Journal of Chromatography B, 990 (2015) 80–83
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
A validated LC–MS/MS method for determination of periplogenin in rat plasma and its application in pharmacokinetic study Fang Bo a , Ting Dou a , Xingrui Wang a , Paul Owusu Donkor a , Huizi Ouyang b , Yanxu Chang a , Yaru Tu a , Xiumei Gao a , Jun He a,∗ a b
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China The First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
a r t i c l e
i n f o
Article history: Received 15 January 2015 Accepted 22 March 2015 Available online 30 March 2015 Keywords: Periplogenin LC–MS/MS Pharmacokinetics Cortex Periplocae
a b s t r a c t A method coupling high performance liquid chromatography with tandem mass spectrometry has been developed and validated for quantifying periplogenin in rat plasma using psoralen as an internal standard (IS). Plasma samples were pretreated using a simple liquid–liquid extraction with ethyl acetate and the chromatographic separation of periplogenin and psoralen was achieved on a Waters XBridgeTM BEH C18 column with 0.1% formic acid and acetonitrile as mobile phase at a flow rate of 0.4 mL/min. The detection was performed on a positive ion mode with electrospray ionization (ESI) source. The optimized ion transition pairs for quantitation were m/z 391.3 → m/z 337.2 for periplogenin and m/z 187.0 → m/z 131.0 for IS. The total run time was 9.0 min. The calibration curve was linear over the range of 0.2–250 ng/mL (r > 0.99) with the lower limit of quantitation (LLOQ) at 0.2 ng/mL. The intra- and inter-day precision were below 9.85% and the mean accuracy were from −10.03% to 10.26%. The average recoveries of periplogenin in plasma ranged from 85.1% to 95.6%. The proposed method was successfully applied in evaluating the pharmacokinetics of periplogenin after an oral dose of 30 mg/kg Cortex Periplocae extract in rats. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Cortex Periplocae is the root bark of Periploca Sepium Bunge which belongs to the Asclepiadaceae family. It has been shown that Cortex Periplocae has therapeutic effects on edema and rheumatism [1]. With the progress in the research, it has been found to improve cardiac function and exhibit cardiotonic effects on the isolated heart of rats [2]. In addition, it has been reported that Cortex Periplocae inhibits tumor cell proliferation by arresting the cell cycle, inducing apoptosis of tumor cells and blocking signaling pathways [3–8]. As is known to all, periplocin is one of the main components in the Cortex Periplocae, and many researches have been widely reported related to its cancer-fighting properties [9,10] and the treatment of heart diseases [11]. Furthermore, periplogenin which is another representative constituent derived from Cortex Periplocae has become a growing concern in recent years. Pharmacological research indicates that periplogenin plays a protective role in regulating hyperthyroidism
∗ Corresponding author. Tel.: +86 22 59596163; fax: +86 22 59596163. E-mail address: [email protected] (J. He). http://dx.doi.org/10.1016/j.jchromb.2015.03.017 1570-0232/© 2015 Elsevier B.V. All rights reserved.
and associated cardiovascular problems [12]. Periplogenin also has outstanding inhibitory effects on histamine release of mast cells either cultured in vitro or in antigen-pulsed rats. Meanwhile, oral administration of periplogenin can result in the marked reduction of histamine release of mast cells in rats. In light of the mast cell degranulation and histamine release in the inflammation effect, periplogenin can be regarded as one of the effective components with anti-inflammatory activity in Cortex Periplocae [13]. Moreover, periplogenin has been reported to possess anti-cancer property [14]. Despite its multiple biological activities, the pharmacokinetic properties of periplogenin have not yet been reported and it is exceedingly necessary to study the regularity of periplogenin concentration changes with time in vivo. The only reported approach to determine periplogenin was by HPLC [15] and no other methods were available for the application to analyze periplogenin in biological samples because of the low sensitivity and poor selectivity. It is well known that LC–MS/MS [16] plays a significant role in the quantification of main components of medicinal products for pharmacokinetic studies. In this study, we developed a LC–MS/MS method for determination of periplogenin in rat plasma. After validation, the method was successfully applied to pharmacokinetic study after oral administration of Cortex Periplocae extract in rats for the first time.
F. Bo et al. / J. Chromatogr. B 990 (2015) 80–83
2. Materials and methods
81
10 min. Five microliters of the supernatant was injected into the LC–MS/MS system.
2.1. Reagents and chemicals Acetonitrile (Merck KGaA, Darmstadt, Germany), methanol (Merck KGaA, Darmstadt, Germany) and formic acid (ROE SCIENTIFIC INC, Newark, USA) were of HPLC grade. Analytical grade ethyl acetate was purchased from Tianjin Concord Science Co. Ltd. (Tianjin, China). Ultrapure water was prepared by a Milli-Q water purification system (Millipore, Milford, MA, USA). Psoralen (>98%) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Periplogenin (>98%) and Cortex Periplocae extract (the purity of periplogenin is 91.2%) were isolated from Cortex Periplocae in our laboratory, the purity and structure were confirmed by 1 H NMR, IR, HPLC, and MS spectra.
2.2. Chromatographic and mass spectrometry conditions The chromatographic separation was achieved on a Waters XBridgeTM BEH C18 column (4.6 mm × 50 mm, 2.5 m). The mobile phase consisted of 0.1% (v/v) formic acid (A) and acetonitrile (B) using a gradient elution of 35–45% B at 0–5 min, 45–55% B at 5–9 min. The flow rate was 0.4 mL/min. The separation temperature was set at 30 ◦ C, and the sample injection volume was 5 L. Mass detection was operated in the positive electrospray ionization mode. The MS parameters were optimized and set as follows: capillary voltage at 4000 V, nebulizer at 15 psi, drying gas flow rate at 11 L/min and temperature at 300 ◦ C. The collision energy (CE) and fragmentor were 10, 135 eV for periplogenin, and 25, 115 eV for IS, respectively. The mass spectrometer was set in MRM mode using target ions at m/z 391.3 → 337.2 for periplogenin and m/z 187.0 → 131.0 for IS. Data acquisition was performed with Masshunter Workstation Software from Agilent Technologies (version B.04.00).
2.3. Preparation of calibration standards and quality control samples Stock solutions of periplogenin and IS were prepared separately in methanol at a concentration of 1 mg/mL and stored at −20 ◦ C. Working solutions at concentrations of 1, 2.5, 5, 25, 125, 250, 625 and 1250 ng/mL and an IS working solution at 1 g/mL were prepared via sequential dilution of the stock solutions of periplogenin and IS with methanol. Calibration standard solutions were prepared by spiking the blank rat plasma (100 L) with an appropriate amount of the working solutions, yielding final concentrations of 0.2, 0.5, 1, 5, 25, 50, 125 and 250 ng/mL of periplogenin and 200 ng/mL of psoralen. Quality control (QC) plasma samples were prepared at three concentrations: 0.5, 25 and 250 ng/mL. All standard stock solutions were stored at −20 ◦ C. 2.4. Plasma sample preparation Hundred microliters of plasma, 20 L of the IS working solution and 20 L of methanol (volume of the corresponding working solution for calibration curve and QC samples) were pipetted into a centrifuge tube and vortexed for 30 s. Ethyl acetate (1.5 mL) was added to the mixture and then the whole vortexed for 3 min to extract compounds from the plasma. After centrifugation at 14,000 rpm for 10 min, 1400 L of the supernatant was transferred into another centrifuge tube and evaporated to dryness. The obtained residue was redissolved in 100 L methanol, then the mixture vortexed for 3 min and centrifuged at 14,000 rpm for
2.5. Method validation 2.5.1. Specificity The specificity of the method was assessed by comparing the chromatograms of the blank plasma with the corresponding blood samples spiked with periplogenin and IS, as well as samples collected from treated rats. The chromatographic interference from endogenous plasma matrix components was estimated using the proposed preparation and LC–MS/MS conditions.
2.5.2. Linearity and sensitivity The linearity was evaluated by constructing a linear regression equation that fits the area ratio response for periplogenin/IS as a function of standard concentration, using 1/x2 as weighting factor. The LLOQ, determined at a signal-to-noise ratio (S/N) of about 10, was defined as the lowest concentration on the calibration curve with the precision (RSD) below 20% and accuracy (RE) within ±20%.
2.5.3. Precision and accuracy To determine intra-day precision and accuracy, six replicates of QC samples at low, medium and high concentration levels (0.5, 25, 250 ng/mL for periplogenin) were prepared and analyzed on the same day. Inter-day precision and accuracy were evaluated on three independent days. The intra- and inter-day precisions were expressed as the RSD value and the accuracy as the RE value.
2.5.4. Extraction recovery and matrix effect The extraction recoveries of periplogenin at 0.5, 25 and 250 ng/mL levels were calculated by comparing the peak area of periplogenin added into blank plasma and extracted using the liquid–liquid extraction procedure to those obtained from postextracted spiked samples. The matrix effects were evaluated as the ratio of the peak areas of samples spiked post-extraction to those in non-extracted samples at equivalent concentrations (n = 6).
2.5.5. Stability The short-term stability was evaluated by determining QC samples at room temperature for 6 h. The auto-sampler stability was detected in auto-sampler after preparation for 12 h. The long-term stability was assessed by storing the QC samples at −20 ◦ C for 30 days. The freeze–thaw stability was determined through 3 complete freeze–thaw cycles on consecutive days.
2.6. Application Six male Sprague–Dawley rats (210 ± 10 g) were used for pharmacokinetic analysis of periplogenin. The rats were kept in plastic cages, in a stable environment, with temperatures between 23–26 ◦ C and relative humidity of 40–60%. They also had free access to standard rat diet and water. After an overnight fast of 12 h, the rats were orally administered Cortex Periplocae extract at a single dose of 30 mg/kg. Blood samples (250 L) were collected into heparinized centrifuge tubes from the fossa orbitalis vein at pre-dose, 0.03, 0.08, 0.16, 0.25, 0.33, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h post administration in rats. All samples were immediately centrifuged at 6000 rpm for 10 min. The supernatant was transferred into another centrifuge tube and stored at −20 ◦ C until analysis.
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Fig. 1. Typical multiple reaction monitoring chromatograms of periplogenin and IS: (A) blank plasma; (B) blank plasma spiked with periplogenin and IS. (C) Real subject sample (63.9 ng/mL of periplogenin).
3. Results and discussion 3.1. Optimization of LC–MS/MS conditions The ion abundances of periplogenin and IS were significantly greater in positive-ion mode than in negative-ion mode. In the fully scanned mass spectra, the positive ESI produced molecular ions ([M+H]+ ) at m/z 391.3 for periplogenin and 187.0 for IS, respectively. The most abundant and stable product ions were generated at m/z 337.2 for periplogenin and m/z 131.0 for IS. After continuous adjustment of the chromatographic separation conditions, it was found that the addition of 0.1% formic acid in the mobile phase could enhance the sensitivity and improve the peak shapes of periplogenin and IS. Meanwhile, gradient elution was discovered to produce better signal response, narrower peaks, exerted fast elution and higher sensitivity. Consequently, periplogenin and IS were eluted within 9 min, without endogenous plasma components interfering with the analyte. The chromatograms of the blank plasma, the blank plasma with periplogenin and IS, and the plasma samples of orally administered rats are shown in Fig. 1.
3.2. Sample preparation Compared with protein precipitation extraction, the simple liquid–liquid extraction (LLE) with ethyl acetate was found to offer satisfactory recovery and efficiency in determining the concentrations of periplogenin. This extraction method was also found to be simple, convenient and time-saving. Eventually, the simple singlestep liquid–liquid extraction with ethyl acetate was adopted for the pharmacokinetic analysis of periplogenin.
3.3.2. Linearity and sensitivity The standard calibration curve for periplogenin was linear over the concentration range of 0.2–250 ng/mL by using weighted least square linear regression analysis with a weigh factor of 1/x2 . The typical equation for the calibration curves for periplogenin was y = 0.221753x + 3.851335 × 10−5 (r = 0.9939), where y represents the peak area ratio of periplogenin to the IS and x represents the concentration of analyte in spiked plasma samples. The LLOQ for periplogenin was established at 0.2 ng/mL, the precision (RSD) and accuracy (RE) were 6.63% and 6.11%, respectively.
3.3.3. Precision and accuracy The intra- and inter-day precision and accuracy of the QC samples at 0.5, 25 and 250 ng/mL are presented in Table 1. At each QC level, the intra- and inter-day precisions (RSD) values were below 9.85%, and the mean accuracy ranged from −10.03% to 10.26%.
3.3.4. Extraction recovery and matrix effect The extraction recoveries of periplogenin using six replicates of QC samples were 85.10 ± 2.45%, 95.62 ± 2.13% and 92.85 ± 3.24% (mean ± SD, n = 6) at three concentrations of 0.5, 25, 250 ng/mL, with RSDs 2.88, 2.23, 3.49%, respectively. The recovery of IS was 86.42 ± 3.71%, and the RSD was 4.29%. The matrix effects of the three QC samples at the three concentrations above were 112.06 ± 4.44%, 91.15 ± 2.22%, 94.50 ± 2.03%, and the RSDs were 3.96%, 2.43%, 2.14%, respectively. In addition, the matrix effect of IS was 95.47 ± 1.27%, and the RSD was 1.34%. The results indicate that the LLE efficiency was acceptable and no coeluting substance could influence the ionization of the periplogenin and the IS.
3.3. Method validation 3.3.1. Specificity In this study, the specificity was investigated by analyzing six different batches of plasma samples. No interference from endogenous substances was observed at the retention time of periplogenin (4.63 min) and IS (7.04 min) due to the high selectivity.
3.3.5. Stability The stability of QC samples was investigated at three concentrations (0.5, 25 and 250 ng/mL) under various conditions during sample collection and processing, and the results are shown in Table 2. These observations suggest that periplogenin is reasonably stable at different conditions.
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Table 1 Precision and accuracy of periplogenin in rat plasma (n = 6). Spiked concentration (ng/mL)
0.5 25 250
Intra-day
Inter-day
Measured concentration (ng/mL)
Precision (RSD, %)
Accuracy (RE, %)
Measured concentration (ng/mL)
Precision (RSD, %)
Accuracy (RE, %)
0.52 ± 0.02 22.49 ± 0.86 231.94 ± 9.72
3.85 3.82 4.19
4.42 −10.03 −7.23
0.49 ± 0.03 22.65 ± 0.95 244.40 ± 24.07
6.14 4.19 9.85
−2.33 −9.39 −2.24
Table 2 Stability of periplogenin in rat plasma (n = 3). Nominal concentration (ng/mL)
0.5 25 250
30 days storage at −20 ◦ C
6 h at room temperature
12 h in autosampler
3 freeze–thaw cycles
Measured concentration
RE (%)
Measured concentration
RE (%)
Measured concentration
RE (%)
Measured concentration
RE (%)
0.53 ± 0.02 23.73 ± 2.03 277.75 ± 4.02
6.37 −5.07 11.10
0.55 ± 0.03 23.62 ± 1.42 268.20 ± 5.40
10.13 −5.52 7.28
0.50 ± 0.06 24.24 ± 3.14 236.15 ± 7.29
−0.53 −3.04 −5.54
0.53 ± 0.03 24.44 ± 2.02 248.49 ± 13.14
6.28 −2.25 −0.60
matrix. In the present study, we developed a LC–MS/MS method with positive-ion mode of MRM. The desired and superior sensitivity with an LLOQ of 0.2 ng/mL was achieved. In addition, the method has shown to have adequate specificity, precision, recovery and stability and has been successfully applied to pharmacokinetic analysis of periplogenin after a single-dose oral administration of Cortex Periplocae extract in rats. This is the first assay regarding periplogenin in the field of pharmacokinetic study and the results might be helpful for application in clinical therapy. Acknowledgments
Fig. 2. Mean plasma profile following oral administration of Cortex Periplocae extract at 30 mg/kg to SD rats (mean ± SD, n = 6). Table 3 Pharmacokinetic parameters of periplogenin following single oral administration at a dose of 30 mg/kg Cortex Periplocae extract to rats (n = 6). Parameter
Mean ± SD
Cmax (ng/mL) Tmax (h) T1/2 (h) Ke (1/h) AUC0–24 (ng h/L) AUC0–∞ (ng h/L)
76.14 0.20 1.03 0.73 124.62 132.41
± ± ± ± ± ±
23.73 0.15 0.36 0.20 30.36 25.56
3.4. Application The validated method was applied to a pharmacokinetic study of periplogenin after a single oral dose of Cortex Periplocae extract at 30 mg/kg to rats. The plasma concentration–time curve of periplogenin is illustrated in Fig. 2. As shown in Table 3, the maximum concentration (Cmax ) of periplogenin in plasma (76.14 ± 23.73 mg/mL) was attained at 0.20 ± 0.15 h (Tmax ), and plasma concentration declined with the T1/2 of 1.03 ± 0.36 h. The results indicate that periplogenin was rapidly absorbed and eliminated in rat plasma after oral administration of Cortex Periplocae extract. 4. Conclusions To the best of our knowledge, there is no validated method available for the determination of periplogenin in biological
This study was supported by National Natural Science Foundation of China (81303140), Doctoral Fund of Ministry of Education of China (20131210120015), Science and Technology Commission of MOST, China (2014ZX09304307001), Tianjin Science and Technology Development Fund for Colleges and Universitie (20110212), Program for Innovative Research Team in Universities of Tianjin. References [1] State Pharmacopoeia Commission, Pharmacopoeia of People’s Republic of China, China Medical Science and Technology Press, Beijing, China, 2010, pp. 240–241. [2] Y.H. Li, X.M. Gao, B.L. Zhang, Q. Xu, H. Liu, G.X. Pan, J. Liaoning Coll. Tradit. Med. 7 (2005) 396–397. [3] J. Zhang, B.E. Shan, G.S. Liu, X.T. Zhao, S.H. Chen, Carcinog. Teratogenesis Mutagen. 18 (2006) 108–111. [4] B.E. Shan, J.X. Li, J. Zhang, W.J. Liang, H.F. Li, Carcinog. Teratogenesis Mutagen. 17 (2005) 265–268. [5] J. Zhang, B.E. Shan, G.S. Liu, S.H. Chen, X.T. Zhao, Tumor 26 (2006) 418–421. [6] L.M. Zhao, B.E. Shan, J. Ai, F.Z. Ren, Y.S. Lian, X.T. Song, Tumor 28 (2008) 203–206. [7] B.E. Shan, J.X. Li, J. Zhang, Chin. Tradit. Herb. Drugs 36 (2005) 1184–1188. [8] Z.J. Lu, Y. Zhou, Q. Song, Z. Qin, H. Zhang, Y.J. Zhou, L.T. Gou, J.L. Yang, F. Luo, Cell. Physiol. Biochem. 26 (2010) 609–618. [9] Z.J. Lu, Q. Song, J.L. Yang, X.F. Zhao, X.H. Zhang, P. Yang, J.B. Kang, Cell. Physiol. Biochem. 33 (2014) 859–868. [10] L.M. Zhao, B.E. Shan, Y.Y. Du, M.X. Wang, L.H. Liu, F.Z. Ren, Oncol. Rep. 24 (2010) 375–383. [11] X.Y. Wang, X.M. Gao, H. Liu, H. Zhang, Y. Liu, M. Jiang, L.M. Hu, B.L. Zhang, Chin. J. Integr. Med. 16 (2010) 33–40. [12] S. Panda, A. Kar, Horm. Metab. Res. 43 (2011) 188–193. [13] W. Gu, L.J. Zhao, A.G. Zhao, China Pharm. 19 (2008) 166–168. [14] Y.B. Han, A.G. Zhao, J. China Pediatr. Blood Cancer 13 (2008) 1–5. [15] H. Liu, M. Wang, H. Yang, G.X. Pan, J.H. Guo, Chin. Tradit. Herb. Drugs 37 (2006) 1355–1356. [16] R.X. Liu, D.A. Guo, M. Ye, Q. Wang, J.L. Zhang, World Science and Technology/Modernization of Traditional Chinese Medicine and Materia, Medica 7 (2005) 33–40.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
A validated LC–MS/MS method for determination of periplogenin in rat plasma and its application in pharmacokinetic study Fang Bo a , Ting Dou a , Xingrui Wang a , Paul Owusu Donkor a , Huizi Ouyang b , Yanxu Chang a , Yaru Tu a , Xiumei Gao a , Jun He a,∗ a b
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China The First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
a r t i c l e
i n f o
Article history: Received 15 January 2015 Accepted 22 March 2015 Available online 30 March 2015 Keywords: Periplogenin LC–MS/MS Pharmacokinetics Cortex Periplocae
a b s t r a c t A method coupling high performance liquid chromatography with tandem mass spectrometry has been developed and validated for quantifying periplogenin in rat plasma using psoralen as an internal standard (IS). Plasma samples were pretreated using a simple liquid–liquid extraction with ethyl acetate and the chromatographic separation of periplogenin and psoralen was achieved on a Waters XBridgeTM BEH C18 column with 0.1% formic acid and acetonitrile as mobile phase at a flow rate of 0.4 mL/min. The detection was performed on a positive ion mode with electrospray ionization (ESI) source. The optimized ion transition pairs for quantitation were m/z 391.3 → m/z 337.2 for periplogenin and m/z 187.0 → m/z 131.0 for IS. The total run time was 9.0 min. The calibration curve was linear over the range of 0.2–250 ng/mL (r > 0.99) with the lower limit of quantitation (LLOQ) at 0.2 ng/mL. The intra- and inter-day precision were below 9.85% and the mean accuracy were from −10.03% to 10.26%. The average recoveries of periplogenin in plasma ranged from 85.1% to 95.6%. The proposed method was successfully applied in evaluating the pharmacokinetics of periplogenin after an oral dose of 30 mg/kg Cortex Periplocae extract in rats. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Cortex Periplocae is the root bark of Periploca Sepium Bunge which belongs to the Asclepiadaceae family. It has been shown that Cortex Periplocae has therapeutic effects on edema and rheumatism [1]. With the progress in the research, it has been found to improve cardiac function and exhibit cardiotonic effects on the isolated heart of rats [2]. In addition, it has been reported that Cortex Periplocae inhibits tumor cell proliferation by arresting the cell cycle, inducing apoptosis of tumor cells and blocking signaling pathways [3–8]. As is known to all, periplocin is one of the main components in the Cortex Periplocae, and many researches have been widely reported related to its cancer-fighting properties [9,10] and the treatment of heart diseases [11]. Furthermore, periplogenin which is another representative constituent derived from Cortex Periplocae has become a growing concern in recent years. Pharmacological research indicates that periplogenin plays a protective role in regulating hyperthyroidism
∗ Corresponding author. Tel.: +86 22 59596163; fax: +86 22 59596163. E-mail address: [email protected] (J. He). http://dx.doi.org/10.1016/j.jchromb.2015.03.017 1570-0232/© 2015 Elsevier B.V. All rights reserved.
and associated cardiovascular problems [12]. Periplogenin also has outstanding inhibitory effects on histamine release of mast cells either cultured in vitro or in antigen-pulsed rats. Meanwhile, oral administration of periplogenin can result in the marked reduction of histamine release of mast cells in rats. In light of the mast cell degranulation and histamine release in the inflammation effect, periplogenin can be regarded as one of the effective components with anti-inflammatory activity in Cortex Periplocae [13]. Moreover, periplogenin has been reported to possess anti-cancer property [14]. Despite its multiple biological activities, the pharmacokinetic properties of periplogenin have not yet been reported and it is exceedingly necessary to study the regularity of periplogenin concentration changes with time in vivo. The only reported approach to determine periplogenin was by HPLC [15] and no other methods were available for the application to analyze periplogenin in biological samples because of the low sensitivity and poor selectivity. It is well known that LC–MS/MS [16] plays a significant role in the quantification of main components of medicinal products for pharmacokinetic studies. In this study, we developed a LC–MS/MS method for determination of periplogenin in rat plasma. After validation, the method was successfully applied to pharmacokinetic study after oral administration of Cortex Periplocae extract in rats for the first time.
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2. Materials and methods
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10 min. Five microliters of the supernatant was injected into the LC–MS/MS system.
2.1. Reagents and chemicals Acetonitrile (Merck KGaA, Darmstadt, Germany), methanol (Merck KGaA, Darmstadt, Germany) and formic acid (ROE SCIENTIFIC INC, Newark, USA) were of HPLC grade. Analytical grade ethyl acetate was purchased from Tianjin Concord Science Co. Ltd. (Tianjin, China). Ultrapure water was prepared by a Milli-Q water purification system (Millipore, Milford, MA, USA). Psoralen (>98%) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Periplogenin (>98%) and Cortex Periplocae extract (the purity of periplogenin is 91.2%) were isolated from Cortex Periplocae in our laboratory, the purity and structure were confirmed by 1 H NMR, IR, HPLC, and MS spectra.
2.2. Chromatographic and mass spectrometry conditions The chromatographic separation was achieved on a Waters XBridgeTM BEH C18 column (4.6 mm × 50 mm, 2.5 m). The mobile phase consisted of 0.1% (v/v) formic acid (A) and acetonitrile (B) using a gradient elution of 35–45% B at 0–5 min, 45–55% B at 5–9 min. The flow rate was 0.4 mL/min. The separation temperature was set at 30 ◦ C, and the sample injection volume was 5 L. Mass detection was operated in the positive electrospray ionization mode. The MS parameters were optimized and set as follows: capillary voltage at 4000 V, nebulizer at 15 psi, drying gas flow rate at 11 L/min and temperature at 300 ◦ C. The collision energy (CE) and fragmentor were 10, 135 eV for periplogenin, and 25, 115 eV for IS, respectively. The mass spectrometer was set in MRM mode using target ions at m/z 391.3 → 337.2 for periplogenin and m/z 187.0 → 131.0 for IS. Data acquisition was performed with Masshunter Workstation Software from Agilent Technologies (version B.04.00).
2.3. Preparation of calibration standards and quality control samples Stock solutions of periplogenin and IS were prepared separately in methanol at a concentration of 1 mg/mL and stored at −20 ◦ C. Working solutions at concentrations of 1, 2.5, 5, 25, 125, 250, 625 and 1250 ng/mL and an IS working solution at 1 g/mL were prepared via sequential dilution of the stock solutions of periplogenin and IS with methanol. Calibration standard solutions were prepared by spiking the blank rat plasma (100 L) with an appropriate amount of the working solutions, yielding final concentrations of 0.2, 0.5, 1, 5, 25, 50, 125 and 250 ng/mL of periplogenin and 200 ng/mL of psoralen. Quality control (QC) plasma samples were prepared at three concentrations: 0.5, 25 and 250 ng/mL. All standard stock solutions were stored at −20 ◦ C. 2.4. Plasma sample preparation Hundred microliters of plasma, 20 L of the IS working solution and 20 L of methanol (volume of the corresponding working solution for calibration curve and QC samples) were pipetted into a centrifuge tube and vortexed for 30 s. Ethyl acetate (1.5 mL) was added to the mixture and then the whole vortexed for 3 min to extract compounds from the plasma. After centrifugation at 14,000 rpm for 10 min, 1400 L of the supernatant was transferred into another centrifuge tube and evaporated to dryness. The obtained residue was redissolved in 100 L methanol, then the mixture vortexed for 3 min and centrifuged at 14,000 rpm for
2.5. Method validation 2.5.1. Specificity The specificity of the method was assessed by comparing the chromatograms of the blank plasma with the corresponding blood samples spiked with periplogenin and IS, as well as samples collected from treated rats. The chromatographic interference from endogenous plasma matrix components was estimated using the proposed preparation and LC–MS/MS conditions.
2.5.2. Linearity and sensitivity The linearity was evaluated by constructing a linear regression equation that fits the area ratio response for periplogenin/IS as a function of standard concentration, using 1/x2 as weighting factor. The LLOQ, determined at a signal-to-noise ratio (S/N) of about 10, was defined as the lowest concentration on the calibration curve with the precision (RSD) below 20% and accuracy (RE) within ±20%.
2.5.3. Precision and accuracy To determine intra-day precision and accuracy, six replicates of QC samples at low, medium and high concentration levels (0.5, 25, 250 ng/mL for periplogenin) were prepared and analyzed on the same day. Inter-day precision and accuracy were evaluated on three independent days. The intra- and inter-day precisions were expressed as the RSD value and the accuracy as the RE value.
2.5.4. Extraction recovery and matrix effect The extraction recoveries of periplogenin at 0.5, 25 and 250 ng/mL levels were calculated by comparing the peak area of periplogenin added into blank plasma and extracted using the liquid–liquid extraction procedure to those obtained from postextracted spiked samples. The matrix effects were evaluated as the ratio of the peak areas of samples spiked post-extraction to those in non-extracted samples at equivalent concentrations (n = 6).
2.5.5. Stability The short-term stability was evaluated by determining QC samples at room temperature for 6 h. The auto-sampler stability was detected in auto-sampler after preparation for 12 h. The long-term stability was assessed by storing the QC samples at −20 ◦ C for 30 days. The freeze–thaw stability was determined through 3 complete freeze–thaw cycles on consecutive days.
2.6. Application Six male Sprague–Dawley rats (210 ± 10 g) were used for pharmacokinetic analysis of periplogenin. The rats were kept in plastic cages, in a stable environment, with temperatures between 23–26 ◦ C and relative humidity of 40–60%. They also had free access to standard rat diet and water. After an overnight fast of 12 h, the rats were orally administered Cortex Periplocae extract at a single dose of 30 mg/kg. Blood samples (250 L) were collected into heparinized centrifuge tubes from the fossa orbitalis vein at pre-dose, 0.03, 0.08, 0.16, 0.25, 0.33, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h post administration in rats. All samples were immediately centrifuged at 6000 rpm for 10 min. The supernatant was transferred into another centrifuge tube and stored at −20 ◦ C until analysis.
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Fig. 1. Typical multiple reaction monitoring chromatograms of periplogenin and IS: (A) blank plasma; (B) blank plasma spiked with periplogenin and IS. (C) Real subject sample (63.9 ng/mL of periplogenin).
3. Results and discussion 3.1. Optimization of LC–MS/MS conditions The ion abundances of periplogenin and IS were significantly greater in positive-ion mode than in negative-ion mode. In the fully scanned mass spectra, the positive ESI produced molecular ions ([M+H]+ ) at m/z 391.3 for periplogenin and 187.0 for IS, respectively. The most abundant and stable product ions were generated at m/z 337.2 for periplogenin and m/z 131.0 for IS. After continuous adjustment of the chromatographic separation conditions, it was found that the addition of 0.1% formic acid in the mobile phase could enhance the sensitivity and improve the peak shapes of periplogenin and IS. Meanwhile, gradient elution was discovered to produce better signal response, narrower peaks, exerted fast elution and higher sensitivity. Consequently, periplogenin and IS were eluted within 9 min, without endogenous plasma components interfering with the analyte. The chromatograms of the blank plasma, the blank plasma with periplogenin and IS, and the plasma samples of orally administered rats are shown in Fig. 1.
3.2. Sample preparation Compared with protein precipitation extraction, the simple liquid–liquid extraction (LLE) with ethyl acetate was found to offer satisfactory recovery and efficiency in determining the concentrations of periplogenin. This extraction method was also found to be simple, convenient and time-saving. Eventually, the simple singlestep liquid–liquid extraction with ethyl acetate was adopted for the pharmacokinetic analysis of periplogenin.
3.3.2. Linearity and sensitivity The standard calibration curve for periplogenin was linear over the concentration range of 0.2–250 ng/mL by using weighted least square linear regression analysis with a weigh factor of 1/x2 . The typical equation for the calibration curves for periplogenin was y = 0.221753x + 3.851335 × 10−5 (r = 0.9939), where y represents the peak area ratio of periplogenin to the IS and x represents the concentration of analyte in spiked plasma samples. The LLOQ for periplogenin was established at 0.2 ng/mL, the precision (RSD) and accuracy (RE) were 6.63% and 6.11%, respectively.
3.3.3. Precision and accuracy The intra- and inter-day precision and accuracy of the QC samples at 0.5, 25 and 250 ng/mL are presented in Table 1. At each QC level, the intra- and inter-day precisions (RSD) values were below 9.85%, and the mean accuracy ranged from −10.03% to 10.26%.
3.3.4. Extraction recovery and matrix effect The extraction recoveries of periplogenin using six replicates of QC samples were 85.10 ± 2.45%, 95.62 ± 2.13% and 92.85 ± 3.24% (mean ± SD, n = 6) at three concentrations of 0.5, 25, 250 ng/mL, with RSDs 2.88, 2.23, 3.49%, respectively. The recovery of IS was 86.42 ± 3.71%, and the RSD was 4.29%. The matrix effects of the three QC samples at the three concentrations above were 112.06 ± 4.44%, 91.15 ± 2.22%, 94.50 ± 2.03%, and the RSDs were 3.96%, 2.43%, 2.14%, respectively. In addition, the matrix effect of IS was 95.47 ± 1.27%, and the RSD was 1.34%. The results indicate that the LLE efficiency was acceptable and no coeluting substance could influence the ionization of the periplogenin and the IS.
3.3. Method validation 3.3.1. Specificity In this study, the specificity was investigated by analyzing six different batches of plasma samples. No interference from endogenous substances was observed at the retention time of periplogenin (4.63 min) and IS (7.04 min) due to the high selectivity.
3.3.5. Stability The stability of QC samples was investigated at three concentrations (0.5, 25 and 250 ng/mL) under various conditions during sample collection and processing, and the results are shown in Table 2. These observations suggest that periplogenin is reasonably stable at different conditions.
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Table 1 Precision and accuracy of periplogenin in rat plasma (n = 6). Spiked concentration (ng/mL)
0.5 25 250
Intra-day
Inter-day
Measured concentration (ng/mL)
Precision (RSD, %)
Accuracy (RE, %)
Measured concentration (ng/mL)
Precision (RSD, %)
Accuracy (RE, %)
0.52 ± 0.02 22.49 ± 0.86 231.94 ± 9.72
3.85 3.82 4.19
4.42 −10.03 −7.23
0.49 ± 0.03 22.65 ± 0.95 244.40 ± 24.07
6.14 4.19 9.85
−2.33 −9.39 −2.24
Table 2 Stability of periplogenin in rat plasma (n = 3). Nominal concentration (ng/mL)
0.5 25 250
30 days storage at −20 ◦ C
6 h at room temperature
12 h in autosampler
3 freeze–thaw cycles
Measured concentration
RE (%)
Measured concentration
RE (%)
Measured concentration
RE (%)
Measured concentration
RE (%)
0.53 ± 0.02 23.73 ± 2.03 277.75 ± 4.02
6.37 −5.07 11.10
0.55 ± 0.03 23.62 ± 1.42 268.20 ± 5.40
10.13 −5.52 7.28
0.50 ± 0.06 24.24 ± 3.14 236.15 ± 7.29
−0.53 −3.04 −5.54
0.53 ± 0.03 24.44 ± 2.02 248.49 ± 13.14
6.28 −2.25 −0.60
matrix. In the present study, we developed a LC–MS/MS method with positive-ion mode of MRM. The desired and superior sensitivity with an LLOQ of 0.2 ng/mL was achieved. In addition, the method has shown to have adequate specificity, precision, recovery and stability and has been successfully applied to pharmacokinetic analysis of periplogenin after a single-dose oral administration of Cortex Periplocae extract in rats. This is the first assay regarding periplogenin in the field of pharmacokinetic study and the results might be helpful for application in clinical therapy. Acknowledgments
Fig. 2. Mean plasma profile following oral administration of Cortex Periplocae extract at 30 mg/kg to SD rats (mean ± SD, n = 6). Table 3 Pharmacokinetic parameters of periplogenin following single oral administration at a dose of 30 mg/kg Cortex Periplocae extract to rats (n = 6). Parameter
Mean ± SD
Cmax (ng/mL) Tmax (h) T1/2 (h) Ke (1/h) AUC0–24 (ng h/L) AUC0–∞ (ng h/L)
76.14 0.20 1.03 0.73 124.62 132.41
± ± ± ± ± ±
23.73 0.15 0.36 0.20 30.36 25.56
3.4. Application The validated method was applied to a pharmacokinetic study of periplogenin after a single oral dose of Cortex Periplocae extract at 30 mg/kg to rats. The plasma concentration–time curve of periplogenin is illustrated in Fig. 2. As shown in Table 3, the maximum concentration (Cmax ) of periplogenin in plasma (76.14 ± 23.73 mg/mL) was attained at 0.20 ± 0.15 h (Tmax ), and plasma concentration declined with the T1/2 of 1.03 ± 0.36 h. The results indicate that periplogenin was rapidly absorbed and eliminated in rat plasma after oral administration of Cortex Periplocae extract. 4. Conclusions To the best of our knowledge, there is no validated method available for the determination of periplogenin in biological
This study was supported by National Natural Science Foundation of China (81303140), Doctoral Fund of Ministry of Education of China (20131210120015), Science and Technology Commission of MOST, China (2014ZX09304307001), Tianjin Science and Technology Development Fund for Colleges and Universitie (20110212), Program for Innovative Research Team in Universities of Tianjin. References [1] State Pharmacopoeia Commission, Pharmacopoeia of People’s Republic of China, China Medical Science and Technology Press, Beijing, China, 2010, pp. 240–241. [2] Y.H. Li, X.M. Gao, B.L. Zhang, Q. Xu, H. Liu, G.X. Pan, J. Liaoning Coll. Tradit. Med. 7 (2005) 396–397. [3] J. Zhang, B.E. Shan, G.S. Liu, X.T. Zhao, S.H. Chen, Carcinog. Teratogenesis Mutagen. 18 (2006) 108–111. [4] B.E. Shan, J.X. Li, J. Zhang, W.J. Liang, H.F. Li, Carcinog. Teratogenesis Mutagen. 17 (2005) 265–268. [5] J. Zhang, B.E. Shan, G.S. Liu, S.H. Chen, X.T. Zhao, Tumor 26 (2006) 418–421. [6] L.M. Zhao, B.E. Shan, J. Ai, F.Z. Ren, Y.S. Lian, X.T. Song, Tumor 28 (2008) 203–206. [7] B.E. Shan, J.X. Li, J. Zhang, Chin. Tradit. Herb. Drugs 36 (2005) 1184–1188. [8] Z.J. Lu, Y. Zhou, Q. Song, Z. Qin, H. Zhang, Y.J. Zhou, L.T. Gou, J.L. Yang, F. Luo, Cell. Physiol. Biochem. 26 (2010) 609–618. [9] Z.J. Lu, Q. Song, J.L. Yang, X.F. Zhao, X.H. Zhang, P. Yang, J.B. Kang, Cell. Physiol. Biochem. 33 (2014) 859–868. [10] L.M. Zhao, B.E. Shan, Y.Y. Du, M.X. Wang, L.H. Liu, F.Z. Ren, Oncol. Rep. 24 (2010) 375–383. [11] X.Y. Wang, X.M. Gao, H. Liu, H. Zhang, Y. Liu, M. Jiang, L.M. Hu, B.L. Zhang, Chin. J. Integr. Med. 16 (2010) 33–40. [12] S. Panda, A. Kar, Horm. Metab. Res. 43 (2011) 188–193. [13] W. Gu, L.J. Zhao, A.G. Zhao, China Pharm. 19 (2008) 166–168. [14] Y.B. Han, A.G. Zhao, J. China Pediatr. Blood Cancer 13 (2008) 1–5. [15] H. Liu, M. Wang, H. Yang, G.X. Pan, J.H. Guo, Chin. Tradit. Herb. Drugs 37 (2006) 1355–1356. [16] R.X. Liu, D.A. Guo, M. Ye, Q. Wang, J.L. Zhang, World Science and Technology/Modernization of Traditional Chinese Medicine and Materia, Medica 7 (2005) 33–40.
