Journal of Chromatography B, 941 (2013) 25–30
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
Development and validation of a quantitative liquid chromatography tandem mass spectrometry assay for pristimerin in rat plasma Xin Luan a,1 , Ying-Yun Guan a,1 , Ya-Rong Liu a , Chao Wang a , Mei Zhao b,c , Qin Lu a , Ya-Bin Tang a , Xiao-Lin Wang a , Chao Fang a,∗ , Hong-Zhuan Chen a,∗∗ a Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China b Department of Pharmacy, Shanghai Institute of Health Sciences, Shanghai 201318, China c Department of Pharmacy, Health School Attached to Shanghai Jiao Tong University School of Medicine, Shanghai 201318, China
a r t i c l e
i n f o
Article history: Received 8 June 2013 Accepted 26 September 2013 Available online 3 October 2013 Keywords: Pristimerin HPLC–MS/MS Pharmacokinetics Rat plasma
a b s t r a c t A sensitive, rapid and simple LC–MS/MS analysis method was developed and validated for the determination of pristimerin (PR) in rat plasma. Protein precipitation with four volumes of acetonitrile as the precipitation reagent was used as the sample preparation method. The analysis process was performed on a Merck ZIC-HILIC column with the mobile phase of acetonitrile–water (containing 5 mM ammonium formate, pH 2.8) (85:15, v/v). PR (m/z 465.3–201.1) and glycyrrhetinic acid (internal standard, m/z 471.5–177.1) were monitored under positive electrospray ionization in multiple reaction monitoring (MRM) mode. Retention time of PR and IS was 2.45 min and 2.4 min, respectively. The limit of detection was 0.5 ng/mL and the linear range was 1–500 ng/mL. The intra-day and inter-day precision were 2.89–6.27% and 4.91–8.98%, and the intra-day and inter-day accuracy ranged from −5.81% to 8.64% and −7.37% to 9.57%, respectively. The matrix effects and absolute recovery ranged from 89.3% to 92.4% and 88.7% to 92.8%, respectively. The method has been successfully applied to the determination of PR concentration in rat plasma after intravenous administration (0.5 mg/kg). © 2013 Elsevier B.V. All rights reserved.
1. Introduction Pristimerin (PR) is a representative of the quinonemethide triterpenoids, which are mainly found in the Celastraceae and Hippocrateaceae families [1]. PR has attracted considerable interests due to its multiple biological properties, including antitumor, antiinflammatory, antioxidant, antimalarial and antifungal activities [2–7]. Recently, the potent antitumor activities of PR have been particularly evaluated, and attractive progress has been made in this field. Extensive studies found that PR exhibits cytotoxicity in several cancer cell lines by apoptosis induction through activating caspase [8–11] and inhibiting proteasome [12] or DNA synthesis [13]. Interestingly, PR is also effective in inhibiting the growth of drug-resistant cancer cells such as vincristine-resistant KB cells and paclitaxel-resistant 1A9 cells [14]. Moreover, PR has also been
∗ Corresponding author. Tel.: +86 21 63846590x778013; fax: +86 21 64674721. ∗∗ Corresponding author. Tel.: +86 21 64674721; fax: +86 21 64674721. E-mail addresses: [email protected] (C. Fang), [email protected] (H.-Z. Chen). 1 These two authors contributed equally to this work. 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.09.038
proved to be able to interfere with tumor metastasis by the inhibition of MDA-MB-231 cell migration and invasion [15] and the antiangiogenic activity through blocking VEGFR2 activation [16]. These findings demonstrated that PR was a promising antitumor entity and a potential leading compound for translational cancer research. In contrast to the favorable pharmacodynamic results, the pharmacokinetic profile of PR has not been investigated before and there is still lack of in vivo validated analytical methods for PR determination. A reverse-phase HPLC–UV method was developed for the determination of PR levels in plant extracts [17]. In another study, HPLC–UV and LC–MS analysis were used to confirm the structures and detectable amounts of PR in the plant extracts; however, the reported limit of detection (LOD) was 40 g/mL [18]. A more sensitive and rapid analysis method needs to be developed and validated for the concentration monitoring of PR in vivo. Compared with HPLC–UV method, LC–MS/MS has the advantages in both qualitative and quantitative basis. The qualitative advantages come from the superior specificity due to resolving co-eluting species by mass. The quantitative advantage is achieved by the low noise, and thus high S/N, observed at specific m/z values for different compounds [19]. In this study,
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a new, rapid, sensitive, and selective LC/MS/MS method was developed, validated and successfully applied in the determination of PR concentration in vivo after intravenous administration (0.5 mg/kg). 2. Experimental 2.1. Reagents and chemicals PR (purity ≥98%) and glycyrrhetinic acid (purity ≥98%) (internal standard, IS) were purchased from Pure-one Bio Technology Co. Ltd. (Shanghai, China). Ammonium formate was purchased from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). HPLCgrade acetonitrile and formic acid were purchased from Merck (Darmstadt, Germany). Water was purified with the Milli-Q Plus system (Millipore, Bedford, MA, USA). Freshly obtained drug free rat plasma was collected from six healthy Sprague-Dawley (SD) rats in our lab and stored at −70 ◦ C prior to use. The heparinized plastic tubes (5 mL, 13 mm × 100 mm) were obtained from Shanghai Kehua Bio-engineering Co. Ltd. (China). The syringes (1 mL, 0.4 mm × 13 mm) were purchased from Kindly Enterprise Development Group (Shanghai, China). 2.2. Animals Female SD rats (200–220 g) were supplied by the SLAC Laboratory Animal Co. Ltd. (Shanghai, China), and were housed in an environmentally controlled quarters (20–25 ◦ C, relative humidity 55–65%, 12 h light/12 h dark cycle) for 3 days before experiment. The rats were fasted overnight before dosing, but the water was available all the time. Animal experiments were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. 2.3. Instrument and conditions Analyses were performed on an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technologies, Inc., USA) equipped with electrospray ionization (ESI) and an Agilent 1200 HPLC system (Agilent Technologies, Inc., USA). The instrumental components of Agilent 1200 HPLC system were G1322A Degasser, G1311A Quat Pump, G1329A ALS Auto Sampler, and G1316A COLCOM Column Oven. A Merck ZIC-HILIC column (2.1 mm × 100 mm, 3.5 m) was used for analyte separation. Isocratic elution with a mobile phase consisting of acetonitrile and water (85:15, v/v, the aqueous phase contained 5 mM ammonium formate, was adjusted to pH 2.8 with formic acid) was used for the separation. The flow rate was 0.1 mL/min, the run time of each sample was 5 min, the injection volume was 2 L and the column temperature was kept at 40 ◦ C. Data were collected under the positive ionization mode with multiple reaction monitoring (MRM). Two MRM transitions, PR (m/z 465.3–201.1, fragmentor 135 eV, collision energy 20 eV) and IS (m/z 471.5–177.1, fragmentor 160 eV, collision energy 30 eV) were monitored. The working parameters of the ESI source were set as follows: temperature 300 ◦ C, capillary voltage 4000 V, drying-gas flow rate 9 L/min and nebulizer pressure 40 psi. Data processing of MS was performed on the MassHunter software package (Version B.04.00, Agilent Technologies, Inc., USA). 2.4. Preparation of calibration standards and quality control (QC) samples The stock solutions of PR and IS each with the exact concentration of 500 g/mL were prepared in acetonitrile and stored at −70 ◦ C. Working solutions for spiking plasma ranging from 5 to
2500 ng/mL were all freshly prepared by step wise dilution of the stock solution with acetonitrile. A series of calibration standards were prepared by spiking blank plasma (80 L) with 20 L aliquots of standard solutions to yield the concentrations of 1, 5, 10, 50, 100, 250 and 500 ng/mL. QC samples were prepared at three levels (low: 5 ng/mL, medium: 100 ng/mL, high: 450 ng/mL) independently in the same way. All QC samples were stored at −70 ◦ C. The IS working solution (2500 ng/mL) was made by diluting the stock solution with acetonitrile. 2.5. Sample preparation All frozen standards and samples were thawed at room temperature (25 ◦ C) and homogenized by vortex. One hundred and fifty microliter acetonitrile and 50 L acetonitrile containing IS (2.5 g/mL) were added to 50 L plasma. The mixed solution was homogenized by vortex for 1 min and centrifuged at the condition of 15,000 × g for 5 min with the supernatant (2 L) injected into the system. 2.6. Method validation The method was validated for selectivity, linearity, precision, accuracy, recovery, matrix effect and stability. Blank plasma from six rats with and without PR and IS were used to evaluate the selectivity. Calibration curves were generated at the range of 1–500 ng/mL by using the peak area ratio (y) of PR to IS vs. nominal concentration of PR (x) with the weighting factor of 1/x. The lower limit of quantification (LLOQ) was defined as the lowest concentration point of the calibration curve. QC samples were processed in five replicates at three concentration levels (low, middle, and high) for PR. The precision was expressed as intra- and inter-day relative standard deviation (%RSD, calculated from the standard deviation divided by the mean) of the QC and LLOQ samples. Both the intra-day and inter-day accuracy (defined as relative error, %RE) were determined by comparing the calculated concentration to the theoretical concentration of the QC and LLOQ samples. Inter- and intra-assay precision and accuracy were determined by repeated analysis of QC and LLOQ samples on the same day (n = 5) and on 5 consecutive days. Matrix effects were defined by calculating the ratio of the peak area for PR in deproteinized plasma relative to the peak area for PR in acetonitrile. Absolute recoveries at QC and LLOQ levels were evaluated by determining the peak area ratios of PR in the post-deproteinized spiked samples to that acquired from pre-deproteinized spiked samples. Long-term stability (30 days), room-temperature (4 h) stability, auto-sampler (24 h) stability and three freeze/thaw stability were examined at three levels of QC samples in five replicates. 2.7. Pharmacokinetic study Six SD rats (200–220 g) were treated with intravenous administration through caudal vein, using plastic syringes (1 mL, 0.4 mm × 13 mm). Blood samples (no more than 0.2 mL) were obtained via retro-orbital puncture with a glass capillary at 0 h (pre-dose) and 0.083, 0.167, 0.33, 0.5, 1, 1.5, 2, 4, 6, 9 and 12 h after administration of 0.5 mg/kg PR dissolved in saline water. The blood sample was collected into the commercial heparinized plastic tubes and separated by 4000 r/min centrifugation (Centrifuge 5417R, Eppendorf, Hamburg, Germany) at 4 ◦ C for 10 min and stored in the refrigerator at −70 ◦ C before the analysis. The pharmacokinetic parameters were calculated with the WinNonlin software (Version 6.1 Pharsight, Mountain View, CA, USA) according to non-compartmental model.
X. Luan et al. / J. Chromatogr. B 941 (2013) 25–30
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Fig. 1. Product ion mass spectra of PR (A) and IS (B) and the chemical structures of both compounds.
3. Results and discussion 3.1. Method development To the best of our knowledge, no LC–MS/MS method has been reported for the determination of PR in plasma. In order to establish a selective and sensitive analysis method, the mass spectrometry and chromatographic conditions should be developed. In our experiment, we selected glycyrrhetinic acid, an analog of PR as IS due to its similar basic structure, physicochemical property and mass spectrometric behavior to those of PR. The MS/MS operation parameters have been carefully optimized for the determination of PR. Positive and negative ion mode were tried for the ionization of PR. Both modes could produce the correct precursor ions, whereas the transition between precursor ion and the ring-opened product ion under positive mode achieved higher response. Thus, [M+H]+ (465.3) was chosen for further fragmentation, and the MassHunter optimizer software (Version B.03.01, Agilent Technologies, Santa Clara, CA, USA) was used to obtain the product ion and optimize ESI source parameters such as fragmentor voltage and collision energy
(CE). The product ion spectra of PR (465.3) and IS (471.5) and their allocation are shown in Fig. 1. The desolvation temperature was set at 300 ◦ C to prevent PR and IS from thermal decomposition at a higher temperature and meanwhile guarantee the effects of desolvation. We found that when the pH of the mobile phase was adjusted to 2.8, the peaks of both PR and IS was symmetrical and sharp. A Merck ZIC-HILIC column was used due to its suited separating capacity for the analyte. The low flow rate (0.1 mL/min) and the high ratio of acetonitrile in mobile phase (85%, v/v) were adopted to obtain an ideal shape of the peaks and appropriate retention time (RT < 5 min) for the purpose of rapid analysis, and it was observed that higher percentage of organic phase can enhance the peak signal by better ionization and shorten the analysis time. The sample preparation method should be optimized to be reproducible, minimized matrix effects and as inexpensive as possible. Thus, protein precipitation with four volumes of acetonitrile was adopted for sample preparation to obtain the balance between better protein deposition effect and higher sample concentration. After centrifugation, the supernatant fluid of the sample can be
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Fig. 2. Representative MRM chromatograms of a double-blank rat plasma sample (A), a blank plasma sample spiked with IS (500 ng/mL) (B), a blank rat plasma sample spiked with PR at LLOQ level (1 ng/mL) and IS (500 ng/mL) (C), and a rat plasma sample at 0.5 h after intravenous injection of PR (0.5 mg/kg) (D), Peak I, IS; Peak II, PR.
directly injected into the analysis system because of its similar composition to that of the mobile phase.
3.2. Method validation The method validation includes several respects such as selectivity, linearity, precision, accuracy, matrix effect, recovery and stability. All the contents were examined according to the US Food and Drug Administration (FDA) bioanalytical method validation guidance [20]. No endogenous interference was observed during the retention time of PR and IS in the chromatograms of different control blank rat plasma. The MRM chromatograms of double-blank rat plasma, plasma sample spiked with IS (500 ng/mL), blank plasma spiked with PR at LLOQ level (1 ng/mL) and IS (500 ng/mL), and the plasma sample obtained at 0.5 h after the intravenous injection of 0.5 mg/kg PR were shown in Fig. 2.
The calibration curve exhibited a good linear correlation within the concentration range of 1–500 ng/mL (y = 0.050424*x − 0.081428) with a weighting factor of 1/x to reduce the effect of large concentrations on the calculation of regression statistics. The coefficient of determination (R2 ) was higher than 0.998. The limit of detection (LOD) for PR, which requires a signal-to-noise ratio above 3, was 0.5 ng/mL. LLOQ was established at 1 ng/mL, and the signal-to-noise ratio (S/N) was 12.4. The relative response factor (RRF) of PR (against the IS) was 25.2. The results of intra-day and inter-day precision, accuracy, matrix effect and absolute recovery of the method for the quantification of PR in rat plasma are summarized in Table 1. The values of S/N at the QC sample concentrations were 74.0 (5 ng/mL), 970.7 (100 ng/mL) and 2596.6 (450 ng/mL), respectively. The values for intra- and inter-day precision were 2.89–6.27% and 4.91–8.98%, and the values for intra- and inter-day accuracy ranged from −5.81% to 8.64% and −7.37% to 9.57%, respectively. The matrix effects of QC samples were 92.4% (5 ng/mL), 91.7% (100 ng/mL) and 89.3%
X. Luan et al. / J. Chromatogr. B 941 (2013) 25–30
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Table 1 Intra-day and inter-day precision, accuracy, matrix effect and absolute recovery of the method for PR quantification in rat plasma. Concentration (ng/mL)
1 5 100 450 a
Intra-day
Matrix effect
Absolute recovery
Precision (%RSD)
Accuracy (%RE)
Inter-day Precision (%RSD)
Accuracy (%RE)
Mean
RSD (%)
Mean
RSD (%)
5.92 3.87 2.89 6.27
−5.81 −3.24 5.63 8.64
6.52 5.29 4.91 8.98
−7.37 −1.83 4.81 9.57
NAa 92.4 91.7 89.3
NA 2.6 4.4 3.8
90.2 92.8 88.7 91.1
4.6 5.8 7.1 3.2
NA, not available.
half-life (T1/2 ) is the time required for PR plasma concentration to fall to half its value as measured at the beginning of the time period. T1/2 is 3.4 h in rats after intravenous administration of PR. 4. Conclusion A sensitive, rapid and specific LC–MS/MS method for the in vivo analysis of PR has been developed and validated for the first time. The LLOQ of 1 ng/mL was achieved in this method, which was sensitive enough for the quantification of PR concentration in rat plasma. This new method has been successfully applied to the pharmacokinetic study of PR in rats, and may be easily extended to the pharmacokinetic study in other species of animal or biological matrixes. Fig. 3. Concentration time profile of PR in rats after intravenous administration at 0.5 mg/kg (mean ± SD, n = 6).
(450 ng/mL) with no statistical difference. Absolute recoveries of PR were 90.2% (1 ng/mL), 92.8% (5 ng/mL), 88.7% (100 ng/mL) and 91.1% (450 ng/mL), respectively. The plasma containing PR was stored at −70 ◦ C, and showed good stability for 30 days and after three freeze/thaw cycles. The processed blood samples were also stable at room temperature for 4 h and in the auto sampler for at least 24 h. All the values of RSD and RE ranged from 1.4% to 9.5% and −9.2% to 3.5%, respectively. The method is claimed to be credible and robust under our experiment conditions. 3.3. Application to a pharmacokinetic study The analysis method was successfully used in the determination of PR plasma concentration in rats after intravenous administration (0.5 mg/kg). The plasma concentration time curve is shown in Fig. 3. The main pharmacokinetic parameters of PR are summarized in Table 2. The apparent volume of distribution at terminal phase (VZ ) is calculated from the total amount of drug divided by the plasma concentration during the terminal phase. Vz was 20.6 L/kg, much bigger than the total body water volume (0.67 L/kg), indicating that PR may be widely distributed in the extravascular tissues. The Table 2 The pharmacokinetic parameters of PR in rats after intravenous administration at 0.5 mg/kg (n = 6, mean ± SD). Parameters
Intravenous
AUC(0–12 h) (h ng/mL) AUC(0→∞) (h ng/mL) MRT(0–12 h) (h) T1/2 (h) Tmax (h) Cmax (ng/mL) VZ (L/kg) CL (mL/min/kg)
104.6 ± 18.7 111.9 ± 22.4 3.8 ± 0.4 3.4 ± 0.5 / 131.6 ± 38.4 20.6 ± 4.8 71.3 ± 17.8
Acknowledgments This work was supported by National Basic Research Program of China (973 Program) (No. 2010CB529806), National Natural Science Foundation of China (Nos. 30873179 and 81272569), Shanghai Rising-Star Program (No. 09QA1403500), Shanghai Pujiang Program (No. 12PJD023), Innovation Program of Shanghai Municipal Education Commission (Nos. 12ZZ200 and 13ZZ087), and “Chen Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (No. 10CGB03). References [1] V.M. Dirsch, A.K. Kiemer, H. Wagner, A.M. Vollmar, Eur. J. Pharmacol. 336 (1997) 211. [2] Y. Wang, Y. Zhou, H. Zhou, G. Jia, J. Liu, B. Han, Z. Cheng, H. Jiang, S. Pan, B. Sun, PLoS ONE 7 (2012) 1. [3] J.Y. Byun, M.J. Kim, D.Y. Eum, C.H. Yoon, W.D. Seo, K.H. Park, J.W. Hyun, Y.S. Lee, J.S. Lee, M.Y. Yoon, S.J. Lee, Mol. Pharmacol. 76 (2009) 734. [4] G. da Silva, M. Tanic¸a, J. Rocha, R. Serrano, E.T. Gomes, B. Sepodes, O. Silva, Hum. Exp. Toxicol. 30 (2011) 693. [5] F.P. Gullo, J.C. Sardi, V.A. Santos, F. Sangalli-Leite, N.S. Pitangui, S.A. Rossi, A.C. de Paula, E. Silva, L.A. Soares, J.F. Silva, H.C. Oliveira, M. Furlan, D.H. Silva, V.S. Bolzani, M.J. Mendes-Giannini, A.M. Fusco-Almeida, Evid. Based Complement. Alternat. Med. 2012 (2012) 1. [6] D.Q. Luo, H. Wang, X. Tian, H.J. Shao, J.K. Liu, Pest Manag. Sci. 61 (2005) 85. [7] H.J. Kim, G.M. Park, J.K. Kim, Arch. Pharm. Res. 36 (2013) 495. [8] A. Petronelli, G. Pannitteri, U. Testa, Anticancer Drugs 20 (2009) 880. [9] H. Yang, K.R. Landis-Piwowar, D. Lu, P. Yuan, L.H. Li, G.P. Reddy, X. Yuan, Q.P. Dou, J. Cell. Biochem. 103 (2008) 234. [10] C.C. Wu, M.L. Chan, W.Y. Chen, C.Y. Tsai, F.R. Chang, Y.C. Wu, Mol. Cancer Ther. 4 (2005) 1277. [11] Y.Y. Yan, J.P. Bai, Y. Xie, J.Z. Yu, C.G. Ma, Oncol. Lett. 5 (2013) 242. [12] R.E. Tiedemann, J. Schmidt, J.J. Keats, C.X. Shi, Y.X. Zhu, S.E. Palmer, X. Mao, A.D. Schimmer, A.K. Stewart, Blood 113 (2009) 4027. [13] P.M. Costa, P.M. Ferreira, Vda.S. Bolzani, M. Furlan, V.A. de Freitas, J. Corsina, M.O. de Moraes, L.V. Costa-Lotufo, R.C. Moutenegro, C. Pessoa, Toxicol. In Vitro 22 (2008) 854. [14] F.R. Chang, K.I. Hayashi, I.H. Chen, C.C. Liaw, K.F. Bastow, Y. Nakanishi, H. Nozaki, G.M. Cragg, Y.C. Wu, K.H. Lee, J. Nat. Prod. 66 (2003) 1416. [15] X.M. Mu, W. Shi, L.X. Sun, H. Li, Y.R. Wang, Z.Z. Jiang, L.Y. Zhang, Asian Pac. J. Cancer Prev. 13 (2012) 1097. [16] X. Mu, W. Shi, L. Sun, H. Li, Z. Jiang, L. Zhang, Molecules 17 (2012) 6854. [17] W.B. Filho, J. Corsino, Vda.S. Boizani, M. Furlan, A.M. Pereira, S.C. Franc¸a, Phytochem. Anal. 13 (2002) 75.
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
Development and validation of a quantitative liquid chromatography tandem mass spectrometry assay for pristimerin in rat plasma Xin Luan a,1 , Ying-Yun Guan a,1 , Ya-Rong Liu a , Chao Wang a , Mei Zhao b,c , Qin Lu a , Ya-Bin Tang a , Xiao-Lin Wang a , Chao Fang a,∗ , Hong-Zhuan Chen a,∗∗ a Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China b Department of Pharmacy, Shanghai Institute of Health Sciences, Shanghai 201318, China c Department of Pharmacy, Health School Attached to Shanghai Jiao Tong University School of Medicine, Shanghai 201318, China
a r t i c l e
i n f o
Article history: Received 8 June 2013 Accepted 26 September 2013 Available online 3 October 2013 Keywords: Pristimerin HPLC–MS/MS Pharmacokinetics Rat plasma
a b s t r a c t A sensitive, rapid and simple LC–MS/MS analysis method was developed and validated for the determination of pristimerin (PR) in rat plasma. Protein precipitation with four volumes of acetonitrile as the precipitation reagent was used as the sample preparation method. The analysis process was performed on a Merck ZIC-HILIC column with the mobile phase of acetonitrile–water (containing 5 mM ammonium formate, pH 2.8) (85:15, v/v). PR (m/z 465.3–201.1) and glycyrrhetinic acid (internal standard, m/z 471.5–177.1) were monitored under positive electrospray ionization in multiple reaction monitoring (MRM) mode. Retention time of PR and IS was 2.45 min and 2.4 min, respectively. The limit of detection was 0.5 ng/mL and the linear range was 1–500 ng/mL. The intra-day and inter-day precision were 2.89–6.27% and 4.91–8.98%, and the intra-day and inter-day accuracy ranged from −5.81% to 8.64% and −7.37% to 9.57%, respectively. The matrix effects and absolute recovery ranged from 89.3% to 92.4% and 88.7% to 92.8%, respectively. The method has been successfully applied to the determination of PR concentration in rat plasma after intravenous administration (0.5 mg/kg). © 2013 Elsevier B.V. All rights reserved.
1. Introduction Pristimerin (PR) is a representative of the quinonemethide triterpenoids, which are mainly found in the Celastraceae and Hippocrateaceae families [1]. PR has attracted considerable interests due to its multiple biological properties, including antitumor, antiinflammatory, antioxidant, antimalarial and antifungal activities [2–7]. Recently, the potent antitumor activities of PR have been particularly evaluated, and attractive progress has been made in this field. Extensive studies found that PR exhibits cytotoxicity in several cancer cell lines by apoptosis induction through activating caspase [8–11] and inhibiting proteasome [12] or DNA synthesis [13]. Interestingly, PR is also effective in inhibiting the growth of drug-resistant cancer cells such as vincristine-resistant KB cells and paclitaxel-resistant 1A9 cells [14]. Moreover, PR has also been
∗ Corresponding author. Tel.: +86 21 63846590x778013; fax: +86 21 64674721. ∗∗ Corresponding author. Tel.: +86 21 64674721; fax: +86 21 64674721. E-mail addresses: [email protected] (C. Fang), [email protected] (H.-Z. Chen). 1 These two authors contributed equally to this work. 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.09.038
proved to be able to interfere with tumor metastasis by the inhibition of MDA-MB-231 cell migration and invasion [15] and the antiangiogenic activity through blocking VEGFR2 activation [16]. These findings demonstrated that PR was a promising antitumor entity and a potential leading compound for translational cancer research. In contrast to the favorable pharmacodynamic results, the pharmacokinetic profile of PR has not been investigated before and there is still lack of in vivo validated analytical methods for PR determination. A reverse-phase HPLC–UV method was developed for the determination of PR levels in plant extracts [17]. In another study, HPLC–UV and LC–MS analysis were used to confirm the structures and detectable amounts of PR in the plant extracts; however, the reported limit of detection (LOD) was 40 g/mL [18]. A more sensitive and rapid analysis method needs to be developed and validated for the concentration monitoring of PR in vivo. Compared with HPLC–UV method, LC–MS/MS has the advantages in both qualitative and quantitative basis. The qualitative advantages come from the superior specificity due to resolving co-eluting species by mass. The quantitative advantage is achieved by the low noise, and thus high S/N, observed at specific m/z values for different compounds [19]. In this study,
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a new, rapid, sensitive, and selective LC/MS/MS method was developed, validated and successfully applied in the determination of PR concentration in vivo after intravenous administration (0.5 mg/kg). 2. Experimental 2.1. Reagents and chemicals PR (purity ≥98%) and glycyrrhetinic acid (purity ≥98%) (internal standard, IS) were purchased from Pure-one Bio Technology Co. Ltd. (Shanghai, China). Ammonium formate was purchased from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). HPLCgrade acetonitrile and formic acid were purchased from Merck (Darmstadt, Germany). Water was purified with the Milli-Q Plus system (Millipore, Bedford, MA, USA). Freshly obtained drug free rat plasma was collected from six healthy Sprague-Dawley (SD) rats in our lab and stored at −70 ◦ C prior to use. The heparinized plastic tubes (5 mL, 13 mm × 100 mm) were obtained from Shanghai Kehua Bio-engineering Co. Ltd. (China). The syringes (1 mL, 0.4 mm × 13 mm) were purchased from Kindly Enterprise Development Group (Shanghai, China). 2.2. Animals Female SD rats (200–220 g) were supplied by the SLAC Laboratory Animal Co. Ltd. (Shanghai, China), and were housed in an environmentally controlled quarters (20–25 ◦ C, relative humidity 55–65%, 12 h light/12 h dark cycle) for 3 days before experiment. The rats were fasted overnight before dosing, but the water was available all the time. Animal experiments were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. 2.3. Instrument and conditions Analyses were performed on an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technologies, Inc., USA) equipped with electrospray ionization (ESI) and an Agilent 1200 HPLC system (Agilent Technologies, Inc., USA). The instrumental components of Agilent 1200 HPLC system were G1322A Degasser, G1311A Quat Pump, G1329A ALS Auto Sampler, and G1316A COLCOM Column Oven. A Merck ZIC-HILIC column (2.1 mm × 100 mm, 3.5 m) was used for analyte separation. Isocratic elution with a mobile phase consisting of acetonitrile and water (85:15, v/v, the aqueous phase contained 5 mM ammonium formate, was adjusted to pH 2.8 with formic acid) was used for the separation. The flow rate was 0.1 mL/min, the run time of each sample was 5 min, the injection volume was 2 L and the column temperature was kept at 40 ◦ C. Data were collected under the positive ionization mode with multiple reaction monitoring (MRM). Two MRM transitions, PR (m/z 465.3–201.1, fragmentor 135 eV, collision energy 20 eV) and IS (m/z 471.5–177.1, fragmentor 160 eV, collision energy 30 eV) were monitored. The working parameters of the ESI source were set as follows: temperature 300 ◦ C, capillary voltage 4000 V, drying-gas flow rate 9 L/min and nebulizer pressure 40 psi. Data processing of MS was performed on the MassHunter software package (Version B.04.00, Agilent Technologies, Inc., USA). 2.4. Preparation of calibration standards and quality control (QC) samples The stock solutions of PR and IS each with the exact concentration of 500 g/mL were prepared in acetonitrile and stored at −70 ◦ C. Working solutions for spiking plasma ranging from 5 to
2500 ng/mL were all freshly prepared by step wise dilution of the stock solution with acetonitrile. A series of calibration standards were prepared by spiking blank plasma (80 L) with 20 L aliquots of standard solutions to yield the concentrations of 1, 5, 10, 50, 100, 250 and 500 ng/mL. QC samples were prepared at three levels (low: 5 ng/mL, medium: 100 ng/mL, high: 450 ng/mL) independently in the same way. All QC samples were stored at −70 ◦ C. The IS working solution (2500 ng/mL) was made by diluting the stock solution with acetonitrile. 2.5. Sample preparation All frozen standards and samples were thawed at room temperature (25 ◦ C) and homogenized by vortex. One hundred and fifty microliter acetonitrile and 50 L acetonitrile containing IS (2.5 g/mL) were added to 50 L plasma. The mixed solution was homogenized by vortex for 1 min and centrifuged at the condition of 15,000 × g for 5 min with the supernatant (2 L) injected into the system. 2.6. Method validation The method was validated for selectivity, linearity, precision, accuracy, recovery, matrix effect and stability. Blank plasma from six rats with and without PR and IS were used to evaluate the selectivity. Calibration curves were generated at the range of 1–500 ng/mL by using the peak area ratio (y) of PR to IS vs. nominal concentration of PR (x) with the weighting factor of 1/x. The lower limit of quantification (LLOQ) was defined as the lowest concentration point of the calibration curve. QC samples were processed in five replicates at three concentration levels (low, middle, and high) for PR. The precision was expressed as intra- and inter-day relative standard deviation (%RSD, calculated from the standard deviation divided by the mean) of the QC and LLOQ samples. Both the intra-day and inter-day accuracy (defined as relative error, %RE) were determined by comparing the calculated concentration to the theoretical concentration of the QC and LLOQ samples. Inter- and intra-assay precision and accuracy were determined by repeated analysis of QC and LLOQ samples on the same day (n = 5) and on 5 consecutive days. Matrix effects were defined by calculating the ratio of the peak area for PR in deproteinized plasma relative to the peak area for PR in acetonitrile. Absolute recoveries at QC and LLOQ levels were evaluated by determining the peak area ratios of PR in the post-deproteinized spiked samples to that acquired from pre-deproteinized spiked samples. Long-term stability (30 days), room-temperature (4 h) stability, auto-sampler (24 h) stability and three freeze/thaw stability were examined at three levels of QC samples in five replicates. 2.7. Pharmacokinetic study Six SD rats (200–220 g) were treated with intravenous administration through caudal vein, using plastic syringes (1 mL, 0.4 mm × 13 mm). Blood samples (no more than 0.2 mL) were obtained via retro-orbital puncture with a glass capillary at 0 h (pre-dose) and 0.083, 0.167, 0.33, 0.5, 1, 1.5, 2, 4, 6, 9 and 12 h after administration of 0.5 mg/kg PR dissolved in saline water. The blood sample was collected into the commercial heparinized plastic tubes and separated by 4000 r/min centrifugation (Centrifuge 5417R, Eppendorf, Hamburg, Germany) at 4 ◦ C for 10 min and stored in the refrigerator at −70 ◦ C before the analysis. The pharmacokinetic parameters were calculated with the WinNonlin software (Version 6.1 Pharsight, Mountain View, CA, USA) according to non-compartmental model.
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Fig. 1. Product ion mass spectra of PR (A) and IS (B) and the chemical structures of both compounds.
3. Results and discussion 3.1. Method development To the best of our knowledge, no LC–MS/MS method has been reported for the determination of PR in plasma. In order to establish a selective and sensitive analysis method, the mass spectrometry and chromatographic conditions should be developed. In our experiment, we selected glycyrrhetinic acid, an analog of PR as IS due to its similar basic structure, physicochemical property and mass spectrometric behavior to those of PR. The MS/MS operation parameters have been carefully optimized for the determination of PR. Positive and negative ion mode were tried for the ionization of PR. Both modes could produce the correct precursor ions, whereas the transition between precursor ion and the ring-opened product ion under positive mode achieved higher response. Thus, [M+H]+ (465.3) was chosen for further fragmentation, and the MassHunter optimizer software (Version B.03.01, Agilent Technologies, Santa Clara, CA, USA) was used to obtain the product ion and optimize ESI source parameters such as fragmentor voltage and collision energy
(CE). The product ion spectra of PR (465.3) and IS (471.5) and their allocation are shown in Fig. 1. The desolvation temperature was set at 300 ◦ C to prevent PR and IS from thermal decomposition at a higher temperature and meanwhile guarantee the effects of desolvation. We found that when the pH of the mobile phase was adjusted to 2.8, the peaks of both PR and IS was symmetrical and sharp. A Merck ZIC-HILIC column was used due to its suited separating capacity for the analyte. The low flow rate (0.1 mL/min) and the high ratio of acetonitrile in mobile phase (85%, v/v) were adopted to obtain an ideal shape of the peaks and appropriate retention time (RT < 5 min) for the purpose of rapid analysis, and it was observed that higher percentage of organic phase can enhance the peak signal by better ionization and shorten the analysis time. The sample preparation method should be optimized to be reproducible, minimized matrix effects and as inexpensive as possible. Thus, protein precipitation with four volumes of acetonitrile was adopted for sample preparation to obtain the balance between better protein deposition effect and higher sample concentration. After centrifugation, the supernatant fluid of the sample can be
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Fig. 2. Representative MRM chromatograms of a double-blank rat plasma sample (A), a blank plasma sample spiked with IS (500 ng/mL) (B), a blank rat plasma sample spiked with PR at LLOQ level (1 ng/mL) and IS (500 ng/mL) (C), and a rat plasma sample at 0.5 h after intravenous injection of PR (0.5 mg/kg) (D), Peak I, IS; Peak II, PR.
directly injected into the analysis system because of its similar composition to that of the mobile phase.
3.2. Method validation The method validation includes several respects such as selectivity, linearity, precision, accuracy, matrix effect, recovery and stability. All the contents were examined according to the US Food and Drug Administration (FDA) bioanalytical method validation guidance [20]. No endogenous interference was observed during the retention time of PR and IS in the chromatograms of different control blank rat plasma. The MRM chromatograms of double-blank rat plasma, plasma sample spiked with IS (500 ng/mL), blank plasma spiked with PR at LLOQ level (1 ng/mL) and IS (500 ng/mL), and the plasma sample obtained at 0.5 h after the intravenous injection of 0.5 mg/kg PR were shown in Fig. 2.
The calibration curve exhibited a good linear correlation within the concentration range of 1–500 ng/mL (y = 0.050424*x − 0.081428) with a weighting factor of 1/x to reduce the effect of large concentrations on the calculation of regression statistics. The coefficient of determination (R2 ) was higher than 0.998. The limit of detection (LOD) for PR, which requires a signal-to-noise ratio above 3, was 0.5 ng/mL. LLOQ was established at 1 ng/mL, and the signal-to-noise ratio (S/N) was 12.4. The relative response factor (RRF) of PR (against the IS) was 25.2. The results of intra-day and inter-day precision, accuracy, matrix effect and absolute recovery of the method for the quantification of PR in rat plasma are summarized in Table 1. The values of S/N at the QC sample concentrations were 74.0 (5 ng/mL), 970.7 (100 ng/mL) and 2596.6 (450 ng/mL), respectively. The values for intra- and inter-day precision were 2.89–6.27% and 4.91–8.98%, and the values for intra- and inter-day accuracy ranged from −5.81% to 8.64% and −7.37% to 9.57%, respectively. The matrix effects of QC samples were 92.4% (5 ng/mL), 91.7% (100 ng/mL) and 89.3%
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Table 1 Intra-day and inter-day precision, accuracy, matrix effect and absolute recovery of the method for PR quantification in rat plasma. Concentration (ng/mL)
1 5 100 450 a
Intra-day
Matrix effect
Absolute recovery
Precision (%RSD)
Accuracy (%RE)
Inter-day Precision (%RSD)
Accuracy (%RE)
Mean
RSD (%)
Mean
RSD (%)
5.92 3.87 2.89 6.27
−5.81 −3.24 5.63 8.64
6.52 5.29 4.91 8.98
−7.37 −1.83 4.81 9.57
NAa 92.4 91.7 89.3
NA 2.6 4.4 3.8
90.2 92.8 88.7 91.1
4.6 5.8 7.1 3.2
NA, not available.
half-life (T1/2 ) is the time required for PR plasma concentration to fall to half its value as measured at the beginning of the time period. T1/2 is 3.4 h in rats after intravenous administration of PR. 4. Conclusion A sensitive, rapid and specific LC–MS/MS method for the in vivo analysis of PR has been developed and validated for the first time. The LLOQ of 1 ng/mL was achieved in this method, which was sensitive enough for the quantification of PR concentration in rat plasma. This new method has been successfully applied to the pharmacokinetic study of PR in rats, and may be easily extended to the pharmacokinetic study in other species of animal or biological matrixes. Fig. 3. Concentration time profile of PR in rats after intravenous administration at 0.5 mg/kg (mean ± SD, n = 6).
(450 ng/mL) with no statistical difference. Absolute recoveries of PR were 90.2% (1 ng/mL), 92.8% (5 ng/mL), 88.7% (100 ng/mL) and 91.1% (450 ng/mL), respectively. The plasma containing PR was stored at −70 ◦ C, and showed good stability for 30 days and after three freeze/thaw cycles. The processed blood samples were also stable at room temperature for 4 h and in the auto sampler for at least 24 h. All the values of RSD and RE ranged from 1.4% to 9.5% and −9.2% to 3.5%, respectively. The method is claimed to be credible and robust under our experiment conditions. 3.3. Application to a pharmacokinetic study The analysis method was successfully used in the determination of PR plasma concentration in rats after intravenous administration (0.5 mg/kg). The plasma concentration time curve is shown in Fig. 3. The main pharmacokinetic parameters of PR are summarized in Table 2. The apparent volume of distribution at terminal phase (VZ ) is calculated from the total amount of drug divided by the plasma concentration during the terminal phase. Vz was 20.6 L/kg, much bigger than the total body water volume (0.67 L/kg), indicating that PR may be widely distributed in the extravascular tissues. The Table 2 The pharmacokinetic parameters of PR in rats after intravenous administration at 0.5 mg/kg (n = 6, mean ± SD). Parameters
Intravenous
AUC(0–12 h) (h ng/mL) AUC(0→∞) (h ng/mL) MRT(0–12 h) (h) T1/2 (h) Tmax (h) Cmax (ng/mL) VZ (L/kg) CL (mL/min/kg)
104.6 ± 18.7 111.9 ± 22.4 3.8 ± 0.4 3.4 ± 0.5 / 131.6 ± 38.4 20.6 ± 4.8 71.3 ± 17.8
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