- Email: [email protected]
MS and its application to a pharmacokinetic study
Accepted Manuscript Title: Determination of parthenolide in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study Author: Ai-qin Zhao Ji-hong Zhao Shu-qing Zhang Yong-yang Pan Xu-lei Huo PII: DOI: Reference:
S0731-7085(15)30258-2 http://dx.doi.org/doi:10.1016/j.jpba.2015.11.039 PBA 10362
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
28-8-2015 20-11-2015 25-11-2015
Please cite this article as: Ai-qin Zhao, Ji-hong Zhao, Shu-qing Zhang, Yong-yang Pan, Xu-lei Huo, Determination of parthenolide in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.11.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination of parthenolide in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study
Ai-qin ZHAO 1, Ji-hong ZHAO 2＊ , Shu-qing ZHANG 1, Yong-yang PAN 2, Xu-lei HUO 2
1. Luoyang Orthopedic Traumatological Hospital of Henan Province, Henan Province Hospital of orthopedics, Luoyang 471002, China 2. Medical College of Henan University of Science and Technology, Luoyang 471003, China
＊ Corresponding author：Ji-hong ZHAO E-mail: [email protected]
1
Graphical Abstract
2
Highlights 1. UPLC-MS/MS assay for parthenolide determination in rat plasma. 2. The method offered shorter run time of 3.0 min. 3. The method is accurate, precise and meets validation requirements by guideline.
3
ABSTRACT A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination and pharmacokinetic investigation of parthenolide in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile containing 30 ng/mL of pirfenidone (IS), and to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.0 min and the elution of parthenolide was at 1.33 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m/z 249.2 → 231.1 for parthenolide and m/z 186.2 → 92.1 for pirfenidone (IS), respectively. The calibration curve was linear over the range of 2.0-500 ng/mL with a lower limit of quantitation (LLOQ) of 2.0 ng/mL. Mean recovery of parthenolide in plasma was in the range of 78.2-86.6%. Intra-day and inter-day precision were both < 8.3%. This method was successfully applied in pharmacokinetic study after oral and intravenous administration of parthenolide in rats.
Keywords: Parthenolide, UPLC-MS/MS, rat plasma, pharmacokinetics.
4
1. Introduction Parthenolide (Fig. 1) is a sesquiterpene lactone that was identified in medicinal plant feverfew (Tanacetum parthenium), a traditional herbal medicine that has been used for the treatment of migraine, fever and arthritis in China [1]. Previous studies have demonstrated that parthenolide exerts strong anti-inflammatory effects and is a potent nuclear factor kappa-B (NF-kB) inhibitor by specifically inhibiting the IkB kinase complex [2-4]. On the other hand, it is emerging as a promising anticancer drug based on its antitumor activity by inducing cell apoptosis in various cancers, such as colorectal cancer [5], cholangiocarcinoma [6, 7], hepatoma [8], and acute myeloid leukemia (AML) [9]. There have several analytical methods been published for determination of parthenolide in feverfew samples, including high-performance liquid chromatography [10, 11], liquid chromatography
coupled with mass spectrometry [12] and gas
chromatography [13]. To our best knowledge, there is only one high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry method for determination of parthenolide in biological fluid, but this method used complicated solid phase extraction procedure and was time-consuming (11.5 mins) [14]. To characterize the pharmacokinetic properties of parthenolide, it is very necessary to develop an accurate and selective bioanalytical method for the determination of parthenolide in plasma. Ultra
performance
liquid
chromatography
tandem
mass
spectrometry
(UPLC-MS/MS) has been evaluated as a faster and more efficient analytical tool compared with current chromatography [15-17]. Here, we report an UPLC-MS/MS method to determinate parthenolide in rat plasma. This method is accurate, reliable and
5
simple and can be used to analyze varied amounts of parthenolide in rat plasma for pharmacokinetic study.
2. Experimental 2.1. Chemicals materials Parthenolide (purity > 98%) was purchased from the Chengdu Mansite Pharmaceutical Company Limited. (Chengdu, China). Pirfenidone (internal standard, IS, purity > 98%) were obtained from Sigma (St. Louis, MO, USA). Acetonitrile and methanol were of HPLC grade and were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was obtained using a Millipore Milli-Q system (Millipore, Bedford, MA, USA).
2.2. UPLC-MS/MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA, USA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) and inline 0.2 μm stainless steel frit filter (Waters Corp.). A gradient program was employed with the mobile phase combining solvent A (0.1% formic acid in water) and solvent B (acetonitrile) as follows: 20-95% B (0-0.3 min), 95-95% B (0.3-1.5 min), 95-20% B (1.5-1.6 min). A subsequent re-equilibration time (1.4 min) should be performed before next injection. The flow rate was 0.40 mL/min and the injection volume was 6 μL. The column and sample temperature were maintained at 40 °C and 4 °C, respectively. A XEVO TQD triple quadruple mass spectrometer equipped with an electro-spray
6
ionization (ESI) source (Waters Corp.) was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution in the mass analyzers. The MRM transitions were m/z 249.2 → 231.1 and m/z 186.2 → 92.1 for parthenolide and IS, respectively. The Masslynx 4.1 software (Waters Corp.) was used for data acquisition and instrument control.
2.3. Standard solutions, calibration standards and quality control (QC) sample The stock solution of parthenolide that was used to make the calibration standards and quality control (QC) samples was prepared by dissolving 10 mg in 10 mL methanol to obtain a concentration of 1.0 mg/mL. The stock solution was further diluted with methanol to obtain working solutions at several concentration levels. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank rat plasma. Final concentrations of the calibration standards were 2.0, 5.0, 10, 20, 50, 100, 200 and 500 ng/mL for parthenolide in rat plasma. The concentrations of QC samples in plasma were 4.0, 40, 400 ng/mL for parthenolide. IS stock solution was made at an initial concentration of 1.0 mg/mL. The IS working solution (30 ng/mL) was made from the stock solution using acetonitrile for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at -20 °C.
2.4. Sample preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 200 µL of the IS working solution (30 ng/mL in
7
acetonitrile) was added to 100 µL of collected plasma sample. The tubes were vortex mixed for 1.0 min and spun in a centrifuge at 13,000 g for 5 min. The supernatant (6 µL) was injected into the UPLC-MS/MS system for analysis.
2.5. Method validation Before using this method to determinate parthenolide in rat plasma, the method was fully validated for specificity, linearity, precision, accuracy, recovery, matrix effect and stability according the United States Food and Drug Administration (US FDA) bioanalytical method validation guidances [18]. Specificity was determined by analysis of blank rat plasma samples from six different volunteers, every blank sample was handled by the procedure described in “Sample preparation” and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of parthenolide to IS were plotted against analyte concentrations, and standard curves were well fitted to the equations by linear regression with a weighting factor of the reciprocal of the concentration (1/x2) in the concentration range of 2.0-500 ng/mL. The lowest limit of quantification (LLOQ) was defined as the lowest concentration of drug in spiked plasma resulting in a signal-to-noise (S/N) ratio of 10:1.0, where the values of the precision met with of less than 20% of the relative standard deviation (RSD), and those of the accuracy were less than 20% of the relative error (RE). The precision and accuracy were evaluated by analyzing six replicates of spiked rat
8
plasma with known concentrations of parthenolide with QC samples at three concentrations (4.0, 40, 400 ng/mL). The intra-day precision and accuracy of the assays were determined in the same day by analyzing six replicates at each concentration level. The inter-day precision and accuracy were evaluated on three consecutive days by analyzing six replicates at each concentration level. The precision values expressed as RSD were required to be below 15%, and the accuracy as RE to be within ± 15%. The recovery of parthenolide was evaluated by comparing peak area ratios of extracted QC samples with those of reference QC solutions reconstituted in blank plasma extracts (n = 6). The recovery of the IS (30 ng/mL) was determined in a similar way. To evaluate the matrix effect, blank rat plasma was extracted and then spiked with the analyte at 4.0, 40 and 400 ng/mL. The corresponding peak areas were then compared with those of neat standard solutions at equivalent concentrations, and this peak area ratio is defined as the matrix effect (ME). The ME of IS was evaluated at the working concentration (30 ng/mL) in the same manner. The stabilities in rat plasma were tested by analyzing five replicates of plasma samples at three concentration levels (4.0, 40, 400 ng/mL) in different conditions. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h, and the ready-to-inject samples (after extraction) in the autosampler at 4 °C for 24 h. The freeze-thaw stability was evaluated after three complete freeze-thaw cycles (-20 to 25 °C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at -20 °C for 30 days. Samples were considered to be stable if assay values were within the acceptable limits of accuracy (RE % ≤ ± 15%) and precision (RSD % ≤ 15%).
9
2.6. Application to a pharmacokinetic study Male Sprague-Dawley rats (180-220 g) were obtained from Laboratory Animal Center of Henan University of Science and Technology (Henan, China) used to study the pharmacokinetics of parthenolide. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Henan University of Science and Technology and were in accordance with the Guide for the Care and Use of Laboratory Animals. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, and 6 h after oral (20 mg/kg) and intravenous (5.0 mg/kg) administration of parthenolide. The samples were immediately centrifuged at 3000 g for 10 min. The plasma obtained (100 µL) was stored at -20 °C until analysis. Plasma parthenolide concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Shanghai University of Traditional Chinese Medicine, China).
3. Results and Discussion 3.1. Method development and optimization Owing to the complex matrices, sample preparation is usually required for the determination of the analytes in biological samples in order to remove the possible interfering matrix components and increase selectivity and sensitivity. In this study, two methods, including a protein precipitation procedure and ethyl acetate extraction, were investigated. Compared to liquid-liquid extraction for the sample preparation, organic
10
solvent precipitation was used for the sample preparation [19]. This simple procedure produced a clean chromatogram for the blank plasma sample and yielded satisfactory recoveries for the analytes. In this work, 200 µL of acetonitrile was added in the process of sample preparation. The sample preparation of this method was simple one-step protein precipitation, which was time- and effort-saving. Thus, this sample preparation meets the requirements of high-throughput bioanalysis. The choice of mobile phase should be concerned based on the consideration of ionization efficiency before the analyte enters the MS/MS system in order to obtain nice resolution and high sensitivity. As for the choice of strong elution mobile phase, methanol and acetonitrile were considered as two candidates. Results showed that the responses of the analyte with acetonitrile as the mobile phase were higher than those with methanol under ESI positive mode. To obtain the maximum sensitivity, we investigated the effects of pH with various mobile phases on the ionization efficiency. Both the analyte and IS were found to have the highest response and the best peak shapes in the mobile phase containing 0.1% formic acid. The LC mobile phase was optimized with varying percentages of organic solvent and different modifiers in water to obtain high sensitivity. Analyte and IS were separated on an Acquity UPLC BEH C18 column with a gradient mobile phase consisting of acetonitrile and 0.1% formic acid in water. The whole separation of the analyte and IS was completed within only 3.0 min per sample.
3.2. Specificity UPLC-MS/MS chromatogram of the analytes in rat plasma samples were shown in Fig. 2. The retention times of parthenolide and IS are 1.33 and 1.14 min, respectively.
11
Compared with chromatogram of blank blood sample, no interference of endogenous peaks was observed.
3.3. Linearity and Sensitivity The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 2.0-500 ng/mL for parthenolide in rat plasma. Typical equation of the calibration curve was: y = 0.008654x + 0.001755, r = 0.996, where y represents the ratios of parthenolide peak area to that of IS and x represents the plasma concentration. The LLOQ of parthenolide in rat plasma was 2.0 ng/mL with the intra- and inter-day RSD less than 8.2%, and the intra- and inter-day accuracy of -6.1% and -4.4%, which was sufficient for the pharmacokinetic studies of parthenolide in rats.
3.4. Precision and Accuracy The intra- and inter-day precision and accuracy of the method were determined from the analysis of QC samples at three different concentrations for each biological matrix. The results are summarized in Table 1. The method was reliable and reproducible since RSD % was below 15% and RE % was between -8.9% and 7.7% for all the investigated concentrations of parthenolide in rat plasma.
3.5. Recovery and Matrix effect As shown in Table 2, the extraction recoveries of parthenolide at concentrations of 4.0, 40 and 400 ng/mL were evaluated by analyzing six replicates, which were 78.2 ± 3.1%, 83.0 ± 3.5%, 86.6 ± 3.0%, respectively. The recovery of IS (30 ng/mL) in plasma
12
was 88.9 ± 4.5%. The ME for parthenolide at concentrations of 4.0, 40 and 400 ng/mL were measured to be 93.8-105.2% (Table 2). The ME for IS (30 ng/mL) was measured to 98.3 ± 2.6% (n = 6). As a result, ME from plasma was negligible in this method.
3.6. Stability Stability tests were performed at the low, medium and high QC samples with five determinations for each under different storage conditions. The RSDs of the mean test responses were within 15% in all stability tests. Table 3 shows the stability data for parthenolide in plasma under different storage and temperature conditions. There was no effect on the quantitation for plasma samples kept at room temperature for 2 h and at 4 °C for 24 h. No significant degradation was observed when samples of parthenolide were taken through three freeze (-20 °C)- thaw (room temperature) cycles. As a result, parthenolide in samples were stable at -20 °C for 30 days.
3.7. Application of the method in a pharmacokinetic study The method was applied to a pharmacokinetic study in rats. The mean plasma concentration-time curve after oral (20 mg/kg) and intravenous (5.0 mg/kg) administration of parthenolide was shown in Fig. 3. The main pharmacokinetic parameters from non-compartment model analysis were summarized in Table 4. The pharmacokinetic profile of parthenolide in rat was characterized for the first time. It helps to better understand pharmacology features of parthenolide. Furthermore, the bioavailability of parthenolide was reported to be 7.78% for first time.
13
4. Conclusions An UPLC-MS/MS method for the determination of parthenolide in rat plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of parthenolide level in rat plasma using an UPLC-MS/MS method. This method offered superior sample preparation with a simple one-step precipitation of plasma protein by acetonitrile and shorter run time of 3.0 min. The method meets the requirement of high sample throughput in bioanalysis and has been successfully applied to the pharmacokinetic study of parthenolide in rats.
14
References [1] D.W. Knight, Feverfew: chemistry and biological activity, Nat Prod Rep 12 (1995) 271-276. [2] Y.J. Nam, H. Lee da, M.S. Lee, C.S. Lee, Sesquiterpene lactone parthenolide attenuates production of inflammatory mediators by suppressing the Toll-like receptor-4-mediated activation of the Akt, mTOR, and NF-kappaB pathways, Naunyn Schmiedebergs Arch Pharmacol 388 (2015) 921-930. [3] X. Zhang, C. Fan, Y. Xiao, X. Mao, Anti-inflammatory and antiosteoclastogenic activities of parthenolide on human periodontal ligament cells in vitro, Evid Based Complement Alternat Med 2014 (2014) 546097. [4] M. Wang, Q. Li, Parthenolide could become a promising and stable drug with anti-inflammatory effects, Nat Prod Res 29 (2015) 1092-1101. [5] S.L. Kim, Y.C. Liu, Y.R. Park, S.Y. Seo, S.H. Kim, I.H. Kim, S.O. Lee, S.T. Lee, D.G. Kim, S.W. Kim, Parthenolide enhances sensitivity of colorectal cancer cells to TRAIL by inducing death receptor 5 and promotes TRAIL-induced apoptosis, Int J Oncol 46 (2015) 1121-1130. [6] B.R. Yun, M.J. Lee, J.H. Kim, I.H. Kim, G.R. Yu, D.G. Kim, Enhancement of parthenolide-induced apoptosis by a PKC-alpha inhibition through heme oxygenase-1 blockage in cholangiocarcinoma cells, Exp Mol Med 42 (2010) 787-797. [7] J.H. Kim, L. Liu, S.O. Lee, Y.T. Kim, K.R. You, D.G. Kim, Susceptibility of cholangiocarcinoma cells to parthenolide-induced apoptosis, Cancer Res 65 (2005) 6312-6320. [8] J. Sun, C. Zhang, Y.L. Bao, Y. Wu, Z.L. Chen, C.L. Yu, Y.X. Huang, Y. Sun, L.H. Zheng, X. Wang, Y.X. Li, Parthenolide-induced apoptosis, autophagy and suppression of proliferation in HepG2 cells, Asian Pac J Cancer Prev 15 (2014) 4897-4902. [9] M.L. Guzman, R.M. Rossi, L. Karnischky, X. Li, D.R. Peterson, D.S. Howard, C.T. Jordan, The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells, Blood 105 (2005) 4163-4169. [10] A.M. El-Shamy, S.S. El-Hawary, M.E. Rateb, Quantitative estimation of parthenolide in Tanacetum parthenium (L.) Schultz-Bip. cultivated in Egypt, J AOAC Int 90 (2007) 21-27. [11] J.Z. Zhou, X. Kou, D. Stevenson, Rapid extraction and high-performance liquid chromatographic determination of parthenolide in feverfew (Tanacetum parthenium), J Agric Food Chem 47 (1999) 1018-1022. [12] B. Avula, A. Navarrete, V.C. Joshi, I.A. Khan, Quantification of parthenolide in Tanacetum species by LC-UV/LC-MS and microscopic comparison of Mexican/US feverfew samples, Pharmazie 61 (2006) 590-594. [13] E.A. Abourashed, I.A. Khan, GC determination of parthenolide in feverfew products, Pharmazie 56 (2001) 971-972. [14] E.A. Curry, 3rd, D.J. Murry, C. Yoder, K. Fife, V. Armstrong, H. Nakshatri, M. O'Connell, C.J. Sweeney, Phase I dose escalation trial of feverfew with standardized doses of parthenolide in patients with cancer, Invest New Drugs 22 (2004) 299-305. [15] A. de Villiers, F. Lestremau, R. Szucs, S. Gelebart, F. David, P. Sandra, Evaluation of ultra performance liquid chromatography. Part I. Possibilities and limitations, J Chromatogr A 1127 (2006) 60-69. [16] X. Qiu, J. Zhao, Z. Wang, Z. Xu, R.A. Xu, Simultaneous determination of bosentan and glimepiride in human plasma by ultra performance liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study, J Pharm Biomed Anal 95 (2014) 207-212. [17] X. Qiu, Z. Wang, B. Wang, H. Zhan, X. Pan, R.A. Xu, Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma by ultra high performance liquid chromatography tandem mass spectrometry and its application to a bioequivalence study, J Chromatogr B Analyt Technol Biomed Life Sci 957 (2014) 110-115. [18] S. Jamalapuram, P.K. Vuppala, A.H. Abdelazeem, C.R. McCurdy, B.A. Avery, Ultra-performance liquid chromatography tandem mass spectrometry method for the determination of AZ66, a sigma receptor ligand, in rat plasma and its application to in vivo pharmacokinetics, Biomed Chromatogr 27 (2013) 1034-1040. [19] Y. Song, S. Zhang, H. Liu, X. Jin, Determination of genkwanin in rat plasma by liquid chromatography-tandem mass spectrometry: application to a bioavailability study, J Pharm Biomed Anal 84 (2013) 129-134. 15
Fig. 1. The chemical structures and ESI full scan mass spectra of parthenolide (A) and pirfenidone (IS, B).
16
Fig. 2. Representative chromatograms of parthenolide and IS in rat plasma samples. (A) a blank plasma sample; (B) a blank plasma sample spiked with parthenolide (50 ng/mL) and IS (30 ng/mL); (C) a rat plasma sample 1.0 h after intravenous administration of single dosage 5.0 mg/kg parthenolide (57.33 ng/mL).
17
Fig. 3. Mean plasma concentration time profile after oral (20 mg/kg) and intravenous (5.0 mg/kg) administration of parthenolide in six rats.
18
Table 1. Precision and accuracy of method for the determination of parthenolide in rat plasma (n = 6). Analyte
Concentration
RSD (%)
RE (%)
(ng/mL)
Parthenolide
Intra-day
Inter-day
Intra-day
Inter-day
4.0
7.2
8.3
7.7
-8.9
40
4.1
6.2
5.8
7.2
400
2.0
1.8
3.1
-4.1
19
Table 2. Recovery and matrix effect of parthenolide and internal standards (n = 6). Analyte
Concentration
Recovery (%)
Matrix effect (%)
(ng/mL)
Parthenolide
IS
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
4.0
78.2 ± 3.1
4.0
105.2 ± 3.9
3.7
40
83.0 ± 3.5
4.2
98.9 ± 3.7
3.8
400
86.6 ± 3.0
3.5
93.8 ± 4.1
4.4
30
88.9 ± 4.5
5.1
98.3 ± 2.6
2.7
20
Table 3. Stability results of parthenolide in rat plasma in different conditions (n = 5). Analyte
Concentration
room temperature, 2 h
4 °C, 24 h
Three freeze-thaw
-20 °C, 30 days
(ng/mL) Mean ± SD
RS
RE
D
(%)
Mean ± SD
RSD
RE
(%)
(%)
Mean ± SD
RSD
RE
(%)
(%)
Mean ± SD
RSD
RE
(%)
(%)
(%)
Parthenolid
4.0
3.9 ± 0.3
8.4
-3.1
4.3 ± 0.3
7.8
6.7
4.0 ± 0.3
6.5
0.7
4.3 ± 0.3
6.5
7.7
40
40.8 ± 3.4
8.4
2.0
40.9 ± 3.5
8.5
2.3
40.8 ± 4.0
9.8
2.0
40.9 ± 2.9
7.2
2.2
400
405.5 ± 25.8
6.4
1.4
377.8 ± 12.8
3.4
-5.5
384.7 ± 16.6
4.3
-3.8
400.7 ± 21.5
5.4
0.2
e
21
Table 4. The main pharmacokinetic parameters after oral (20 mg/kg) and intravenous (5.0 mg/kg) administration of parthenolide in six rats. Parameters
Oral administration
Intravenous administration
t1/2 (h)
1.38 ± 0.14
1.13 ± 0.32
Cmax (ng/mL)
13.63 ± 2.69
139.29 ± 26.25
C0 (ng/mL)
162.71 ± 37.70
Vz (L/kg)
47.65 ± 15.53
AUC0→t (ng/mL•h)
52.18 ± 12.95
162.83 ± 27.94
AUC0→∞ (ng/mL•h)
53.85 ± 14.29
173.09 ± 30.73
Bioavailability, F
7.78%
22
S0731-7085(15)30258-2 http://dx.doi.org/doi:10.1016/j.jpba.2015.11.039 PBA 10362
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
28-8-2015 20-11-2015 25-11-2015
Please cite this article as: Ai-qin Zhao, Ji-hong Zhao, Shu-qing Zhang, Yong-yang Pan, Xu-lei Huo, Determination of parthenolide in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.11.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination of parthenolide in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study
Ai-qin ZHAO 1, Ji-hong ZHAO 2＊ , Shu-qing ZHANG 1, Yong-yang PAN 2, Xu-lei HUO 2
1. Luoyang Orthopedic Traumatological Hospital of Henan Province, Henan Province Hospital of orthopedics, Luoyang 471002, China 2. Medical College of Henan University of Science and Technology, Luoyang 471003, China
＊ Corresponding author：Ji-hong ZHAO E-mail: [email protected]
1
Graphical Abstract
2
Highlights 1. UPLC-MS/MS assay for parthenolide determination in rat plasma. 2. The method offered shorter run time of 3.0 min. 3. The method is accurate, precise and meets validation requirements by guideline.
3
ABSTRACT A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination and pharmacokinetic investigation of parthenolide in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile containing 30 ng/mL of pirfenidone (IS), and to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.0 min and the elution of parthenolide was at 1.33 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m/z 249.2 → 231.1 for parthenolide and m/z 186.2 → 92.1 for pirfenidone (IS), respectively. The calibration curve was linear over the range of 2.0-500 ng/mL with a lower limit of quantitation (LLOQ) of 2.0 ng/mL. Mean recovery of parthenolide in plasma was in the range of 78.2-86.6%. Intra-day and inter-day precision were both < 8.3%. This method was successfully applied in pharmacokinetic study after oral and intravenous administration of parthenolide in rats.
Keywords: Parthenolide, UPLC-MS/MS, rat plasma, pharmacokinetics.
4
1. Introduction Parthenolide (Fig. 1) is a sesquiterpene lactone that was identified in medicinal plant feverfew (Tanacetum parthenium), a traditional herbal medicine that has been used for the treatment of migraine, fever and arthritis in China [1]. Previous studies have demonstrated that parthenolide exerts strong anti-inflammatory effects and is a potent nuclear factor kappa-B (NF-kB) inhibitor by specifically inhibiting the IkB kinase complex [2-4]. On the other hand, it is emerging as a promising anticancer drug based on its antitumor activity by inducing cell apoptosis in various cancers, such as colorectal cancer [5], cholangiocarcinoma [6, 7], hepatoma [8], and acute myeloid leukemia (AML) [9]. There have several analytical methods been published for determination of parthenolide in feverfew samples, including high-performance liquid chromatography [10, 11], liquid chromatography
coupled with mass spectrometry [12] and gas
chromatography [13]. To our best knowledge, there is only one high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry method for determination of parthenolide in biological fluid, but this method used complicated solid phase extraction procedure and was time-consuming (11.5 mins) [14]. To characterize the pharmacokinetic properties of parthenolide, it is very necessary to develop an accurate and selective bioanalytical method for the determination of parthenolide in plasma. Ultra
performance
liquid
chromatography
tandem
mass
spectrometry
(UPLC-MS/MS) has been evaluated as a faster and more efficient analytical tool compared with current chromatography [15-17]. Here, we report an UPLC-MS/MS method to determinate parthenolide in rat plasma. This method is accurate, reliable and
5
simple and can be used to analyze varied amounts of parthenolide in rat plasma for pharmacokinetic study.
2. Experimental 2.1. Chemicals materials Parthenolide (purity > 98%) was purchased from the Chengdu Mansite Pharmaceutical Company Limited. (Chengdu, China). Pirfenidone (internal standard, IS, purity > 98%) were obtained from Sigma (St. Louis, MO, USA). Acetonitrile and methanol were of HPLC grade and were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was obtained using a Millipore Milli-Q system (Millipore, Bedford, MA, USA).
2.2. UPLC-MS/MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA, USA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) and inline 0.2 μm stainless steel frit filter (Waters Corp.). A gradient program was employed with the mobile phase combining solvent A (0.1% formic acid in water) and solvent B (acetonitrile) as follows: 20-95% B (0-0.3 min), 95-95% B (0.3-1.5 min), 95-20% B (1.5-1.6 min). A subsequent re-equilibration time (1.4 min) should be performed before next injection. The flow rate was 0.40 mL/min and the injection volume was 6 μL. The column and sample temperature were maintained at 40 °C and 4 °C, respectively. A XEVO TQD triple quadruple mass spectrometer equipped with an electro-spray
6
ionization (ESI) source (Waters Corp.) was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution in the mass analyzers. The MRM transitions were m/z 249.2 → 231.1 and m/z 186.2 → 92.1 for parthenolide and IS, respectively. The Masslynx 4.1 software (Waters Corp.) was used for data acquisition and instrument control.
2.3. Standard solutions, calibration standards and quality control (QC) sample The stock solution of parthenolide that was used to make the calibration standards and quality control (QC) samples was prepared by dissolving 10 mg in 10 mL methanol to obtain a concentration of 1.0 mg/mL. The stock solution was further diluted with methanol to obtain working solutions at several concentration levels. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank rat plasma. Final concentrations of the calibration standards were 2.0, 5.0, 10, 20, 50, 100, 200 and 500 ng/mL for parthenolide in rat plasma. The concentrations of QC samples in plasma were 4.0, 40, 400 ng/mL for parthenolide. IS stock solution was made at an initial concentration of 1.0 mg/mL. The IS working solution (30 ng/mL) was made from the stock solution using acetonitrile for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at -20 °C.
2.4. Sample preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 200 µL of the IS working solution (30 ng/mL in
7
acetonitrile) was added to 100 µL of collected plasma sample. The tubes were vortex mixed for 1.0 min and spun in a centrifuge at 13,000 g for 5 min. The supernatant (6 µL) was injected into the UPLC-MS/MS system for analysis.
2.5. Method validation Before using this method to determinate parthenolide in rat plasma, the method was fully validated for specificity, linearity, precision, accuracy, recovery, matrix effect and stability according the United States Food and Drug Administration (US FDA) bioanalytical method validation guidances [18]. Specificity was determined by analysis of blank rat plasma samples from six different volunteers, every blank sample was handled by the procedure described in “Sample preparation” and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of parthenolide to IS were plotted against analyte concentrations, and standard curves were well fitted to the equations by linear regression with a weighting factor of the reciprocal of the concentration (1/x2) in the concentration range of 2.0-500 ng/mL. The lowest limit of quantification (LLOQ) was defined as the lowest concentration of drug in spiked plasma resulting in a signal-to-noise (S/N) ratio of 10:1.0, where the values of the precision met with of less than 20% of the relative standard deviation (RSD), and those of the accuracy were less than 20% of the relative error (RE). The precision and accuracy were evaluated by analyzing six replicates of spiked rat
8
plasma with known concentrations of parthenolide with QC samples at three concentrations (4.0, 40, 400 ng/mL). The intra-day precision and accuracy of the assays were determined in the same day by analyzing six replicates at each concentration level. The inter-day precision and accuracy were evaluated on three consecutive days by analyzing six replicates at each concentration level. The precision values expressed as RSD were required to be below 15%, and the accuracy as RE to be within ± 15%. The recovery of parthenolide was evaluated by comparing peak area ratios of extracted QC samples with those of reference QC solutions reconstituted in blank plasma extracts (n = 6). The recovery of the IS (30 ng/mL) was determined in a similar way. To evaluate the matrix effect, blank rat plasma was extracted and then spiked with the analyte at 4.0, 40 and 400 ng/mL. The corresponding peak areas were then compared with those of neat standard solutions at equivalent concentrations, and this peak area ratio is defined as the matrix effect (ME). The ME of IS was evaluated at the working concentration (30 ng/mL) in the same manner. The stabilities in rat plasma were tested by analyzing five replicates of plasma samples at three concentration levels (4.0, 40, 400 ng/mL) in different conditions. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h, and the ready-to-inject samples (after extraction) in the autosampler at 4 °C for 24 h. The freeze-thaw stability was evaluated after three complete freeze-thaw cycles (-20 to 25 °C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at -20 °C for 30 days. Samples were considered to be stable if assay values were within the acceptable limits of accuracy (RE % ≤ ± 15%) and precision (RSD % ≤ 15%).
9
2.6. Application to a pharmacokinetic study Male Sprague-Dawley rats (180-220 g) were obtained from Laboratory Animal Center of Henan University of Science and Technology (Henan, China) used to study the pharmacokinetics of parthenolide. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Henan University of Science and Technology and were in accordance with the Guide for the Care and Use of Laboratory Animals. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, and 6 h after oral (20 mg/kg) and intravenous (5.0 mg/kg) administration of parthenolide. The samples were immediately centrifuged at 3000 g for 10 min. The plasma obtained (100 µL) was stored at -20 °C until analysis. Plasma parthenolide concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Shanghai University of Traditional Chinese Medicine, China).
3. Results and Discussion 3.1. Method development and optimization Owing to the complex matrices, sample preparation is usually required for the determination of the analytes in biological samples in order to remove the possible interfering matrix components and increase selectivity and sensitivity. In this study, two methods, including a protein precipitation procedure and ethyl acetate extraction, were investigated. Compared to liquid-liquid extraction for the sample preparation, organic
10
solvent precipitation was used for the sample preparation [19]. This simple procedure produced a clean chromatogram for the blank plasma sample and yielded satisfactory recoveries for the analytes. In this work, 200 µL of acetonitrile was added in the process of sample preparation. The sample preparation of this method was simple one-step protein precipitation, which was time- and effort-saving. Thus, this sample preparation meets the requirements of high-throughput bioanalysis. The choice of mobile phase should be concerned based on the consideration of ionization efficiency before the analyte enters the MS/MS system in order to obtain nice resolution and high sensitivity. As for the choice of strong elution mobile phase, methanol and acetonitrile were considered as two candidates. Results showed that the responses of the analyte with acetonitrile as the mobile phase were higher than those with methanol under ESI positive mode. To obtain the maximum sensitivity, we investigated the effects of pH with various mobile phases on the ionization efficiency. Both the analyte and IS were found to have the highest response and the best peak shapes in the mobile phase containing 0.1% formic acid. The LC mobile phase was optimized with varying percentages of organic solvent and different modifiers in water to obtain high sensitivity. Analyte and IS were separated on an Acquity UPLC BEH C18 column with a gradient mobile phase consisting of acetonitrile and 0.1% formic acid in water. The whole separation of the analyte and IS was completed within only 3.0 min per sample.
3.2. Specificity UPLC-MS/MS chromatogram of the analytes in rat plasma samples were shown in Fig. 2. The retention times of parthenolide and IS are 1.33 and 1.14 min, respectively.
11
Compared with chromatogram of blank blood sample, no interference of endogenous peaks was observed.
3.3. Linearity and Sensitivity The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 2.0-500 ng/mL for parthenolide in rat plasma. Typical equation of the calibration curve was: y = 0.008654x + 0.001755, r = 0.996, where y represents the ratios of parthenolide peak area to that of IS and x represents the plasma concentration. The LLOQ of parthenolide in rat plasma was 2.0 ng/mL with the intra- and inter-day RSD less than 8.2%, and the intra- and inter-day accuracy of -6.1% and -4.4%, which was sufficient for the pharmacokinetic studies of parthenolide in rats.
3.4. Precision and Accuracy The intra- and inter-day precision and accuracy of the method were determined from the analysis of QC samples at three different concentrations for each biological matrix. The results are summarized in Table 1. The method was reliable and reproducible since RSD % was below 15% and RE % was between -8.9% and 7.7% for all the investigated concentrations of parthenolide in rat plasma.
3.5. Recovery and Matrix effect As shown in Table 2, the extraction recoveries of parthenolide at concentrations of 4.0, 40 and 400 ng/mL were evaluated by analyzing six replicates, which were 78.2 ± 3.1%, 83.0 ± 3.5%, 86.6 ± 3.0%, respectively. The recovery of IS (30 ng/mL) in plasma
12
was 88.9 ± 4.5%. The ME for parthenolide at concentrations of 4.0, 40 and 400 ng/mL were measured to be 93.8-105.2% (Table 2). The ME for IS (30 ng/mL) was measured to 98.3 ± 2.6% (n = 6). As a result, ME from plasma was negligible in this method.
3.6. Stability Stability tests were performed at the low, medium and high QC samples with five determinations for each under different storage conditions. The RSDs of the mean test responses were within 15% in all stability tests. Table 3 shows the stability data for parthenolide in plasma under different storage and temperature conditions. There was no effect on the quantitation for plasma samples kept at room temperature for 2 h and at 4 °C for 24 h. No significant degradation was observed when samples of parthenolide were taken through three freeze (-20 °C)- thaw (room temperature) cycles. As a result, parthenolide in samples were stable at -20 °C for 30 days.
3.7. Application of the method in a pharmacokinetic study The method was applied to a pharmacokinetic study in rats. The mean plasma concentration-time curve after oral (20 mg/kg) and intravenous (5.0 mg/kg) administration of parthenolide was shown in Fig. 3. The main pharmacokinetic parameters from non-compartment model analysis were summarized in Table 4. The pharmacokinetic profile of parthenolide in rat was characterized for the first time. It helps to better understand pharmacology features of parthenolide. Furthermore, the bioavailability of parthenolide was reported to be 7.78% for first time.
13
4. Conclusions An UPLC-MS/MS method for the determination of parthenolide in rat plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of parthenolide level in rat plasma using an UPLC-MS/MS method. This method offered superior sample preparation with a simple one-step precipitation of plasma protein by acetonitrile and shorter run time of 3.0 min. The method meets the requirement of high sample throughput in bioanalysis and has been successfully applied to the pharmacokinetic study of parthenolide in rats.
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References [1] D.W. Knight, Feverfew: chemistry and biological activity, Nat Prod Rep 12 (1995) 271-276. [2] Y.J. Nam, H. Lee da, M.S. Lee, C.S. Lee, Sesquiterpene lactone parthenolide attenuates production of inflammatory mediators by suppressing the Toll-like receptor-4-mediated activation of the Akt, mTOR, and NF-kappaB pathways, Naunyn Schmiedebergs Arch Pharmacol 388 (2015) 921-930. [3] X. Zhang, C. Fan, Y. Xiao, X. Mao, Anti-inflammatory and antiosteoclastogenic activities of parthenolide on human periodontal ligament cells in vitro, Evid Based Complement Alternat Med 2014 (2014) 546097. [4] M. Wang, Q. Li, Parthenolide could become a promising and stable drug with anti-inflammatory effects, Nat Prod Res 29 (2015) 1092-1101. [5] S.L. Kim, Y.C. Liu, Y.R. Park, S.Y. Seo, S.H. Kim, I.H. Kim, S.O. Lee, S.T. Lee, D.G. Kim, S.W. Kim, Parthenolide enhances sensitivity of colorectal cancer cells to TRAIL by inducing death receptor 5 and promotes TRAIL-induced apoptosis, Int J Oncol 46 (2015) 1121-1130. [6] B.R. Yun, M.J. Lee, J.H. Kim, I.H. Kim, G.R. Yu, D.G. Kim, Enhancement of parthenolide-induced apoptosis by a PKC-alpha inhibition through heme oxygenase-1 blockage in cholangiocarcinoma cells, Exp Mol Med 42 (2010) 787-797. [7] J.H. Kim, L. Liu, S.O. Lee, Y.T. Kim, K.R. You, D.G. Kim, Susceptibility of cholangiocarcinoma cells to parthenolide-induced apoptosis, Cancer Res 65 (2005) 6312-6320. [8] J. Sun, C. Zhang, Y.L. Bao, Y. Wu, Z.L. Chen, C.L. Yu, Y.X. Huang, Y. Sun, L.H. Zheng, X. Wang, Y.X. Li, Parthenolide-induced apoptosis, autophagy and suppression of proliferation in HepG2 cells, Asian Pac J Cancer Prev 15 (2014) 4897-4902. [9] M.L. Guzman, R.M. Rossi, L. Karnischky, X. Li, D.R. Peterson, D.S. Howard, C.T. Jordan, The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells, Blood 105 (2005) 4163-4169. [10] A.M. El-Shamy, S.S. El-Hawary, M.E. Rateb, Quantitative estimation of parthenolide in Tanacetum parthenium (L.) Schultz-Bip. cultivated in Egypt, J AOAC Int 90 (2007) 21-27. [11] J.Z. Zhou, X. Kou, D. Stevenson, Rapid extraction and high-performance liquid chromatographic determination of parthenolide in feverfew (Tanacetum parthenium), J Agric Food Chem 47 (1999) 1018-1022. [12] B. Avula, A. Navarrete, V.C. Joshi, I.A. Khan, Quantification of parthenolide in Tanacetum species by LC-UV/LC-MS and microscopic comparison of Mexican/US feverfew samples, Pharmazie 61 (2006) 590-594. [13] E.A. Abourashed, I.A. Khan, GC determination of parthenolide in feverfew products, Pharmazie 56 (2001) 971-972. [14] E.A. Curry, 3rd, D.J. Murry, C. Yoder, K. Fife, V. Armstrong, H. Nakshatri, M. O'Connell, C.J. Sweeney, Phase I dose escalation trial of feverfew with standardized doses of parthenolide in patients with cancer, Invest New Drugs 22 (2004) 299-305. [15] A. de Villiers, F. Lestremau, R. Szucs, S. Gelebart, F. David, P. Sandra, Evaluation of ultra performance liquid chromatography. Part I. Possibilities and limitations, J Chromatogr A 1127 (2006) 60-69. [16] X. Qiu, J. Zhao, Z. Wang, Z. Xu, R.A. Xu, Simultaneous determination of bosentan and glimepiride in human plasma by ultra performance liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study, J Pharm Biomed Anal 95 (2014) 207-212. [17] X. Qiu, Z. Wang, B. Wang, H. Zhan, X. Pan, R.A. Xu, Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma by ultra high performance liquid chromatography tandem mass spectrometry and its application to a bioequivalence study, J Chromatogr B Analyt Technol Biomed Life Sci 957 (2014) 110-115. [18] S. Jamalapuram, P.K. Vuppala, A.H. Abdelazeem, C.R. McCurdy, B.A. Avery, Ultra-performance liquid chromatography tandem mass spectrometry method for the determination of AZ66, a sigma receptor ligand, in rat plasma and its application to in vivo pharmacokinetics, Biomed Chromatogr 27 (2013) 1034-1040. [19] Y. Song, S. Zhang, H. Liu, X. Jin, Determination of genkwanin in rat plasma by liquid chromatography-tandem mass spectrometry: application to a bioavailability study, J Pharm Biomed Anal 84 (2013) 129-134. 15
Fig. 1. The chemical structures and ESI full scan mass spectra of parthenolide (A) and pirfenidone (IS, B).
16
Fig. 2. Representative chromatograms of parthenolide and IS in rat plasma samples. (A) a blank plasma sample; (B) a blank plasma sample spiked with parthenolide (50 ng/mL) and IS (30 ng/mL); (C) a rat plasma sample 1.0 h after intravenous administration of single dosage 5.0 mg/kg parthenolide (57.33 ng/mL).
17
Fig. 3. Mean plasma concentration time profile after oral (20 mg/kg) and intravenous (5.0 mg/kg) administration of parthenolide in six rats.
18
Table 1. Precision and accuracy of method for the determination of parthenolide in rat plasma (n = 6). Analyte
Concentration
RSD (%)
RE (%)
(ng/mL)
Parthenolide
Intra-day
Inter-day
Intra-day
Inter-day
4.0
7.2
8.3
7.7
-8.9
40
4.1
6.2
5.8
7.2
400
2.0
1.8
3.1
-4.1
19
Table 2. Recovery and matrix effect of parthenolide and internal standards (n = 6). Analyte
Concentration
Recovery (%)
Matrix effect (%)
(ng/mL)
Parthenolide
IS
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
4.0
78.2 ± 3.1
4.0
105.2 ± 3.9
3.7
40
83.0 ± 3.5
4.2
98.9 ± 3.7
3.8
400
86.6 ± 3.0
3.5
93.8 ± 4.1
4.4
30
88.9 ± 4.5
5.1
98.3 ± 2.6
2.7
20
Table 3. Stability results of parthenolide in rat plasma in different conditions (n = 5). Analyte
Concentration
room temperature, 2 h
4 °C, 24 h
Three freeze-thaw
-20 °C, 30 days
(ng/mL) Mean ± SD
RS
RE
D
(%)
Mean ± SD
RSD
RE
(%)
(%)
Mean ± SD
RSD
RE
(%)
(%)
Mean ± SD
RSD
RE
(%)
(%)
(%)
Parthenolid
4.0
3.9 ± 0.3
8.4
-3.1
4.3 ± 0.3
7.8
6.7
4.0 ± 0.3
6.5
0.7
4.3 ± 0.3
6.5
7.7
40
40.8 ± 3.4
8.4
2.0
40.9 ± 3.5
8.5
2.3
40.8 ± 4.0
9.8
2.0
40.9 ± 2.9
7.2
2.2
400
405.5 ± 25.8
6.4
1.4
377.8 ± 12.8
3.4
-5.5
384.7 ± 16.6
4.3
-3.8
400.7 ± 21.5
5.4
0.2
e
21
Table 4. The main pharmacokinetic parameters after oral (20 mg/kg) and intravenous (5.0 mg/kg) administration of parthenolide in six rats. Parameters
Oral administration
Intravenous administration
t1/2 (h)
1.38 ± 0.14
1.13 ± 0.32
Cmax (ng/mL)
13.63 ± 2.69
139.29 ± 26.25
C0 (ng/mL)
162.71 ± 37.70
Vz (L/kg)
47.65 ± 15.53
AUC0→t (ng/mL•h)
52.18 ± 12.95
162.83 ± 27.94
AUC0→∞ (ng/mL•h)
53.85 ± 14.29
173.09 ± 30.73
Bioavailability, F
7.78%
22