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Year : 2014  |  Volume : 10  |  Issue : 1  |  Page : 84-88

Validation of a high-performance liquid chromatographic ultraviolet detection method for the quantification of vandetanib in rat plasma and its application to pharmacokinetic studies

1 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China
2 Department of Oncology, Chengdu Fifth People's Hospital, Chengdu 611130, China

Date of Web Publication23-Apr-2014

Correspondence Address:
Yinglan Zhao
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.131393

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 > Abstract 

Aim: To develop a simple and sensitive high-performance liquid chromatography (HPLC) assay with ultraviolet detection method of vandetanib in rat plasma.
Materials and Methods: Samples were extracted with methanol and acetonitrile, evaporated, and then the residue was reconstituted in mobile phase. Vandetanib and the internal standard (I.S.) trazodone hydrochloride were separated with gradient elution (on a C18 Atlantis® column using a mobile phase of acetonitrile/0.5% triethylamine, pH 3.0, with a flow rate of 1.0 ml/min), then detected at 341 nm.
Results: A linear curve over the concentration range of 80-4000 ng/ml (R 2 = 0.9998) was obtained. Intra- and inter-assay accuracy ranged from 98.80% to 103.08% and 95.32% to 98.40%, with high precision (R.S.D. % <5%), respectively. The mean absolute recovery was 96.65%.
Conclusion: A simple and sensitive HPLC assay with ultraviolet detection method was developed for the determination of vandetanib in rat plasma. This method is sufficient for pharmacokinetic studies of vandetanib in small animals and may be applied to human pharmacokinetic studies.

 > Abstract in Chinese 

材料和方法:用甲醇和乙腈提取样本、蒸馏,残留物再构成移动相。凡德他尼和内部标准的曲唑酮氢氯化物被梯度洗提分离(在C18亚特兰蒂斯®列使用乙腈/0.5%三乙胺移动相, pH 3.0,流动率 1.0ml/min),检测到341nm。

Keywords: High-performance liquid chromatography-ultraviolet detection, pharmacokinetic, vandetanib

How to cite this article:
Lin H, Cui D, Cao Z, Bu Q, Xu Y, Zhao Y. Validation of a high-performance liquid chromatographic ultraviolet detection method for the quantification of vandetanib in rat plasma and its application to pharmacokinetic studies. J Can Res Ther 2014;10:84-8

How to cite this URL:
Lin H, Cui D, Cao Z, Bu Q, Xu Y, Zhao Y. Validation of a high-performance liquid chromatographic ultraviolet detection method for the quantification of vandetanib in rat plasma and its application to pharmacokinetic studies. J Can Res Ther [serial online] 2014 [cited 2021 Jul 28];10:84-8. Available from: https://www.cancerjournal.net/text.asp?2014/10/1/84/131393

Hongjun Lin, Dandan Cui.
This authors contributed equally to this work

 > Introduction Top

Vandetanib (ZD6474) (N-(4-bromo-2- fluorophenyl)- )6-methoxy-7- [(1-methyl-4-pipe ridinyl) methoxy]- 4-quinazolinamine) is a recently identified small molecule inhibitor, which shows antitumor efficacy by inhibiting tumor cell proliferation and survival via epidermal growth factor receptor (EGFR) and RET inhibition, as well as inhibiting tumor angiogenesis via vascular EGFR-2 (VEGFR-2) inhibition. [1],[2] Its preclinical and clinical activity against several tumor types including advanced and metastatic papillary thyroid cancer, non-small cell lung cancer (NSCLC), and advanced colorectal cancer (CRC) either in monotherapy or in combination with other anti-cancer agent as first or second-line therapy has been demonstrated. [3],[4]

The pharmacokinetic study of vandetanib in humans has been performed in both phase I dose escalation studies [5] and in phase II trials at doses of 100 and 300 mg/day. [6] The results from these studies have shown that vandetanib is slowly absorbed (in relatively small Ka), widely distributed (large volume of distribution), and is slowly eliminated with an elimination half-life of approximately 120 h and a minimum of 28 days of continuous dosing being required to achieve a steady-state plasma concentration. Recently, longer half-life (approaching 200 h) of vandetanib was reported in pharmacokinetic studies in humans. [5],[6] Because of these properties, there is a large intersubject variation in peak concentration and in area under the curve (AUC) after the same oral dose of this drug. [7] Hence, a specific, sensitive, and accurate analytical method for the determination of vandetanib in human plasma was considered mandatory.

To our knowledge, only one liquid chromatography with tandem mass spectrometry (LC-MS/MS) method has been reported for the determination of vandetanib concentration in mouse plasma. [8] In the present study, we developed a novel analytical method based on HPLC with UV detection for the determination of vandetanib in rat plasma samples.

 > Materials and Methods Top

Vandetanib (purity, 98% by HPLC) and trazodone hydrochloride (internal standard [I.S], purity, 95% by HPLC) [Figure 1] were purchased from Yingxuan Chempharm Co. (Shanghai, China). HPLC grade acetonitrile and methanol were obtained from Fisher Scientific (Fairlawn, NJ, USA). All other chemicals and solvents were of analytical grade or HPLC grade and were commercially available. Water was purified and deionized by the Milli-Q-UF system (Millipore, Milford, MA, USA) and used throughout.
Figure 1: Structure of vandetanib and trazodone hydrochloride

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Vandetanib and trazodone hydrochloride stock solutions were prepared by exact weighing of the respective compounds and dissolving them in methanol and water to the concentration of 500 μg/ml and 1 mg/ml, respectively. Both stock solutions were stored at − 20°C and prepared freshly every month.

With each analytical run, calibration standards in methanol were prepared freshly by step-wise dilution of the above solution with methanol to obtain final concentrations of 80, 100, 200, 400, 800, 2000, and 4000 ng/ml. A 60 μl volume of each prepared sample was transferred to a polypropylene tube (1.5 ml) and then dried under a constant stream of nitrogen at 45°C. Next, 60 μl volume of rat plasma was added to the tubes to reconstitute the residue and vandetanib working standard solutions selected for the calibration curve at concentration of 80, 100, 200, 400, 800, 2000, and 4000 ng/ml were obtained. The I.S solution was prepared from the stock by dilution with water to 20 μg/ml. Pools of quality control (QC) samples of vandetanib in methanol were prepared similarly in polypropylene tubes at concentrations of 200, 1000, and 2000 ng/ml and then stored in batch at − 20°C for the duration of the validation procedure.

A 60 μl volume of each concentrations of vandetanib in plasma (described in section 2.2) was transferred to a polypropylene tube (1.5 ml) and then 40 μl trazodone hydrochloride was added. After vortex-mixing for 10 s, 300 μl methanol followed by 300 μl acetonitrile was added to precipitate protein, followed by another round of vortex-mixing for another 20 s. Subsequently, samples were centrifuged at 18,000 × g for 10 min. The supernatant was collected and evaporated to dryness as before. The residue was reconstituted in 60 μl mobile phase.

The analysis of calibration standards, QCs, and samples were performed on a Waters 2695 alliance system and a Waters 2996 PDA detector (Milford, MA, USA). Data acquisition and analysis was performed using the Empower 2 software package. The analyte was carried out on a reversed phase C18 column (Atlantis® C18, 150 mm × 4.6 mm, 5 μm), followed by specific measurement at wavelength of 341 nm. A 15-min linear gradient started at 15% eluent A (100% acetonitrile) and 85% eluent B (0.5% triethylamine, adjusted to pH 3 by phosphoric acid) to 60% eluent A and 40% eluent B. The temperature of the injector and column were maintained at 10°C and 25°C, respectively. The injection volume was 20 μl. The column was equilibrated for 10 min with the initial mobile phase at a flow rate of 1.0 ml/min before the next injection.

Male Sprague-Dawley rats (260-280 g body weight) were obtained from Animal Center of Si Chuan University (Cheng Du, China). The rats were housed under controlled environmental conditions (temperature of 23 ± 1°C and humidity of 55 ± 5%) with a commercial food diet and water freely available. All animal experiments were carried out in accordance with the guidelines of Sichuan Province Experimental Animal Management Committee and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

For oral pharmacokinetic study, rats under light anesthesia with sodium pentobarbital (25 mg/kg) were cannulated with polyethylene tube into the jugular vein for blood collection. After abrosia for 12 h, rats were orally administrated with vandetanib, which was suspended in 1% Tween 80 at a dose of 25 mg/kg. [9] Blood samples (200 μl) were collected in a polypropylene tube (1.5 ml) containing heparin as an anticoagulant at appropriate intervals after the administration of vandetanib 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 h. Blood samples were centrifuged at 3,000 g for 15 min at 4°C to yield plasma samples and plasma samples were stored at − 20°C until analysis. Samples were analyzed as described above.

 > Results Top

The method was validated based on the principles provided by the "Guidance for Industry" under the section of "Bioanalytical Method Validation" by Food and Drug Administration of USA. [10]

Representative chromatograms of an extract of blank rat plasma (A), an extract of a plasma sample spiked with vandetanib and trazodone hydrochloride at a concentration of 800 ng/ml and 8 μg/ml (B) are displayed in [Figure 2]. An excellent chromatographic specificity was observed with the sharp and symmetrical resolution of the peaks, with no significant interfering peaks for all compounds in blank rat plasma samples. Under the chromatographic conditions used in the present study, the retention times for vandetanib and I.S trazodone hydrochloride were 7.7 and 8.5 min, respectively. The total chromatography run time was 15 min. Our HPLC assay was found to be selective and free from other possible interferences.
Figure 2: Plasma log concentration vs. time curve of vandetanib after single oral dose of 25 mg/kg in SD rats. Data are expressed as the mean ± SD (n = 3)

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To evaluate the linearity of the calibration curves, six calibration standards containing vandetanib at the concentrations of 80-4000 ng/ml were prepared as described above. The peak area ratios of vandetanib versus I.S. were measured and plotted against the concentration of vandetanib. The slope, intercept, and correlation coefficient were conducted from these peak area ratios. Calibration curves were linear over the concentration range used with a mean r2 of 0.9998. The normalized intercept/slope of the regression line and the coefficient of correlation were calculated for the whole data set. Representative results are displayed in [Table 1]. The limit of quantification (LOQ), defined in the presented experiment as the lowest plasma concentration in the calibration curve that can be measured routinely with acceptable precision (R.S.D. >20%) and accuracy (80-120%), was 80 ng/ml [Table 2].
Table 1: Linearity data for vandetanib, calibration standard response values for a calibration curve range of 80-4000 ng/ml (S.D.: Standard deviation of the mean)

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Table 2: The limit of quantitation of the method for determination of vandetanib in rat plasma samples. (S.D.: Standard deviation, R.S.D.: Relative standard deviation)

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The precision and accuracy of the method was assessed by determining the coefficients of variation of the 3 QC samples (concentrations of 200, 1000, and 2000 ng/ml) within the same analysis (n = 5, intra-day precision) and over a series of analyses (n = 6, inter-day precision). Both intra-day and inter-day precisions were calculated with the following formula: RSD (%) = (standard deviation)/(mean) ×100. The accuracy was calculated with the following formula: Accuracy (%) = (mean measured concentration)/(nominal concentration) ×100. As shown in [Table 3], the intra-day and inter-day accuracy were 95.32-103.08% for vandetanib at different concentrations, and the precision was estimated to be within 5%.
Table 3: Precision and accuracy of the method for determination of vandetanib in rat plasma samples. (S.D.: Standard deviation, R.S.D.: Relative standard deviation)

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To determine extraction efficiency, plasma samples were spiked with vandetanib to achieve a final concentration of 200, 1000, and 2000 ng/ml. Five samples were extracted and analyzed for each concentration. Extraction efficiency was calculated with the following equation: Extraction efficiency (%) = (peak area extracted sample for × ng/ml/peak area neat sample for × ng/ml) ×100. The absolute recoveries of vandetanib from rat plasma ranged from 94.35 to 97.99% and %RSD of recovery was below 15% for each concentration [Table 4].
Table 4: Absolute recovery of the method for determining the concentration of vandetanib in rat plasma samples (n=5). (S.D.: Standard deviation, R.S.D.: Relative standard deviation)

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To prove that the developed analytical method is applicable to plasma samples of rats treated with vandetanib, the pharmacokinetic study was performed in rats. According to preclinical research, at least 25 mg/kg/once daily vandetanib provide statistically significant antitumor activity; [11] therefore, the dose of 25 mg/kg was chosen for in vivo pharmacokinetic study. The log concentration vs. time curve of vandetanib through p.o. route is shown in [Figure 3]. The curve represents a composite picture of absorption through the stomach and intestine superimposed on to the distribution and finally the log-linear metabolism/elimination phase. The peak concentration of vandetanib was 446 ng/ml and was 135 ng/ml at the point of 72 h. This result was supported by previous study [9] that stated the lowest concentration as above 100 ng/ml. The long half-life in plasma (54 h) is consistent with mice and phase I data in humans that has shown a terminal half-life approaching 27 h and 100 h for vandetanib, respectively. [5],[9] [Table 5] lists the mean pharmacokinetic parameters. From above results, we can draw the conclusion that the LOQ of 80 ng/ml was enough for pharmacokinetic studies and that this analytical method is suitable for further studies.
Figure 3: HPLC chromatograms of blank rat plasma at 341 nm (A) and rat plasma with 800 ng/ml vandetanib and 8 ìg/ml trazodone hydrochloride. The retention times for vandetanib and trazodone hydrochloride were 7.7 and 8.5 min, respectively

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Table 5: Pharmacokinetic parameters of vandetanib in SD rats following single oral dose of 25 mg/kg

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 > Discussion Top

In the present study, trazodone hydrochloride was selected as I.S at a concentration of 8 μg/ml during the validation procedure, as reported previously. [8] To optimize vandetanib extraction, sample preparation of vandetanib in plasma was initially performed by a one-step solvent extraction with a specific solvent extraction technique involving pentane and ethyl acetate. However, this simple extraction resulted in a major interfering peak around the retention time of the I.S. In order to avoid this, we tried other organic solvent such as acetoacetate and methyl tertiary butyl ether (MTBE). But these methods were aborted neither for its low extraction efficiency nor for bad precision. Eventually, vandetanib and the I.S were isolated with a single step extraction involving a mixture of acetonitrile and methanol, which produced satisfactory recovery, precision, and clean chromatogram.

In order to ensure sufficient selectivity and analyte separation, we have also slightly modified the mobile phase composition as compared to an earlier method [12] by increasing the organic modifier content from 15% to 60%. In fact, low contents of acetonitrile or the use of methanol in the mobile phase resulted in poor accuracy and precision because of severe front-tailing bands (asymmetry factor ≥ 2.0). To increase the accuracy and precision, a linear gradient was used in this study that started at 15% eluent A (100% acetonitrile) and 85% eluent B (0.5% triethylamine, pH 3.0) to 60% eluent A and 40% eluent B. This mobile phase for the separation of vandetanib showed optimum peak shape and the detector sensitivity was increased as compared with that of isocratic elution. Blank blood showed no peaks of interfering endogenous compounds around the retention time of vandetanib and I.S. In addition, the retention time of vandetanib was short enough so that it is suitable for routine analysis.

 > Conclusion Top

In conclusion, the method presented for the determination of vandetanib in small animal plasma is specific, accurate, and precise, and it is selective enough to be used in preclinical trials without any extrapolation. Thus, this method is applicable for pharmacokinetic monitoring of vandetanib in small animals, and it may as well be applied to extensive human pharmacokinetic studies.

 > Acknowledgments Top

The authors thank Dr. Minghai Tang and Dr. Xianhuo Wang (State Key Laboratory of Biotherapy and Cancer Center, West China hospital, Sichuan University) for their excellent technical assistance during this experiment.

 > References Top

1.Hennequin LF, Sstokes ES, Thomas AP, Johnstone C, Pié PA, Ogilvie DJ, et al. Novel 4-anilinoquinazolines with C-7 basic side chains: Design and structure activity relationship of a series of potent, orally active, VEGF receptor tyrosine kinase inhibitors. J Med Chem 2002;45:1300-12.  Back to cited text no. 1
2.Carlomagno F, Vitagliano D, Guida T, Ciardiello F, Tortora G, Vecchio G, et al. ZD6474, an orally available inhibitor of KDR tyrosine kinase activity, efficiently blocks oncogenic RET kinases. Cancer Res 2002;62:7284-90.  Back to cited text no. 2
3.Morabito A, Piccirillo MC, Falasconi F, De Fio G, Del Giudice A, Bryce J, et al. Vandetanib (ZD6474), a dual inhibitor of vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) tyrosine kinases: Current status and future directions. Oncologist 2009;14:378-90.  Back to cited text no. 3
4.Michael M, Gibbs P, Smith R, Godwood A, Oliver S, Tebbutt N. Open-label phase I trial of vandetanib in combination with mFOLFOX6 in patients with advanced colorectal cancer. Invest New Drugs 2009;27:253-61.  Back to cited text no. 4
5.Holden SN, Eckhardt SG, Basser R, de Boer R, Rischin D, Green M. Clinical evaluation of ZD6474, an orally active inhibitor of VEGF and EGF receptor signaling, in patients with solid, malignant tumors. Ann Oncol 2005;16:1391-7.  Back to cited text no. 5
6.Miller KD, Trigo JM, Wheeler C, Bargr A, Rowbottom J, Sledge G, et al. A multicenter phase II trial of ZD6474, a vascular endothelial growth factor receptor-2 and epidermal growth factor receptor tyrosine kinase inhibitor, in patients with previously treated metastatic breast cancer. Clin Cancer Res 2005;11:3369-76.  Back to cited text no. 6
7.Tamura T, Minami H, Yamada Y, Yamamoto N, Shimoyama T, Murakami H, et al. A phase I dose-escalation study of ZD6474 in Japanese patients with solid, malignant tumors. J Thorac Oncol 2006;1:1002-9.  Back to cited text no. 7
8.Zirrolli JA, Bradshaw EL, Long ME, Gustafson DL. Rapid and sensitive LC/MS/MS analysis of the novel tyrosine kinase inhibitor ZD6474 in mouse plasma and tissues. J Pharm Biomed Anal 2005;39:705-11.  Back to cited text no. 8
9.Gustafson DL, Bradshaw-Pierce EL, Merz AL, Zirrolli JA. Tissue distribution and metabolism of the tyrosine kinase inhibitor ZD6474 (Zactima) in tumor-bearing nude mice following oral dosing. J Pharmacol Exp Ther 2006;318:872-80.  Back to cited text no. 9
10.Center for Drug Evaluation and Research (FDA), Guidance for industry: Bioanalytical Method Validation. Available from: http://www.fda.gov/cder/guidance/4252fnl.pdf 2001.  Back to cited text no. 10
11.Wedge SR, Ogilvie DJ, Dukes M, Kendrew J, Chester R, Jackson JA, et al. ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration. Cancer Res 2002;62:4645-55.  Back to cited text no. 11
12.Azzariti A, Porcelli L, Xu JM, Simone GM, Paradiso A. Prolonged exposure of coloncancer cells to the epidermal growth factor receptor inhibitor gefitinib (Iressa (TM)) and to the antiangiogenic agent ZD6474: Cytotoxic and biomolecular effects. World J Gastroenterol 2006;12:5140-7.  Back to cited text no. 12


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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