was reliable and reproducible for the quantitative determination of Lx2-32c. 3.2.4. Matrix effect and recovery The matrix effect values were 107.2%, 103.6% and 101.0% for Lx232c at low, medium and high concentration levels, respectively. These results suggested that the matrix effect on the ionization of Lx2-32c was not obvious under our experimental conditions. The recoveries in rat plasma were 91.5–101.1% for Lx2-32c at three concentration levels. 3.2.5. Stability The stability of Lx2-32c in rat plasma is summarized in Table 2. Lx2-32c in rat plasma was found to be stable after being placed at room temperature for 24 h, stored at −20 ◦ C for 30 days or through three freeze–thaw cycles. Furthermore, samples after treatment were stable at 15 ◦ C in auto-sampler for a period of 24 h, which indicated that a large number of samples could be determined in each analytical run. 3.3. Pharmacokinetic study The present method was successfully applied to the pharmacokinetic studies of Lx2-32c in rats after i.p. or i.v. injection. A plot of plasma concentration versus time for Lx2-32c in rats is presented in Fig. 3. The corresponding pharmacokinetic parameters calculated using non-compartmental analysis are listed as mean ± SD in Table 3. The plasma peak of Lx2-32c reached at 2 h after a single i.p. dose at 30 mg/kg, and the values of Cmax and AUC0−t were 0.6 g/ml and 5.4(g/ml) h, respectively. The AUC0−t of Lx2-32c after an i.v. dosing of 3 mg/kg was 1.6(g/ml) h. Lx2-32c displayed much smaller volume of distribution and lower clearance after i.v.
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Table 3 Mean pharmacokinetic parameters of Lx2-32c in rats following a single i.p. dose of 30 mg/kg and i.v. dose of 3 mg/kg of Lx2-32c (n = 5). Parameter
AUC(0−t) (mg/l h) MRT(0−t) (h) tl/2z (h) Tmax (h) CLz/F (l/h kg) Vz/F (l/kg) Cmax (mg/l) F (%)
Mean ± SD i.p. 30 mg/kg
i.v. 3 mg/kg
4.5 ± 2.4 7.3 ± 2.0 5.9 ± 2.5 2.6 ± 2.4 9.4 ± 8.9 85.5 ± 94.3 0.6 ± 0.4 28.1
1.6 5.1 10.9 – 1.7 24.8 3.3
± 0.2 ± 0.3 ± 6.0 ± 0.4 ± 10.4 ± 0.4
Fig. 3. Plasma concentration–time profile of Lx2-32c in rats following a single i.p. dose of 30 mg/kg and i.v. dose of 3 mg/kg of Lx2-32c (n = 5). Data are expressed as mean ± SD.
dosing than that of i.p. administration. The bioavailability of Lx232c after i.p. dosing was 28.1%. Acknowledgments This work was supported by the National Science & Technology Major Project, China (2012ZX09103101-054, 2012ZX09301002001-007, and 2012ZX09301002-006). We thank Professor Weishuo Fang for providing Lx2-32c compound.
References [1] C. Ferlini, D. Gallo, G. Scambia, New taxanes in development, Expert Opin. Investig. Drugs 17 (2008) 335–347. [2] B.T. McGrogan, B. Gilmartin, D.N. Carney, A. McCann., Taxanes, microtubules and chemoresistant breast cancer, Biochim. Biophys. Acta 1785 (2008) 96–132. [3] A. Moreno-Aspitia, E.A. Perez, Anthracycline- and/or taxane-resistant breast cancer: results of a literature review to determine the clinical challenges and current treatment trends, Clin. Ther. 31 (2009) 1619–1640. [4] E. Galletti, M. Magnani, M.L. Renzulli, M. Botta, Paclitaxel and docetaxel resistance: molecular mechanisms and development of new generation taxanes, Chem. Med. Chem. 2 (2007) 920–942. [5] G.A. Orr, P. Verdier-Pinard, H. McDaid, S.B. Horwitz, Mechanisms of Taxol resistance related to microtubules, Oncogene 22 (2003) 7280–7295. [6] W.S. Fang, X.T. Liang, Recent progress in structure activity relationship and mechanistic studies of taxol analogues, Mini Rev. Med. Chem. 5 (2005) 1–12. [7] X. Xiao, J. Wu, C. Trigili, H. Chen, J.W. Chu, Y. Zhao, P. Lu, L. Sheng, Y. Li, F.J. Sharom, I. Barasoain, J.F. Diaz, W.S. Fang, Effects of C7 substitutions in a high affinity microtubule-binding taxane on antitumor activity and drug transport, Bioorg. Med. Chem. Lett. 21 (2011) 4852–4856. [8] H. Wang, H. Li, M. Zuo, Y. Zhang, H. Liu, W. Fang, X. Chen, Lx2-32c a novel taxane and its antitumor activities in vitro and in vivo, Cancer Lett. 268 (2008) 89–97. [9] Q. Zhou, Y. Li, J. Jin, L. Lang, Z. Zhu, W. Fang, X. Chen., Lx2-32c, a novel taxane derivative, exerts anti-resistance activity by initiating intrinsic apoptosis pathway in vitro and inhibits the growth of resistant tumor in vivo, Biol. Pharm. Bull. 35 (2012) 2170–2179. [10] P. Guo, J. Ma, S. Li, J.M. Gallo, Determination of paclitaxel in mouse plasma and brain tissue by liquid chromatography–mass spectrometry, J. Chromatogr. B 798 (2003) 79–86. [11] R.A. Parise, R.K. Ramanathan, W.C. Zamboni, M.J. Egorin, Sensitive liquidchromatography–mass spectrometry assay for quantitation of docetaxel and paclitaxel in human plasma, J. Chromatogr. B 783 (2003) 231–236. [12] A. Schellen, B. Ooms, M. van Gils, O. Halmingh, E. van der Vlies, D. van de Lagemaat, E. Verheij, High throughput on-line solid phase extraction/tandem mass spectrometric determination of paclitaxel in human serum, Rapid Commun. Mass Spectrom. 14 (2000) 230–233. [13] M.S. Alexander, M.M. Kiser, T. Culley, J.R. Kern, J.W. Dolan, J.D. McChesney, J. Zygmunt, S.J. Bannister, Measurement of paclitaxel in biological matrices: high-throughput liquid chromatographic–tandem mass spectrometric quantification of paclitaxel and metabolites in human and dog plasma, J. Chromatogr. B 785 (2003) 253–261. [14] G. Basileo, M. Breda, G. Fonte, R. Pisano, C.A. James, Quantitative determination of paclitaxel in human plasma using semi-automated liquid extraction in conjunction with liquid chromatography/tandem mass spectrometry, J. Pharm. Biomed. Anal. 32 (2003) 591–600. [15] E. Stokvis, M. Ouwehand, L.G. Nan, E.M. Kemper, O. van Tellingen, H. Rosing, J.H. Beijnen, Liquid chromatography–mass spectrometry for the quantitative bioanalysis of anticancer drugs, J. Mass Spectrom. 39 (2004) 1506–1512. [16] W. Zhang, G.E. Dutschman, X. Li, Y.C. Cheng, Quantitation of paclitaxel and its two major metabolites using a liquid chromatography–electrospray ionization tandem mass spectrometry, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 879 (2011) 2018–2022.
