Research article Received: 12 July 2013,
Revised: 23 September 2013,
Accepted: 29 September 2013
Published online in Wiley Online Library: 13 November 2013
(wileyonlinelibrary.com) DOI 10.1002/bmc.3076
Simultaneous analysis of polygala acid, senegenin and 3,6′-disinapoylsucrose in rat plasma by liquid chromatography– tandem mass spectrometry: application to a pharmacokinetic study after oral administration Longfei Lin, Lei Yan, Hui Zhang, Xuechun Li, Jin Zhang, Haoran Dou, Mingrui Shen, Xingbin Yin, Changhai Qu and Jian Ni* ABSTRACT: A rapid and specific LC-MS/MS method has been developed for the simultaneous analysis of polygala acid, senegenin and 3,6′-disinapoylsucrose (DSS) in rat plasma. The method was applied to the pharmacokinetics studies of polygala acid, senegenin and DSS. The analysis was carried out on an Agilent Eclipse plus C18 reversed-phase column (100 × 4.6 mm, 3.5 μm) by gradient elution with methanol and ammonia (0.01%, v/v). The flow rate was 0.4 mL/min. All analytes including internal standard (IS) were monitored by selected reaction monitoring with an electrospray ionization source. Linear responses were obtained for polygala acid and DSS ranging from 2.5 to 2000 ng/mL, and senegenin ranging from 5 to 2000 ng/mL. The intra- and inter-day precisions (relative standard deviation) were 70.89 ± 4.60% and the recovery of the IS was 77.23 ± 3.68%. No matrix components in plasma caused significant changes in the MS/MS responses to polygala acid, senegenin, DSS and IS. Overall, these results indicated that no endogenous substances significantly influenced recovery or ionization of the IS and analytes.
Table 5. Recoveries and matrix effects of polygala acid, senegenin, DSS and IS (data are means ± SD, n = 6) Compound concentration (ng/mL)
10 50 500 1500 10 50 500 1500 10 50 500 1500
Polygala acid
Senegenin
598 wileyonlinelibrary.com/journal/bmc
DSS
Senegenin
DSS Polygala acid
Added concentration (ng/mL)
Compound
Compound
Table 4. Accuracy and precision for the analysis of polygala acid, senegenin and DSS in rat plasma
Inter-day (n = 18)
L. Lin et al.
IS
Copyright © 2013 John Wiley & Sons, Ltd.
10 50 500 1500 10 50 500 1500 10 50 500 1500 200
Recovery (%) 70.89 ± 4.60 78.17 ± 2.75 83.98 ± 9.10 83.30 ± 2.90 75.17 ± 4.63 79.37 ± 6.38 77.80 ± 6.99 84.16 ± 3.79 88.49 ± 3.26 83.85 ± 3.90 87.30 ± 6.70 86.73 ± 4.91 77.23 ± 3.68
Matrix effect (%) 98.80 ± 4.00 97.60 ± 3.84 100.16 ± 5.49 97.33 ± 3.74 94.51 ± 5.51 93.06 ± 7.65 96.54 ± 5.14 97.16 ± 3.69 97.55 ± 5.80 98.53 ± 2.04 96.15 ± 4.31 95.90 ± 6.27 98.19 ± 3.75
Biomed. Chromatogr. 2014; 28: 594–600
Analysis of polygala acid, senegenin and DSS in rat plasma by LC/MS Table 6. Stability of polygala acid, senegenin and DSS under various storage conditions (data are means ± SD, n = 6) Compound concentration (ng/mL)
Long-term ( 20°C)
Short-term ( 6°C)
Freeze–thaw
Polygala acid 10 50 500 1500
9.84 ± 0.74 50.81 ± 2.09 503.54 ± 6.73 1488.54 ± 114
9.11 ± 0.57 50.94 ± 1.37 496.54 ± 16.97 1512.67 ± 28.99
10.36 ± 0.54 50.2 ± 1.49 486.02 ± 23 1559.35 ± 30.94
Senegenin 10 50 500 1500
9.09 ± 0.65 50.54 ± 1.35 495.61 ± 29.05 1536.05 ± 92.89
9.93 ± 1.12 47.84 ± 3.81 491.96 ± 12.84 1478.63 ± 47.04
9.89 ± 0.45 48.26 ± 2.3 491.7 ± 8.14 1485.65 ± 65.3
DSS 10 50 500 1500
10.02 ± 1.07 51.37 ± 1.87 480.39 ± 23.74 1425.86 ± 41.26
10.04 ± 0.8 50.66 ± 2.95 501.84 ± 14.46 1483.34 ± 43.82
10.09 ± 0.57 49.12 ± 3.38 494.17 ± 10.76 1511.65 ± 56.72
Stability. Stability study showed that the concentrations of polygala acid, senegenin and DSS were not significantly changed in plasma stored in 20°C for 1 week and 6°C for 24 h and experienced three freeze–thaw cycles from 20 to 6°C. All the values are shown in Table 6.
Figure 3. Mean (±SD) plasma concentration–time profiles of polygala acid, senegenin and DSS after oral administration of mixed standard to six rats.
Pharmacokinetic study In the present study, the method described above was applied to the analysis of plasma samples obtained from rats administered orally with polygala acid and senegenin (25 mg/kg, n = 6) and DSS (50 mg/kg, n = 6). The mean concentrations of different times are shown in Fig. 3, and the main pharmacokinetic parameters, as derived from these data, are summarized in Table 7. Polygala acid plasma concentration reached a maximum at 1.21 ± 0.89 h after administration with an average maximum concentration (Cmax) of 90.02 ± 23.38 ng/mL. The area under the curve (AUC0–∞) was 958.82 ± 76.28 (h ng/mL) and mean residence time (MRT) was 16.38 ± 6.74 h. Senegenin plasma concentration reached a maximum at 0.83 ± 0.13 h after administration with an average Cmax of 0.75 ± 0.22 ng/mL. The area under the curve (AUC0–∞) was 751.7 ± 40.65 (h.ng/mL) and MRT was 19.05 ± 7.54 h. DSS plasma concentration reached a maximum at 0.75 ± 0.22 h after administration with an average Cmax of 1.08 ± 0.47 ng/mL. The area under the curve (AUC0–∞) was 632.92 ± 96.64 (h.ng/mL) and MRT was 13.75 ± 7.21 h. From the mean plasma concentration–time profiles, we can see that each concentration–time curve has double peaks. This may be caused by the appearance of a lipophilic drug after administration, which may assimilate into the organs quickly,
Table 7. Pharmacokinetic parameters of polygala acid, senegenin and DSS after oral administration of mixed compounds (data are means ± SD, n = 6) Parameters Tmax Cmax AUC0-24 AUC0–∞ T1/2 MRT
Unit h ng/mL ng.h/mL ng.h/mL h h
Polygala acid 1.21 ± 0.89 90.02 ± 23.38 768.66 ± 156.09 958.82 ± 76.28 10.6 ± 6.01 16.38 ± 6.74
Senegenin 0.75 ± 0.22 89.35 ± 36.3 507.26 ± 150.19 751.7 ± 40.65 12.66 ± 5.29 19.05 ± 7.54
DSS 1.08 ± 0.47 120.14 ± 37.68 494.9 ± 143.71 632.92 ± 96.64 9.61 ± 5.65 13.75 ± 7.21
Biomed. Chromatogr. 2014; 28: 594–600
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
599
Tmax, Time to reach peak drug concentration in plasma; Cmax, peak drug concentration; AUC, area under the plasma concentration– time curve; T1/2, half-life; MRT, mean residence time.
L. Lin et al. and then is released again from organs. It also possiblely caused by enterohepatic circulation.
Conclusion In conclusion, for the first time, a rapid and specific LC-MS/MS method was developed for the analysis of polygala acid and senegenin in rat plasma after oral administration of mixed compounds, which had high sensitivity, precision, accuracy and recovery, Although there has been a method published to determine the concentration of DSS in rat plasma by LC-MS/MS, this method shows better sensitivity and a broadened linear range. This method will be useful for the determination of polygala acid, senegenin and DSS in other bio-samples in future studies. Moreover, the pharmacokinetic parameters of polygala acid, senegenin and DSS following oral dosing are a suitable reference in clinical application.
References Arai M, Hayashi A, Sobou M, Ishida S, Kawachi T, Kotoku N and Kobayashi M. Anti-angiogenic effect of triterpenoidal saponins from Polygala senega. Journal of Natural Medicine 2011; 65(1): 149–156. Chen Y, Liu X-M, Pan R-L, Wang G-N, Fu Y and Chang Q. An LC-MS/MS method for determination of 3,6′-disinapoylsucrose in rat plasma and its application to a pharmacokinetic study. Biomedical Chromatography 2009; 23(12): 1326–1332. Choi JG, Kim HG, Kim MC, Yang WM, Huh Y, Kim SY and Oh MS. Polygalae radix inhibits toxin-induced neuronal death in the Parkinson’s disease models. Journal of Ethnopharmacology 2011; 134(2): 414–421. Egashira N, Yuzurihara M, Hattori N, Sakakibara I and Ishige A. Ninjinyoei-to (Ren-Shen-Yang-Rong-Tang) and Polygalae radix improves
scopolamine-induced impairment of passive avoidance response in mice. Phytomedicine 2003; 10(6–7): 467–473. Hu Y, Li J, Liu P, Chen X, Guo DH, Li QS and Rahman K. Protection of SH-SY5Y neuronal cells from glutamate-induced apoptosis by 3,6′-disinapoyl sucrose, a bioactive compound isolated from Radix Polygala. Journal of Biomedicine and Biotechnology 2012; 2012: 1–5. Ikeya Y, Takeda S, Tunakawa M, Karakida H, Toda K, Yamaguchi T and Aburada M. Cognitive improving and cerebral protective effects of acylated oligosaccharides in Polygala tenuifolia. Biological and Pharmeutical Bulletin 2004; 27(7): 1081–1085. Lin Z, Gu J, Xiu J, Mi T, Dong J and Tiwari JK. Traditional Chinese medicine for senile dementia. Evidence-based Complementary and Alternative Medicine 2012; 692621. doi: 10.1155/2012/692621 Liu P, Hu Y, Guo DH, Wang DX, Tu HH, Ma L, Xie TT and Kong LY. Potential antidepressant properties of Radix Polygalae (Yuan Zhi). Phytomedicine 2010; 17(10): 794–799. Naito R and Tohda C. Biological characterization of anti-neurodegenerative effects of Polygala tenuifolia in Abeta(25–35)-treated cortical neurons. Biological and Pharmeutical Bulletin 2006; 29(9): 1892–1896. Park CH, Choi SH, Koo JW, Seo JH, Kim HS, Jeong SJ and Suh YH. Novel cognitive improving and neuroprotective activities of Polygala tenuifolia Willdenow extract, BT-11. Journal of Neuroscience Research 2002; 70(3): 484–492. Wang YP, Yang JS, Zhang YM and Chen JM. Chemical constituents of Polygala tenuifolia. Zhong Cao Yao 2005; 36: 1291–1393. Xie W, Yang Y, Gu X, Zheng Y, Sun YE, Liang Y, Bo J and Ma Z. Senegenin attenuates hepatic ischemia–reperfusion induced cognitive dysfunction by increasing hippocampal NR2B expression in rats. PLoS One 2012; 7(9): e45575. Yin X, Qu C, Li Z, Zhai Y, Cao S, Lin L, Feng L, Yan L and Ni J. Simultaneous determination and pharmacokinetic study of polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII in beagle dog plasma after oral administration of Rhizoma Paridis extracts by LC-MS/MS. Biomedical Chromatography 2013; 27(3): 343–348. Yoo KY and Park SY. Terpenoids as potential anti-Alzheimer’s disease therapeutics. Molecules 2012; 17(3): 3524–3538.
600 wileyonlinelibrary.com/journal/bmc
Copyright © 2013 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 594–600
Revised: 23 September 2013,
Accepted: 29 September 2013
Published online in Wiley Online Library: 13 November 2013
(wileyonlinelibrary.com) DOI 10.1002/bmc.3076
Simultaneous analysis of polygala acid, senegenin and 3,6′-disinapoylsucrose in rat plasma by liquid chromatography– tandem mass spectrometry: application to a pharmacokinetic study after oral administration Longfei Lin, Lei Yan, Hui Zhang, Xuechun Li, Jin Zhang, Haoran Dou, Mingrui Shen, Xingbin Yin, Changhai Qu and Jian Ni* ABSTRACT: A rapid and specific LC-MS/MS method has been developed for the simultaneous analysis of polygala acid, senegenin and 3,6′-disinapoylsucrose (DSS) in rat plasma. The method was applied to the pharmacokinetics studies of polygala acid, senegenin and DSS. The analysis was carried out on an Agilent Eclipse plus C18 reversed-phase column (100 × 4.6 mm, 3.5 μm) by gradient elution with methanol and ammonia (0.01%, v/v). The flow rate was 0.4 mL/min. All analytes including internal standard (IS) were monitored by selected reaction monitoring with an electrospray ionization source. Linear responses were obtained for polygala acid and DSS ranging from 2.5 to 2000 ng/mL, and senegenin ranging from 5 to 2000 ng/mL. The intra- and inter-day precisions (relative standard deviation) were 70.89 ± 4.60% and the recovery of the IS was 77.23 ± 3.68%. No matrix components in plasma caused significant changes in the MS/MS responses to polygala acid, senegenin, DSS and IS. Overall, these results indicated that no endogenous substances significantly influenced recovery or ionization of the IS and analytes.
Table 5. Recoveries and matrix effects of polygala acid, senegenin, DSS and IS (data are means ± SD, n = 6) Compound concentration (ng/mL)
10 50 500 1500 10 50 500 1500 10 50 500 1500
Polygala acid
Senegenin
598 wileyonlinelibrary.com/journal/bmc
DSS
Senegenin
DSS Polygala acid
Added concentration (ng/mL)
Compound
Compound
Table 4. Accuracy and precision for the analysis of polygala acid, senegenin and DSS in rat plasma
Inter-day (n = 18)
L. Lin et al.
IS
Copyright © 2013 John Wiley & Sons, Ltd.
10 50 500 1500 10 50 500 1500 10 50 500 1500 200
Recovery (%) 70.89 ± 4.60 78.17 ± 2.75 83.98 ± 9.10 83.30 ± 2.90 75.17 ± 4.63 79.37 ± 6.38 77.80 ± 6.99 84.16 ± 3.79 88.49 ± 3.26 83.85 ± 3.90 87.30 ± 6.70 86.73 ± 4.91 77.23 ± 3.68
Matrix effect (%) 98.80 ± 4.00 97.60 ± 3.84 100.16 ± 5.49 97.33 ± 3.74 94.51 ± 5.51 93.06 ± 7.65 96.54 ± 5.14 97.16 ± 3.69 97.55 ± 5.80 98.53 ± 2.04 96.15 ± 4.31 95.90 ± 6.27 98.19 ± 3.75
Biomed. Chromatogr. 2014; 28: 594–600
Analysis of polygala acid, senegenin and DSS in rat plasma by LC/MS Table 6. Stability of polygala acid, senegenin and DSS under various storage conditions (data are means ± SD, n = 6) Compound concentration (ng/mL)
Long-term ( 20°C)
Short-term ( 6°C)
Freeze–thaw
Polygala acid 10 50 500 1500
9.84 ± 0.74 50.81 ± 2.09 503.54 ± 6.73 1488.54 ± 114
9.11 ± 0.57 50.94 ± 1.37 496.54 ± 16.97 1512.67 ± 28.99
10.36 ± 0.54 50.2 ± 1.49 486.02 ± 23 1559.35 ± 30.94
Senegenin 10 50 500 1500
9.09 ± 0.65 50.54 ± 1.35 495.61 ± 29.05 1536.05 ± 92.89
9.93 ± 1.12 47.84 ± 3.81 491.96 ± 12.84 1478.63 ± 47.04
9.89 ± 0.45 48.26 ± 2.3 491.7 ± 8.14 1485.65 ± 65.3
DSS 10 50 500 1500
10.02 ± 1.07 51.37 ± 1.87 480.39 ± 23.74 1425.86 ± 41.26
10.04 ± 0.8 50.66 ± 2.95 501.84 ± 14.46 1483.34 ± 43.82
10.09 ± 0.57 49.12 ± 3.38 494.17 ± 10.76 1511.65 ± 56.72
Stability. Stability study showed that the concentrations of polygala acid, senegenin and DSS were not significantly changed in plasma stored in 20°C for 1 week and 6°C for 24 h and experienced three freeze–thaw cycles from 20 to 6°C. All the values are shown in Table 6.
Figure 3. Mean (±SD) plasma concentration–time profiles of polygala acid, senegenin and DSS after oral administration of mixed standard to six rats.
Pharmacokinetic study In the present study, the method described above was applied to the analysis of plasma samples obtained from rats administered orally with polygala acid and senegenin (25 mg/kg, n = 6) and DSS (50 mg/kg, n = 6). The mean concentrations of different times are shown in Fig. 3, and the main pharmacokinetic parameters, as derived from these data, are summarized in Table 7. Polygala acid plasma concentration reached a maximum at 1.21 ± 0.89 h after administration with an average maximum concentration (Cmax) of 90.02 ± 23.38 ng/mL. The area under the curve (AUC0–∞) was 958.82 ± 76.28 (h ng/mL) and mean residence time (MRT) was 16.38 ± 6.74 h. Senegenin plasma concentration reached a maximum at 0.83 ± 0.13 h after administration with an average Cmax of 0.75 ± 0.22 ng/mL. The area under the curve (AUC0–∞) was 751.7 ± 40.65 (h.ng/mL) and MRT was 19.05 ± 7.54 h. DSS plasma concentration reached a maximum at 0.75 ± 0.22 h after administration with an average Cmax of 1.08 ± 0.47 ng/mL. The area under the curve (AUC0–∞) was 632.92 ± 96.64 (h.ng/mL) and MRT was 13.75 ± 7.21 h. From the mean plasma concentration–time profiles, we can see that each concentration–time curve has double peaks. This may be caused by the appearance of a lipophilic drug after administration, which may assimilate into the organs quickly,
Table 7. Pharmacokinetic parameters of polygala acid, senegenin and DSS after oral administration of mixed compounds (data are means ± SD, n = 6) Parameters Tmax Cmax AUC0-24 AUC0–∞ T1/2 MRT
Unit h ng/mL ng.h/mL ng.h/mL h h
Polygala acid 1.21 ± 0.89 90.02 ± 23.38 768.66 ± 156.09 958.82 ± 76.28 10.6 ± 6.01 16.38 ± 6.74
Senegenin 0.75 ± 0.22 89.35 ± 36.3 507.26 ± 150.19 751.7 ± 40.65 12.66 ± 5.29 19.05 ± 7.54
DSS 1.08 ± 0.47 120.14 ± 37.68 494.9 ± 143.71 632.92 ± 96.64 9.61 ± 5.65 13.75 ± 7.21
Biomed. Chromatogr. 2014; 28: 594–600
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
599
Tmax, Time to reach peak drug concentration in plasma; Cmax, peak drug concentration; AUC, area under the plasma concentration– time curve; T1/2, half-life; MRT, mean residence time.
L. Lin et al. and then is released again from organs. It also possiblely caused by enterohepatic circulation.
Conclusion In conclusion, for the first time, a rapid and specific LC-MS/MS method was developed for the analysis of polygala acid and senegenin in rat plasma after oral administration of mixed compounds, which had high sensitivity, precision, accuracy and recovery, Although there has been a method published to determine the concentration of DSS in rat plasma by LC-MS/MS, this method shows better sensitivity and a broadened linear range. This method will be useful for the determination of polygala acid, senegenin and DSS in other bio-samples in future studies. Moreover, the pharmacokinetic parameters of polygala acid, senegenin and DSS following oral dosing are a suitable reference in clinical application.
References Arai M, Hayashi A, Sobou M, Ishida S, Kawachi T, Kotoku N and Kobayashi M. Anti-angiogenic effect of triterpenoidal saponins from Polygala senega. Journal of Natural Medicine 2011; 65(1): 149–156. Chen Y, Liu X-M, Pan R-L, Wang G-N, Fu Y and Chang Q. An LC-MS/MS method for determination of 3,6′-disinapoylsucrose in rat plasma and its application to a pharmacokinetic study. Biomedical Chromatography 2009; 23(12): 1326–1332. Choi JG, Kim HG, Kim MC, Yang WM, Huh Y, Kim SY and Oh MS. Polygalae radix inhibits toxin-induced neuronal death in the Parkinson’s disease models. Journal of Ethnopharmacology 2011; 134(2): 414–421. Egashira N, Yuzurihara M, Hattori N, Sakakibara I and Ishige A. Ninjinyoei-to (Ren-Shen-Yang-Rong-Tang) and Polygalae radix improves
scopolamine-induced impairment of passive avoidance response in mice. Phytomedicine 2003; 10(6–7): 467–473. Hu Y, Li J, Liu P, Chen X, Guo DH, Li QS and Rahman K. Protection of SH-SY5Y neuronal cells from glutamate-induced apoptosis by 3,6′-disinapoyl sucrose, a bioactive compound isolated from Radix Polygala. Journal of Biomedicine and Biotechnology 2012; 2012: 1–5. Ikeya Y, Takeda S, Tunakawa M, Karakida H, Toda K, Yamaguchi T and Aburada M. Cognitive improving and cerebral protective effects of acylated oligosaccharides in Polygala tenuifolia. Biological and Pharmeutical Bulletin 2004; 27(7): 1081–1085. Lin Z, Gu J, Xiu J, Mi T, Dong J and Tiwari JK. Traditional Chinese medicine for senile dementia. Evidence-based Complementary and Alternative Medicine 2012; 692621. doi: 10.1155/2012/692621 Liu P, Hu Y, Guo DH, Wang DX, Tu HH, Ma L, Xie TT and Kong LY. Potential antidepressant properties of Radix Polygalae (Yuan Zhi). Phytomedicine 2010; 17(10): 794–799. Naito R and Tohda C. Biological characterization of anti-neurodegenerative effects of Polygala tenuifolia in Abeta(25–35)-treated cortical neurons. Biological and Pharmeutical Bulletin 2006; 29(9): 1892–1896. Park CH, Choi SH, Koo JW, Seo JH, Kim HS, Jeong SJ and Suh YH. Novel cognitive improving and neuroprotective activities of Polygala tenuifolia Willdenow extract, BT-11. Journal of Neuroscience Research 2002; 70(3): 484–492. Wang YP, Yang JS, Zhang YM and Chen JM. Chemical constituents of Polygala tenuifolia. Zhong Cao Yao 2005; 36: 1291–1393. Xie W, Yang Y, Gu X, Zheng Y, Sun YE, Liang Y, Bo J and Ma Z. Senegenin attenuates hepatic ischemia–reperfusion induced cognitive dysfunction by increasing hippocampal NR2B expression in rats. PLoS One 2012; 7(9): e45575. Yin X, Qu C, Li Z, Zhai Y, Cao S, Lin L, Feng L, Yan L and Ni J. Simultaneous determination and pharmacokinetic study of polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII in beagle dog plasma after oral administration of Rhizoma Paridis extracts by LC-MS/MS. Biomedical Chromatography 2013; 27(3): 343–348. Yoo KY and Park SY. Terpenoids as potential anti-Alzheimer’s disease therapeutics. Molecules 2012; 17(3): 3524–3538.
600 wileyonlinelibrary.com/journal/bmc
Copyright © 2013 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 594–600
