Journal of Chromatography B, 967 (2014) 57–62
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of chrysotoxine in rat plasma by liquid chromatography–tandem mass spectrometry and its application to a rat pharmacokinetic study Jingjing Fan a , Li Guan b , Zeqi Kou b , Feng Feng b , Yanbo Zhang c,∗ , Wenyuan Liu a,∗∗ a
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, China Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, 210009, China c School of Chinese Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, China b
a r t i c l e
i n f o
Article history: Received 25 January 2014 Accepted 9 July 2014 Available online 16 July 2014 Keywords: Chrysotoxine HPLC–MS/MS Pharmacokinetics Method validation Bioavailability
a b s t r a c t Chrysotoxine (CTX), a naturally occurring bibenzyl compound isolated from Dendrobium species, has been reported to have neuroprotective effects. To evaluate its pharmacokinetics in rats, a rapid, sensitive and specific high performance liquid chromatography–tandem mass spectrometric (HPLC–MS/MS) method has been developed and validated for the quantification of CTX in rat plasma. Samples were pretreated using a simple liquid–liquid extraction with ethyl acetate and the chromatographic separation was performed on a C18 column with acetonitrile–water (90:10, v/v) as the mobile phase. CTX and the internal standard (wogonin) were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. Method validation revealed excellent linearity over the range 0.5–1000 ng/mL together with satisfactory intra- and inter-day precision, accuracy and recovery. Stability testing showed that CTX spiked into rat plasma was stable for 8 h at room temperature, for up to two weeks at −20 ◦ C, and during three freeze–thaw cycles. Extracted samples were also observed to be stable over 24 h in an auto-sampler. The method was successfully used to investigate the pharmacokinetic profile of CTX after oral (100 mg/kg) and intravenous (25 mg/kg) administration in rats. CTX showed rapid excretion and low bioavailability in rats. © 2014 Published by Elsevier B.V.
1. Introduction Dendrobium species, such as Dendrobium nobile Lindl., Dendrobium chrysotoxum Lindl. and Dendrobium fimbriatum Hook, play an important role in traditional Chinese medicine. Activities recorded in the Chinese pharmacopoeia [1] include hypoglycemic, antitumor, anti-aging and immunomodulatory effects [2–5]. In recent years, bibenzyls in Dendrobium have attracted much attention for their unique structures and biological activities [6–10]. Chrysotoxine (CTX) is a bibenzyl isolated from Dendrobium species. Recent studies have revealed that CTX can inhibit dopaminergic cell death in SH-SY5Y cells induced by 6-hydroxydopamine and 1-methyl-4phenyl pyridinium, suggesting a potential protective effect against neurodegeneration [11,12]. To evaluate the potential of CTX as
a drug candidate, we have now investigated its pharmacokinetic properties in rats. Several methods for the separation and analysis of CTX in raw herbs have been reported; most of these use mass spectrometry (MS), ultraviolet (UV) spectroscopy, infra-red (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy [13], high performance liquid chromatography (HPLC) or high performance liquid chromatography–diode array detection (HPLC–DAD) [14–16]. However, none of these methods has been used for pharmacokinetic studies and the aim of the present work was to develop a rapid, sensitive and specific analytical method for the determination of CTX in biological samples. Using our newly developed method, we then evaluated the bioavailability of CTX in rats.
2. Experimental ∗ Corresponding author. Tel.: +852 9506 3396. ∗∗ Corresponding author. Tel.: +86 25 8327 1038. E-mail addresses: [email protected] (Y. Zhang), [email protected] (W. Liu). http://dx.doi.org/10.1016/j.jchromb.2014.07.011 1570-0232/© 2014 Published by Elsevier B.V.
2.1. Drug and chemicals Chrysotoxine (CTX, purity >96.0%) and wogonin (purity >99.5%, internal standard, IS) were prepared by the Department of Natural
58
J. Fan et al. / J. Chromatogr. B 967 (2014) 57–62
Medicinal Chemistry, China Pharmaceutical University (Nanjing, China) and their structures were confirmed by comprehensive analysis using high resolution mass spectrometry, UV and IR spectroscopy and 1D and 2D NMR spectroscopy. Acetonitrile (HPLC grade; Merck) and ultra pure water from a Milli-Q system (Millipore) were used to prepare the mobile phase. All other reagents were of analytical grade and obtained from conventional commercial sources.
Each drug-free rat plasma sample (50 L) was spiked with IS solution (5 L) and serial standard solutions of CTX (5 L) to prepare calibration standards in the concentration range 0.5–1000 ng/mL. QC samples were prepared by spiking control rat plasma in bulk at appropriate concentrations, and then dividing into small aliquots (around 80 L) in different tubes. These samples were stored under the same conditions as the experimental samples and underwent the same pretreatment procedure (described below).
2.2. Instrumentation and conditions 2.5. Sample pretreatment procedure A Shimadzu LC-2010 series HPLC system (Kyoto, Japan), equipped with a quaternary pump, a vacuum degasser, an auto-sampler and a column heater-cooler was connected by an electrospray ionization (ESI) interface to a Thermo Finnigan TSQ AM tandem mass spectrometer (Thermo Finnigan, San Jose, CA, USA). Samples were separated on a YMC-Pack ODS-A column (50 mm × 4.6 mm, 3 m, YMC Co. Ltd., Kyoto, Japan) with the column temperature set at 30 ◦ C. The mobile phase consisted of acetonitrile–water (90:10, v/v), delivered at a flow rate of 0.7 mL/min with a split ratio of 1:3. The injection volume was 10 L and the run time was 3 min. Solvent eluted from chromatography column during the first minute was switched to waste before it entered the ion source. The MS was set in positive multiple reaction monitoring (MRM) mode. MS transitions were m/z 318.8 → 165.0 for CTX and m/z 284.8 → 269.9 for the IS in MRM mode. The MS operating conditions were optimized and set as follows: sheath gas pressure, 35 arbitrary units; the auxiliary gas pressure, 5 arbitrary units; capillary temperature, 350 ◦ C; spray voltage, 5 kV and source collision-induces dissociation (CID), 10 V. The collision gas was argon and the collision energies were 20 eV for CTX and 26 eV for the IS, respectively. Data were processed using Xcalibur software (Thermo Finnigan, San Jose, CA, USA). 2.3. Animals Healthy male Sprague-Dawley rats (200–240 g) (Certificate No. SCXK2008-0016) were purchased from Shanghai Super-B&K Laboratory Animal Co., Ltd. (Shanghai, China). Experiments using animals were approved by the Animal Ethics Committee of the China Pharmaceutical University (Nanjing, China) and conformed to the Guide for Care and Use of Laboratory Animals published by the US National Institute of Health [17]. Animals were housed in a room at a controlled temperature of 23–26 ◦ C and a relative humidity of 40–60%, with access to food and water ad libitum. All animals were acclimated in the laboratory for at least seven days prior to the experiment and fasted for 12 h but allowed water ad libitum before experiments. 2.4. Preparation of standard and quality control (QC) samples Stock solutions of CTX (1 mg/mL) and IS (1 mg/mL) in acetonitrile were prepared separately. In order to ensure weighing precision, two weighings, as long as their concentrations agreed within 5%, were prepared, then one was used for calibrators and the other for QC samples. Primary stock solutions for calibration curve standards and QC samples were prepared using separate weighings. Standard solutions of CTX for the preparation of calibration curves were obtained by further dilution of the stock solution with acetonitrile to give final concentrations of 5, 10, 20, 50, 100, 500, 2000, 5000 and 10,000 ng/mL. QC solutions were prepared at concentrations of 10, 500, 8000 ng/mL by dilution of the primary stock solution with acetonitrile. A solution containing the IS (50 ng/mL) was also obtained by dilution of the IS stock solution with acetonitrile. All standard solutions were stored at 4 ◦ C.
All samples were thawed to room temperature before analysis. The plasma sample (50 L) was transferred into a 1.5 mL centrifuge tube and IS working solution (5 L) was then added. The mixture was vortex-mixed for 1 min and then extracted with ethyl acetate (1 mL) by vortex-mixing for 3 min. The sample was then centrifuged at 16,000 rpm for 5 min and the supernatant was transferred into another test tube and evaporated to dryness at 37 ◦ C. The residue was reconstituted in mobile phase (50 L) by vortex-mixing for 2 min and centrifuging at 16,000 rpm for 3 min. Finally, the supernatant (10 L) was injected for HPLC–MS/MS analysis. 2.6. Method validation Method validation for determination of CTX in rat plasma was performed according to the US Food and Drug Administration (FDA) guidance [18]. Selectivity was evaluated by comparing chromatograms of blank plasma samples collected from six different rats with the chromatogram of a plasma sample spiked with CTX and the IS. A least-squares linear regression method (1/x2 weighting) was used to determine the slope, intercept and square regression coefficient (r2 ) of the linear regression equation. The calibration curve was established using the Bioavailability Program Package software (BAPP, Version 2.2, Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University). Precision and accuracy were calculated by determining QC samples at three concentration levels. Precision is expressed as the relative standard deviation (RSD) and accuracy as the relative error (RE). Precision and accuracy were also assessed at the lowest concentration of the standards (0.5 ng/mL), representing the lower limit of quantification (LLOQ) for the assay. A blank sample was placed immediately after the upper limit of quantification (ULOQ) standard to evaluate carryover in the LC–MS/MS method. The recovery of analytes was determined by comparing the responses of the analytes from QC samples with analytes spiked in post-extracted blank rat plasma at equivalent concentrations. The matrix effect was measured by comparing the responses obtained from post-extraction blank rat plasma spiked samples with mobile phase spiked with low, middle and high concentrations of analyte. The dilution effect was estimated by analysis of rat plasma spiked with analyte at 5000 ng/mL and diluted 10-fold with blank rat plasma. The stability of the analyte in rat plasma was evaluated by analyzing QC samples stored under the following conditions: at 4 ◦ C in an auto-sampler for 24 h, at room temperature for 8 h and at −20 ◦ C for two weeks. The effect of three freeze/thaw cycles on the analyte was also examined. 2.7. Pharmacokinetic study Ten rats were randomly divided into two groups. Five rats received CTX (100 mg/kg) by oral administration (p.o.) and the other group received CTX (25 mg/kg) by intravenous injection (i.v.). After dosing, the rats were fasted for the first 2 h but had free access to water. Blood samples (∼200 L) taken from the tail vein were collected in heparinized tubes at the following time points: 0, 0.083, 0.17, 0.25 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4 and 6 h after the p.o. dose and
J. Fan et al. / J. Chromatogr. B 967 (2014) 57–62
59
Fig. 1. Chemical structures and mass spectra of CTX (A) and IS (B).
0, 0.083, 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 4 and 6 h after the i.v. dose. The blood samples were immediately centrifuged at 3500 rpm for 10 min and the plasma removed and stored at −20 ◦ C until analysis. Pharmacokinetic parameters for CTX were calculated with the Drug and Statistics (DAS) Software (version 2.1, Mathematical Pharmacology Professional Committee of China, Shanghai, China) using a non-compartmental model. Data was expressed as mean ± SD.
3. Results and discussion
blank plasma, blank plasma spiked with CTX and the IS, a carryover blank sample and a rat plasma sample are shown in Fig. 2. 3.3. Sample preparation Liquid–liquid extraction and protein precipitation were compared as methods of sample preparation; the former produced a cleaner background. Ethyl acetate, diethyl ether, methyl tert-butyl ether and dichloromethane were investigated as extraction solvents; ethyl acetate was selected as the extraction agent since it gave stable recovery and is more environmentally friendly.
3.1. Optimization of MS conditions 3.4. Method validation Operating parameters for MS detection of CTX and the IS were optimized by flow injection using the standard solutions. The MS spectra were recorded and are shown in Fig. 1. CTX and the IS showed the [M+H]+ ions at m/z 318.8 and 284.8, respectively. These [M+H]+ ions were used as the precursors to select product ions formed by CID. The strongest fragment for CTX was the ion at m/z 165.0, as reported previously [19]. The transition m/z 318.8 → 165.0 was then used to optimize the CID and other MS parameters for CTX. Parameters for the IS transition m/z 284.8 → 269.9 were optimized in the same way as those for CTX.
3.2. Optimization of chromatographic conditions Mixtures of methanol and acetonitrile with water and different buffers, such as formic acid, ammonium formate and acetic acid, were investigated as chromatography solvents. Acetonitrile, rather than methanol, was chosen for the quantification of CTX because it produced more symmetrical peaks for CTX and the IS. Different buffers showed no obvious advantage over water. Isocratic elution using acetonitrile–water (90:10, v/v) was found to achieve suitable resolution and a short run time. Representative chromatograms of
3.4.1. Specificity Typical chromatograms (Fig. 2) showed that, under the chromatographic conditions described above, there were no obvious endogenous interferences at the retention time of CTX and the IS. 3.4.2. Linearity, LLOQ and carryover effect The calibration curve for CTX exhibited good linearity over the concentration range 0.5–1000 ng/mL. The typical standard curve was described by the equation y = 0.1678x − 0.05151, where y is the peak area ratio of the component to the IS, and x is the concentration of CTX. The square regression coefficient (r2 ) was found to be >0.99. The LLOQ of CTX was 0.5 ng/mL (n = 5, S/N was 297.6 ± 40.6, RE = 6.0%) in this study. No carryover effect was detected with the chosen settings; a chromatogram of a carryover blank sample is presented in Fig. 2. 3.4.3. Precision, accuracy and dilution effect The precision and accuracy of the method were assessed using QC samples (1, 50 and 800 ng/mL). The method was found to be highly precise with intra-day precision ≤6.5% and inter-day precision ≤8.7%, and highly accurate with ≤12.5% deviation from
60
J. Fan et al. / J. Chromatogr. B 967 (2014) 57–62
Fig. 2. Chromatograms of blank rat plasma (A); LLOQ (B); a calibration standard (50 ng/mL CTX) (C); a carryover blank sample (D) and a rat plasma sample at 30 min after oral dose of 100 mg/kg CTX (E).
J. Fan et al. / J. Chromatogr. B 967 (2014) 57–62
61
Table 1 Intra- and inter-batch precision and accuracy for CTX in rat plasma (n = 5). Added concentration (ng/mL)
Precision (RSD, %)
0.5 1 50 800
Accuracy (RE, %)
Intra-day
Inter-day
Intra-day
Inter-day
7.9 6.5 5.7 5.3
9.8 8.7 5.7 8.1
6.0 12.5 −2.1 10.9
5.4 6.7 4.3 4.8
Table 2 Matrix effects and recoveries for determination of CTX in plasma (n = 5). Concentration (ng/mL)
Recovery (mean ± SD (%))
Matrix effecta (mean ± SD (%))
1 50 800
112.2 ± 4.3 98.6 ± 4.4 110.0 ± 5.8
90.4 ± 6.8 93.9 ± 3.7 105.4 ± 8.4
a
100% means no matrix effect.
Table 3 Results of stability test for determination of CTX in rat plasma (n = 5). Stability
Spiked concentration (ng/mL)
Remaining (mean ± SD, ng/mL)
Accuracy (%)a
24 h at 4 ◦ C (in an auto-sampler)
1 50 800
1.09 ± 0.07 55.5 ± 5.0 765.0 ± 51.9
109.0 111.0 95.6
8 h (bench-top)
1 50 800
1.05 ± 0.1 47.5 ± 2.2 770.1 ± 34.5
105.0 95.0 96.3
Three freeze–thaw cycles
1 50 800
0.97 ± 0.08 52.4 ± 4.5 814.3 ± 57.6
97.0 104.8 101.8
Two weeks at −20 ◦ C a
1 50 800
1.10 ± 0.06 46.4 ± 3.7 774.5 ± 33.6
110.0 92.8 96.8
(Mean assayed concentration/nominal value) × 100%.
the nominal values at each QC sample concentration (Table 1). Dilution integrity samples at 5000 ng/mL were calculated as 4289.0 ± 274.0 ng/mL; the RE was −14.2% (n = 5).
Fig. 3. Mean plasma concentration–time profiles of CTX after oral (100 mg/kg) and intravenous (25 mg/kg) administration in rats (n = 5).
Table 4 The pharmacokinetic parameters of CTX in rats after intravenous or oral administration (n = 5, mean ± SD). Parameters
Intravenous
AUC0–t (g h/L) AUC0–∞ (g h/L) MRT0–t (g h/L) MRT0–∞ (g h/L) t1/2Z (h) Tmax (h) CLZ /F (L/h/kg) VZ /F (L/kg) Cmax (g/L) F (%)
1257.6 ± 1270.1 ± 0.467 ± 0.59 ± 1.4 ± / 22.9 ± 55.3 ± 4961.2 ± /
570.7 560.6 0.056 0.21 0.76 11.2 54.1 3254.8
Oral 172.8 202.5 1.2 2.4 1.7 0.098 668.7 1443.2 408.8 3.4
± ± ± ± ± ± ± ± ± ±
118.9 123.8 0.46 1.8 1.1 0.040 396.9 943.0 160.5 2.4
3.4.4. Recovery, matrix effect and stability The extraction recovery was in the range 98.6–112.2% (Table 2). The ratio of the peak area resolved in the post-extraction blank sample with that resolved in the mobile phase showed no significant matrix effects. CTX spiked into rat plasma was found to be stable for 8 h at room temperature, for up to two weeks at −20 ◦ C, and during three freeze–thaw cycles (Table 3). Extracted samples were also stable over 24 h in an auto-sampler. The stability was thus satisfactory for a routine pharmacokinetic study.
Based on the area under the concentration–time curve (AUC) values, the absolute bioavailability (F) was calculated as: F = (AUCp.o. × Dosei.v. )/(AUCi.v. × Dosep.o. ) × 100%. The absolute bioavailability of CTX in rats was 3.4 ± 2.4%, suggesting poor absorption via the gastrointestinal segment. CTX thus showed rapid excretion and low bioavailability in rats.
3.5. Pharmacokinetic application
4. Conclusion
The mean plasma concentration–time profiles of CTX in rats are illustrated in Fig. 3. The main pharmacokinetic parameters were calculated with DAS 2.1 software using a non-compartmental model and are presented in Table 4. The plasma elimination half life (t1/2Z ) of CTX was 1.4 ± 0.76 h following the i.v. dose. Total plasma clearance (CLZ ) was 22.9 ± 11.2 L/h/kg and the mean volume of distribution (VZ ) was 55.3 ± 54.1 L/kg. Following p.o. administration, CTX showed rapid absorption (tmax 0.098 ± 0.040 h), with a Cmax of 408.8 ± 160.5 ng/mL. CLZ /F (clearance) and T1/2Z (elimination halflife) were 668.7 ± 396.9 L/h/kg and 1.7 ± 1.1 h, respectively.
A rapid, sensitive and specific HPLC–MS/MS has been developed and validated for a pharmacokinetic study of CTX in rats. The samples were pretreated by a simple liquid–liquid extraction with ethyl acetate and the chromatographic separation was performed on a C18 column using acetonitrile–water (90:10, v/v) as the mobile phase. Tandem MS detection was set in positive MRM mode. The method was validated and successfully used to determine the pharmacokinetic profiles of CTX after oral (100 mg/kg) and intravenous (25 mg/kg) administration in rats. CTX was found to be rapidly excreted and to have low bioavailability in rats.
62
J. Fan et al. / J. Chromatogr. B 967 (2014) 57–62
Acknowledgements This study was financially supported by Seed Funding Program for Basic Research from HKU (201111159043), and the Natural Science Foundation of China (nos. 81274064 and 81373956). References [1] China Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of China, Peoples Medicinal Publishing House, Beijing, 2010, p. 85. [2] Z.B. Guan, Z.L. Li, E. Li, Chin Wild Plant Resour. 21 (2002) 36. [3] G.N. Zhang, Z.M. Bi, Z.T. Wang, L.S. Luo, G.J. Xu, Chin. Tradit. Herb. Drug 34 (2003) 5. [4] J. Xu, Q.B. Han, S.L. Li, X.J. Chen, X.N. Wang, Z.Z. Zhao, H.B. Chen, Phytochem. Rev. 12 (2013) 341. [5] L. Xiao, T.B. Ng, Y.B. Feng, T. Yao, J.H. Wong, R.M. Yao, L. Li, F.Z. Mo, Y. Xiao, P.C. Shaw, Z.M. Li, S.C.W. Sze, K.Y. Zhang, Phytomedicine 18 (2011) 194. [6] Z.Q. Kou, D.B. Yan, F. Feng, Strait. Pharm. J. 25 (2013) 1. [7] J.B. Qu, L.M. Sun, H.X. Lou, Chin. Chem. Lett. 24 (2013) 801.
[8] H. Sawada, M. Okazaki, D. Morita, T. Kuroda, K. Matsuno, Y. Hashimoto, H. Miyachi, Bioorg. Med. Chem. Lett. 22 (2012) 7444. [9] J.B. Qu, C.F. Xie, H.F. Guo, W.T. Yu, H.X. Lou, Phytochemistry 68 (2007) 1767. [10] H. Sawada, K. Onoda, D. Morita, E. Ishitsubo, K. Matsubo, K. Matsuno, H. Tokiwa, T. Kuroda, H. Miyachi, Bioorg. Med. Chem. Lett. 23 (2013) 6563. [11] J.X. Song, P.C. Shaw, C.W. Sze, Y. Tong, X.S. Yao, T.B. Ng, Y.B. Zhang, Neurochem. Int. 57 (2010) 676. [12] J.X. Song, P.C. Shaw, N.S. Wong, C.W. Sze, X.S. Yao, C.W. Tang, Y. Tong, Y.B. Zhang, Neurosci. Lett. 521 (2012) 76. [13] G.X. Ma, G.J. Xu, L.S. Xu, Z.T. Wang, C.C. Xu, Acta Pharm. Sin. 31 (1996) 222. [14] G.X. Ma, G.J. Xu, L.S. Xu, Z.T. Wang, J. Chin. Pharm. Univ. 25 (1994) 103. [15] Q. Ding, Z.M. Bi, Z.T. Wang, L.S. Xu, Chin. Tradit. Herb. Drugs 39 (2008) 610. [16] L. Yang, Z.T. Wang, L.S. Xu, J. Chromatogr. A 1104 (2006) 230. [17] US NIH, Guide for the Care and Use of Laboratory Animals, http://grants. nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf [18] Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research Center for Veterinary Medicine, May 2001, http://www.fda.gov/cder/guidance/index.htm [19] L. Yang, Y. Wang, G.N. Zhang, F. Zhang, Z.J. Zhang, Z.T. Wang, L.S. Xu, Biomed. Chromatogr. 21 (2007) 687.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of chrysotoxine in rat plasma by liquid chromatography–tandem mass spectrometry and its application to a rat pharmacokinetic study Jingjing Fan a , Li Guan b , Zeqi Kou b , Feng Feng b , Yanbo Zhang c,∗ , Wenyuan Liu a,∗∗ a
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, China Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, 210009, China c School of Chinese Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, China b
a r t i c l e
i n f o
Article history: Received 25 January 2014 Accepted 9 July 2014 Available online 16 July 2014 Keywords: Chrysotoxine HPLC–MS/MS Pharmacokinetics Method validation Bioavailability
a b s t r a c t Chrysotoxine (CTX), a naturally occurring bibenzyl compound isolated from Dendrobium species, has been reported to have neuroprotective effects. To evaluate its pharmacokinetics in rats, a rapid, sensitive and specific high performance liquid chromatography–tandem mass spectrometric (HPLC–MS/MS) method has been developed and validated for the quantification of CTX in rat plasma. Samples were pretreated using a simple liquid–liquid extraction with ethyl acetate and the chromatographic separation was performed on a C18 column with acetonitrile–water (90:10, v/v) as the mobile phase. CTX and the internal standard (wogonin) were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. Method validation revealed excellent linearity over the range 0.5–1000 ng/mL together with satisfactory intra- and inter-day precision, accuracy and recovery. Stability testing showed that CTX spiked into rat plasma was stable for 8 h at room temperature, for up to two weeks at −20 ◦ C, and during three freeze–thaw cycles. Extracted samples were also observed to be stable over 24 h in an auto-sampler. The method was successfully used to investigate the pharmacokinetic profile of CTX after oral (100 mg/kg) and intravenous (25 mg/kg) administration in rats. CTX showed rapid excretion and low bioavailability in rats. © 2014 Published by Elsevier B.V.
1. Introduction Dendrobium species, such as Dendrobium nobile Lindl., Dendrobium chrysotoxum Lindl. and Dendrobium fimbriatum Hook, play an important role in traditional Chinese medicine. Activities recorded in the Chinese pharmacopoeia [1] include hypoglycemic, antitumor, anti-aging and immunomodulatory effects [2–5]. In recent years, bibenzyls in Dendrobium have attracted much attention for their unique structures and biological activities [6–10]. Chrysotoxine (CTX) is a bibenzyl isolated from Dendrobium species. Recent studies have revealed that CTX can inhibit dopaminergic cell death in SH-SY5Y cells induced by 6-hydroxydopamine and 1-methyl-4phenyl pyridinium, suggesting a potential protective effect against neurodegeneration [11,12]. To evaluate the potential of CTX as
a drug candidate, we have now investigated its pharmacokinetic properties in rats. Several methods for the separation and analysis of CTX in raw herbs have been reported; most of these use mass spectrometry (MS), ultraviolet (UV) spectroscopy, infra-red (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy [13], high performance liquid chromatography (HPLC) or high performance liquid chromatography–diode array detection (HPLC–DAD) [14–16]. However, none of these methods has been used for pharmacokinetic studies and the aim of the present work was to develop a rapid, sensitive and specific analytical method for the determination of CTX in biological samples. Using our newly developed method, we then evaluated the bioavailability of CTX in rats.
2. Experimental ∗ Corresponding author. Tel.: +852 9506 3396. ∗∗ Corresponding author. Tel.: +86 25 8327 1038. E-mail addresses: [email protected] (Y. Zhang), [email protected] (W. Liu). http://dx.doi.org/10.1016/j.jchromb.2014.07.011 1570-0232/© 2014 Published by Elsevier B.V.
2.1. Drug and chemicals Chrysotoxine (CTX, purity >96.0%) and wogonin (purity >99.5%, internal standard, IS) were prepared by the Department of Natural
58
J. Fan et al. / J. Chromatogr. B 967 (2014) 57–62
Medicinal Chemistry, China Pharmaceutical University (Nanjing, China) and their structures were confirmed by comprehensive analysis using high resolution mass spectrometry, UV and IR spectroscopy and 1D and 2D NMR spectroscopy. Acetonitrile (HPLC grade; Merck) and ultra pure water from a Milli-Q system (Millipore) were used to prepare the mobile phase. All other reagents were of analytical grade and obtained from conventional commercial sources.
Each drug-free rat plasma sample (50 L) was spiked with IS solution (5 L) and serial standard solutions of CTX (5 L) to prepare calibration standards in the concentration range 0.5–1000 ng/mL. QC samples were prepared by spiking control rat plasma in bulk at appropriate concentrations, and then dividing into small aliquots (around 80 L) in different tubes. These samples were stored under the same conditions as the experimental samples and underwent the same pretreatment procedure (described below).
2.2. Instrumentation and conditions 2.5. Sample pretreatment procedure A Shimadzu LC-2010 series HPLC system (Kyoto, Japan), equipped with a quaternary pump, a vacuum degasser, an auto-sampler and a column heater-cooler was connected by an electrospray ionization (ESI) interface to a Thermo Finnigan TSQ AM tandem mass spectrometer (Thermo Finnigan, San Jose, CA, USA). Samples were separated on a YMC-Pack ODS-A column (50 mm × 4.6 mm, 3 m, YMC Co. Ltd., Kyoto, Japan) with the column temperature set at 30 ◦ C. The mobile phase consisted of acetonitrile–water (90:10, v/v), delivered at a flow rate of 0.7 mL/min with a split ratio of 1:3. The injection volume was 10 L and the run time was 3 min. Solvent eluted from chromatography column during the first minute was switched to waste before it entered the ion source. The MS was set in positive multiple reaction monitoring (MRM) mode. MS transitions were m/z 318.8 → 165.0 for CTX and m/z 284.8 → 269.9 for the IS in MRM mode. The MS operating conditions were optimized and set as follows: sheath gas pressure, 35 arbitrary units; the auxiliary gas pressure, 5 arbitrary units; capillary temperature, 350 ◦ C; spray voltage, 5 kV and source collision-induces dissociation (CID), 10 V. The collision gas was argon and the collision energies were 20 eV for CTX and 26 eV for the IS, respectively. Data were processed using Xcalibur software (Thermo Finnigan, San Jose, CA, USA). 2.3. Animals Healthy male Sprague-Dawley rats (200–240 g) (Certificate No. SCXK2008-0016) were purchased from Shanghai Super-B&K Laboratory Animal Co., Ltd. (Shanghai, China). Experiments using animals were approved by the Animal Ethics Committee of the China Pharmaceutical University (Nanjing, China) and conformed to the Guide for Care and Use of Laboratory Animals published by the US National Institute of Health [17]. Animals were housed in a room at a controlled temperature of 23–26 ◦ C and a relative humidity of 40–60%, with access to food and water ad libitum. All animals were acclimated in the laboratory for at least seven days prior to the experiment and fasted for 12 h but allowed water ad libitum before experiments. 2.4. Preparation of standard and quality control (QC) samples Stock solutions of CTX (1 mg/mL) and IS (1 mg/mL) in acetonitrile were prepared separately. In order to ensure weighing precision, two weighings, as long as their concentrations agreed within 5%, were prepared, then one was used for calibrators and the other for QC samples. Primary stock solutions for calibration curve standards and QC samples were prepared using separate weighings. Standard solutions of CTX for the preparation of calibration curves were obtained by further dilution of the stock solution with acetonitrile to give final concentrations of 5, 10, 20, 50, 100, 500, 2000, 5000 and 10,000 ng/mL. QC solutions were prepared at concentrations of 10, 500, 8000 ng/mL by dilution of the primary stock solution with acetonitrile. A solution containing the IS (50 ng/mL) was also obtained by dilution of the IS stock solution with acetonitrile. All standard solutions were stored at 4 ◦ C.
All samples were thawed to room temperature before analysis. The plasma sample (50 L) was transferred into a 1.5 mL centrifuge tube and IS working solution (5 L) was then added. The mixture was vortex-mixed for 1 min and then extracted with ethyl acetate (1 mL) by vortex-mixing for 3 min. The sample was then centrifuged at 16,000 rpm for 5 min and the supernatant was transferred into another test tube and evaporated to dryness at 37 ◦ C. The residue was reconstituted in mobile phase (50 L) by vortex-mixing for 2 min and centrifuging at 16,000 rpm for 3 min. Finally, the supernatant (10 L) was injected for HPLC–MS/MS analysis. 2.6. Method validation Method validation for determination of CTX in rat plasma was performed according to the US Food and Drug Administration (FDA) guidance [18]. Selectivity was evaluated by comparing chromatograms of blank plasma samples collected from six different rats with the chromatogram of a plasma sample spiked with CTX and the IS. A least-squares linear regression method (1/x2 weighting) was used to determine the slope, intercept and square regression coefficient (r2 ) of the linear regression equation. The calibration curve was established using the Bioavailability Program Package software (BAPP, Version 2.2, Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University). Precision and accuracy were calculated by determining QC samples at three concentration levels. Precision is expressed as the relative standard deviation (RSD) and accuracy as the relative error (RE). Precision and accuracy were also assessed at the lowest concentration of the standards (0.5 ng/mL), representing the lower limit of quantification (LLOQ) for the assay. A blank sample was placed immediately after the upper limit of quantification (ULOQ) standard to evaluate carryover in the LC–MS/MS method. The recovery of analytes was determined by comparing the responses of the analytes from QC samples with analytes spiked in post-extracted blank rat plasma at equivalent concentrations. The matrix effect was measured by comparing the responses obtained from post-extraction blank rat plasma spiked samples with mobile phase spiked with low, middle and high concentrations of analyte. The dilution effect was estimated by analysis of rat plasma spiked with analyte at 5000 ng/mL and diluted 10-fold with blank rat plasma. The stability of the analyte in rat plasma was evaluated by analyzing QC samples stored under the following conditions: at 4 ◦ C in an auto-sampler for 24 h, at room temperature for 8 h and at −20 ◦ C for two weeks. The effect of three freeze/thaw cycles on the analyte was also examined. 2.7. Pharmacokinetic study Ten rats were randomly divided into two groups. Five rats received CTX (100 mg/kg) by oral administration (p.o.) and the other group received CTX (25 mg/kg) by intravenous injection (i.v.). After dosing, the rats were fasted for the first 2 h but had free access to water. Blood samples (∼200 L) taken from the tail vein were collected in heparinized tubes at the following time points: 0, 0.083, 0.17, 0.25 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4 and 6 h after the p.o. dose and
J. Fan et al. / J. Chromatogr. B 967 (2014) 57–62
59
Fig. 1. Chemical structures and mass spectra of CTX (A) and IS (B).
0, 0.083, 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 4 and 6 h after the i.v. dose. The blood samples were immediately centrifuged at 3500 rpm for 10 min and the plasma removed and stored at −20 ◦ C until analysis. Pharmacokinetic parameters for CTX were calculated with the Drug and Statistics (DAS) Software (version 2.1, Mathematical Pharmacology Professional Committee of China, Shanghai, China) using a non-compartmental model. Data was expressed as mean ± SD.
3. Results and discussion
blank plasma, blank plasma spiked with CTX and the IS, a carryover blank sample and a rat plasma sample are shown in Fig. 2. 3.3. Sample preparation Liquid–liquid extraction and protein precipitation were compared as methods of sample preparation; the former produced a cleaner background. Ethyl acetate, diethyl ether, methyl tert-butyl ether and dichloromethane were investigated as extraction solvents; ethyl acetate was selected as the extraction agent since it gave stable recovery and is more environmentally friendly.
3.1. Optimization of MS conditions 3.4. Method validation Operating parameters for MS detection of CTX and the IS were optimized by flow injection using the standard solutions. The MS spectra were recorded and are shown in Fig. 1. CTX and the IS showed the [M+H]+ ions at m/z 318.8 and 284.8, respectively. These [M+H]+ ions were used as the precursors to select product ions formed by CID. The strongest fragment for CTX was the ion at m/z 165.0, as reported previously [19]. The transition m/z 318.8 → 165.0 was then used to optimize the CID and other MS parameters for CTX. Parameters for the IS transition m/z 284.8 → 269.9 were optimized in the same way as those for CTX.
3.2. Optimization of chromatographic conditions Mixtures of methanol and acetonitrile with water and different buffers, such as formic acid, ammonium formate and acetic acid, were investigated as chromatography solvents. Acetonitrile, rather than methanol, was chosen for the quantification of CTX because it produced more symmetrical peaks for CTX and the IS. Different buffers showed no obvious advantage over water. Isocratic elution using acetonitrile–water (90:10, v/v) was found to achieve suitable resolution and a short run time. Representative chromatograms of
3.4.1. Specificity Typical chromatograms (Fig. 2) showed that, under the chromatographic conditions described above, there were no obvious endogenous interferences at the retention time of CTX and the IS. 3.4.2. Linearity, LLOQ and carryover effect The calibration curve for CTX exhibited good linearity over the concentration range 0.5–1000 ng/mL. The typical standard curve was described by the equation y = 0.1678x − 0.05151, where y is the peak area ratio of the component to the IS, and x is the concentration of CTX. The square regression coefficient (r2 ) was found to be >0.99. The LLOQ of CTX was 0.5 ng/mL (n = 5, S/N was 297.6 ± 40.6, RE = 6.0%) in this study. No carryover effect was detected with the chosen settings; a chromatogram of a carryover blank sample is presented in Fig. 2. 3.4.3. Precision, accuracy and dilution effect The precision and accuracy of the method were assessed using QC samples (1, 50 and 800 ng/mL). The method was found to be highly precise with intra-day precision ≤6.5% and inter-day precision ≤8.7%, and highly accurate with ≤12.5% deviation from
60
J. Fan et al. / J. Chromatogr. B 967 (2014) 57–62
Fig. 2. Chromatograms of blank rat plasma (A); LLOQ (B); a calibration standard (50 ng/mL CTX) (C); a carryover blank sample (D) and a rat plasma sample at 30 min after oral dose of 100 mg/kg CTX (E).
J. Fan et al. / J. Chromatogr. B 967 (2014) 57–62
61
Table 1 Intra- and inter-batch precision and accuracy for CTX in rat plasma (n = 5). Added concentration (ng/mL)
Precision (RSD, %)
0.5 1 50 800
Accuracy (RE, %)
Intra-day
Inter-day
Intra-day
Inter-day
7.9 6.5 5.7 5.3
9.8 8.7 5.7 8.1
6.0 12.5 −2.1 10.9
5.4 6.7 4.3 4.8
Table 2 Matrix effects and recoveries for determination of CTX in plasma (n = 5). Concentration (ng/mL)
Recovery (mean ± SD (%))
Matrix effecta (mean ± SD (%))
1 50 800
112.2 ± 4.3 98.6 ± 4.4 110.0 ± 5.8
90.4 ± 6.8 93.9 ± 3.7 105.4 ± 8.4
a
100% means no matrix effect.
Table 3 Results of stability test for determination of CTX in rat plasma (n = 5). Stability
Spiked concentration (ng/mL)
Remaining (mean ± SD, ng/mL)
Accuracy (%)a
24 h at 4 ◦ C (in an auto-sampler)
1 50 800
1.09 ± 0.07 55.5 ± 5.0 765.0 ± 51.9
109.0 111.0 95.6
8 h (bench-top)
1 50 800
1.05 ± 0.1 47.5 ± 2.2 770.1 ± 34.5
105.0 95.0 96.3
Three freeze–thaw cycles
1 50 800
0.97 ± 0.08 52.4 ± 4.5 814.3 ± 57.6
97.0 104.8 101.8
Two weeks at −20 ◦ C a
1 50 800
1.10 ± 0.06 46.4 ± 3.7 774.5 ± 33.6
110.0 92.8 96.8
(Mean assayed concentration/nominal value) × 100%.
the nominal values at each QC sample concentration (Table 1). Dilution integrity samples at 5000 ng/mL were calculated as 4289.0 ± 274.0 ng/mL; the RE was −14.2% (n = 5).
Fig. 3. Mean plasma concentration–time profiles of CTX after oral (100 mg/kg) and intravenous (25 mg/kg) administration in rats (n = 5).
Table 4 The pharmacokinetic parameters of CTX in rats after intravenous or oral administration (n = 5, mean ± SD). Parameters
Intravenous
AUC0–t (g h/L) AUC0–∞ (g h/L) MRT0–t (g h/L) MRT0–∞ (g h/L) t1/2Z (h) Tmax (h) CLZ /F (L/h/kg) VZ /F (L/kg) Cmax (g/L) F (%)
1257.6 ± 1270.1 ± 0.467 ± 0.59 ± 1.4 ± / 22.9 ± 55.3 ± 4961.2 ± /
570.7 560.6 0.056 0.21 0.76 11.2 54.1 3254.8
Oral 172.8 202.5 1.2 2.4 1.7 0.098 668.7 1443.2 408.8 3.4
± ± ± ± ± ± ± ± ± ±
118.9 123.8 0.46 1.8 1.1 0.040 396.9 943.0 160.5 2.4
3.4.4. Recovery, matrix effect and stability The extraction recovery was in the range 98.6–112.2% (Table 2). The ratio of the peak area resolved in the post-extraction blank sample with that resolved in the mobile phase showed no significant matrix effects. CTX spiked into rat plasma was found to be stable for 8 h at room temperature, for up to two weeks at −20 ◦ C, and during three freeze–thaw cycles (Table 3). Extracted samples were also stable over 24 h in an auto-sampler. The stability was thus satisfactory for a routine pharmacokinetic study.
Based on the area under the concentration–time curve (AUC) values, the absolute bioavailability (F) was calculated as: F = (AUCp.o. × Dosei.v. )/(AUCi.v. × Dosep.o. ) × 100%. The absolute bioavailability of CTX in rats was 3.4 ± 2.4%, suggesting poor absorption via the gastrointestinal segment. CTX thus showed rapid excretion and low bioavailability in rats.
3.5. Pharmacokinetic application
4. Conclusion
The mean plasma concentration–time profiles of CTX in rats are illustrated in Fig. 3. The main pharmacokinetic parameters were calculated with DAS 2.1 software using a non-compartmental model and are presented in Table 4. The plasma elimination half life (t1/2Z ) of CTX was 1.4 ± 0.76 h following the i.v. dose. Total plasma clearance (CLZ ) was 22.9 ± 11.2 L/h/kg and the mean volume of distribution (VZ ) was 55.3 ± 54.1 L/kg. Following p.o. administration, CTX showed rapid absorption (tmax 0.098 ± 0.040 h), with a Cmax of 408.8 ± 160.5 ng/mL. CLZ /F (clearance) and T1/2Z (elimination halflife) were 668.7 ± 396.9 L/h/kg and 1.7 ± 1.1 h, respectively.
A rapid, sensitive and specific HPLC–MS/MS has been developed and validated for a pharmacokinetic study of CTX in rats. The samples were pretreated by a simple liquid–liquid extraction with ethyl acetate and the chromatographic separation was performed on a C18 column using acetonitrile–water (90:10, v/v) as the mobile phase. Tandem MS detection was set in positive MRM mode. The method was validated and successfully used to determine the pharmacokinetic profiles of CTX after oral (100 mg/kg) and intravenous (25 mg/kg) administration in rats. CTX was found to be rapidly excreted and to have low bioavailability in rats.
62
J. Fan et al. / J. Chromatogr. B 967 (2014) 57–62
Acknowledgements This study was financially supported by Seed Funding Program for Basic Research from HKU (201111159043), and the Natural Science Foundation of China (nos. 81274064 and 81373956). References [1] China Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of China, Peoples Medicinal Publishing House, Beijing, 2010, p. 85. [2] Z.B. Guan, Z.L. Li, E. Li, Chin Wild Plant Resour. 21 (2002) 36. [3] G.N. Zhang, Z.M. Bi, Z.T. Wang, L.S. Luo, G.J. Xu, Chin. Tradit. Herb. Drug 34 (2003) 5. [4] J. Xu, Q.B. Han, S.L. Li, X.J. Chen, X.N. Wang, Z.Z. Zhao, H.B. Chen, Phytochem. Rev. 12 (2013) 341. [5] L. Xiao, T.B. Ng, Y.B. Feng, T. Yao, J.H. Wong, R.M. Yao, L. Li, F.Z. Mo, Y. Xiao, P.C. Shaw, Z.M. Li, S.C.W. Sze, K.Y. Zhang, Phytomedicine 18 (2011) 194. [6] Z.Q. Kou, D.B. Yan, F. Feng, Strait. Pharm. J. 25 (2013) 1. [7] J.B. Qu, L.M. Sun, H.X. Lou, Chin. Chem. Lett. 24 (2013) 801.
[8] H. Sawada, M. Okazaki, D. Morita, T. Kuroda, K. Matsuno, Y. Hashimoto, H. Miyachi, Bioorg. Med. Chem. Lett. 22 (2012) 7444. [9] J.B. Qu, C.F. Xie, H.F. Guo, W.T. Yu, H.X. Lou, Phytochemistry 68 (2007) 1767. [10] H. Sawada, K. Onoda, D. Morita, E. Ishitsubo, K. Matsubo, K. Matsuno, H. Tokiwa, T. Kuroda, H. Miyachi, Bioorg. Med. Chem. Lett. 23 (2013) 6563. [11] J.X. Song, P.C. Shaw, C.W. Sze, Y. Tong, X.S. Yao, T.B. Ng, Y.B. Zhang, Neurochem. Int. 57 (2010) 676. [12] J.X. Song, P.C. Shaw, N.S. Wong, C.W. Sze, X.S. Yao, C.W. Tang, Y. Tong, Y.B. Zhang, Neurosci. Lett. 521 (2012) 76. [13] G.X. Ma, G.J. Xu, L.S. Xu, Z.T. Wang, C.C. Xu, Acta Pharm. Sin. 31 (1996) 222. [14] G.X. Ma, G.J. Xu, L.S. Xu, Z.T. Wang, J. Chin. Pharm. Univ. 25 (1994) 103. [15] Q. Ding, Z.M. Bi, Z.T. Wang, L.S. Xu, Chin. Tradit. Herb. Drugs 39 (2008) 610. [16] L. Yang, Z.T. Wang, L.S. Xu, J. Chromatogr. A 1104 (2006) 230. [17] US NIH, Guide for the Care and Use of Laboratory Animals, http://grants. nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf [18] Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research Center for Veterinary Medicine, May 2001, http://www.fda.gov/cder/guidance/index.htm [19] L. Yang, Y. Wang, G.N. Zhang, F. Zhang, Z.J. Zhang, Z.T. Wang, L.S. Xu, Biomed. Chromatogr. 21 (2007) 687.
