Journal of Chromatography B, 972 (2014) 22–28
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
The metabolism of YiGan San and subsequent pharmacokinetic evaluation of four metabolites in rat based on liquid chromatography with tandem mass spectrometry Yanan Gai a,b , Han Chen a , Wenyuan Liu a,c,∗ , Feng Feng d,∗∗ , Ning Xie e a
Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China The Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China c Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, China d Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China e Jiangxi Qingfeng Pharmaceutical Corporation, Ganzhou, China b
a r t i c l e
i n f o
Article history: Received 4 May 2014 Accepted 23 September 2014 Available online 30 September 2014 Keywords: YiGan San Pharmacokinetics Metabolites High performance liquid chromatography Quadrupole mass spectrometry Time-of-flight mass spectrometry
a b s t r a c t A new method based on liquid chromatography-tandem time-of-flight mass spectrometry was developed to identify the metabolites in rat urine after oral administration of YiGan San (YGS). Eighteen prototype compounds and four metabolites named 11-hydroxyhirsuteine, 19-carbonylhirsutine, 19-carbonyldihydrocorynantheine, and 18-hydroxy-geissoschizine methyl ether were identified. Subsequently, a method of high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry was established for pharmacokinetic study of YGS in rat plasma. The concentration–time curves of four prototype compounds, senkyunolide I, ajmalicine, isocorynoxeine and rhynchophylline were constructed after an oral (9.1 g YGS per kilogram of body weight) administration in rats. Method validation revealed excellent linearity over the range 220.00–0.55, 220.00–0.55, 21.40–0.05, and 19.80–0.05 ng/mL for the four prototype compounds respectively. The stabilities results indicate that all of the analytes were stable in rat plasma in the autosampler for 24 h, under freeze/thaw cycles (4 times in 24 h), and at −20 ◦ C for one week. Residual analysis, heteroskedasticity test, and goodness-of-fit test were also performed to determine the accuracy of the linear regression method. The pharmacokinetic parameters were obtained. Four hours after administration, compound 11-hydroxyhirsuteine can be detected in rat plasma. Compared with purified ligustilide, YGS required a slightly longer period to reach maximum concentration (Cmax ) in rat plasma. © 2014 Elsevier B.V. All rights reserved.
1. Introduction YiGan San (YGS), a traditional formula, is used to treat insomnia and irritability in children. An increasing number of pharmacological and clinical studies have proven its ability to improve the neuropsychiatric symptoms of Alzheimer’s disease [1] and Parkinson’s disease [2]. This ability may be associated with attenuated neuronal activity in the medial prefrontal cortex and amygdala [3], extracellular glutamate levels, and glutamate transporter-1 (GLT-1) mRNA expression in the striatum [4]. YGS is composed of
∗ Corresponding author at: Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Tel.: +86 25 83271038; fax: +86 25 83271038. ∗∗ Corresponding author. Tel.: +86 25 83271038; fax: +86 25 83271038. E-mail addresses: [email protected] (W. Liu), [email protected] (F. Feng). http://dx.doi.org/10.1016/j.jchromb.2014.09.033 1570-0232/© 2014 Elsevier B.V. All rights reserved.
seven ingredient herbals, Atractylodis rhizome, Poria sclerotium, Chuanxiong rhizome, Ramulus uncariae cum uncis, Angelicae radix, Bupleuri radix, and Glycyrrhizae radix et rhizome in the proportions of 4:4:3:3:3:2:1.5. Ramulus uncariae cum uncis, Glycyrrhizae radix et rhizome and their constituents (geissoschizine methyl ether, rhynchophylline, and isoliquiritigenin) [5–7], were found to contribute to the highly protective effects [8]. However, there are few studies on the metabolism and disposition of YGS formula, except that pharmacokinetics of four essential components (rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine) has been investigated [9]. A recent study indicated that the levels of rhynchophylline and isorhynchophylline in rat brain after administration of YGS were higher than administrating with Ramulus uncariae cum uncis singly. This difference is attributed to drug synergism, which is a beneficial effect of the traditional formula [10]. It is suggested that, administration of the formula instead of single herb may be more beneficial.
Y. Gai et al. / J. Chromatogr. B 972 (2014) 22–28
Therefore, in the present study, a high-performance liquid chromatography–electrospray ionization-hybrid quadrupole/ time-of-flight mass spectrometry (HPLC–Q-TOF-MS) method was developed to profile the metabolism of YGS formula. In vivo fingerprint chromatogram was obtained and compared with that of YGS. As a result, 18 prototype compounds and 4 metabolites were identified. Subsequently, a sensitive high-performance liquid chromatography–electrospray ionization–triple quadrupole mass spectrometry (HPLC–ESI-QqQ-MS) method with multiple reaction monitoring (MRM) mode was established to quantify senkyunolide I, ajmalicine, isocorynoxeine, and rhynchophylline in rat plasma. The quantitative method was applied in the pharmacokinetic study, and concentration–time curves of senkyunolide I, ajmalicine, isocorynoxeine, and rhynchophylline were plotted by DAS 2.0.
2. Experimental 2.1. Chemicals and reagents Reference standards of senkyunolide I, ajmalicine, rhynchophylline, isocorynoxeine, corynoxeine, and colchicine (internal standard, IS) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of the standards was >98.0%. Acetonitrile and HPLCgrade methanol were purchased from Tedia (Fair-field, OH, USA). Ultrahigh-purity water prepared by using a Milli-Q water purification system (Millipore Corporation, MA, USA) was used for HPLC. Analytically pure acetic acid, ammonium acetate, and ethyl acetate were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). 2.2. Preparation of YGS Seven medicinal herbs were purchased from local herb stores in Jiangsu Province (Jiangsu, China). 126 g herbal mixture: Atractylodis rhizome, Poria sclerotium, Chuanxiong rhizome, Ramulus uncariae cum uncis, Angelicae radix, Bupleuri radix, and Glycyrrhizae radix in the proportions of 4:4:3:3:3:2:1.5 was pulverized, soaked overnight and extracted thrice with 1 L of purified water for 60 min. 1 h of conventional heat refluxing was conducted for each extraction. The liquid was filtered, combined, and then evaporated under reduced pressure to obtain 45 g of dry extract (YGS). 1.2 g YGS was dissolved in 8 mL of purified water. The resulting mixture was centrifuged at 16,000 rpm for 5 min and the supernatant was filtered through a 0.45 m membrane. A 10 L aliquot of the supernatant was used to obtain the chemical profile by HPLC–Q-TOF-MS. 2.3. Preparation of urine samples Male Sprague–Dawley rats (200–240 g) were purchased from the Comparative Medicine Center of Yangzhou University (Jiangsu, China). The rats were housed in an environment with 25 ◦ C temperature, 45–55% humidity, and 12 h/12 h light–dark cycle. The animals had free access to food and water. All procedures used in the animal experiments complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. After 3 days of acclimatization, blank urine samples and urine samples after oral administration of YGS (9.1 g YGS per kilogram of body weight) [11] were collected by using metabolism cages. The urine was then extracted with ethyl acetate at a ratio of 1:2 (urine/ethyl acetate, v/v). The ethyl acetate extract was centrifuged at 16,000 rpm for 5 min. The supernatant was dried with a stream of N2 and then dissolved in methanol. A 10 L aliquot of the
23
methanol solution was analyzed by HPLC–Q-TOF-MS to screen for compounds that enter the systemic circulation. 2.4. Preparation of plasma samples Blood samples (500 L) were collected into heparinized 1.5 mL polyethylene centrifuge tubes before administration and at 0.08, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 20 h after administration. All of the samples were centrifuged immediately at 5000 rpm for 10 min at 4 ◦ C. Plasma from each sample was collected and frozen at −20 ◦ C until analysis. 2.5. Preparation of the calibration standards and quality control samples Standard stock solutions were prepared by separately dissolving 1.0 mg of senkyunolide I, ajmalicine, rhynchophylline, and isocorynoxeine in 10 mL of methanol to obtain a nominal concentration of 0.1 mg/mL. All of the stock solutions were kept at 4 ◦ C before use. A series of working solutions of the standard solutions were freshly prepared by diluting the standard stock solutions with methanol to appropriate concentrations. A methanolic solution of the IS (0.1 mg/mL) was prepared and then further diluted to 100 ng/mL to prepare a working solution. Calibration curves were constructed upon analysis of 200 L plasma samples spiked with 10 L of the appropriate working solution of each standard. Quality control (QC) samples were independently prepared. Each spiked sample contained all of the standards at three specified concentrations. To a 200 L aliquot of rat plasma, 10 L of IS solution and 500 L of ethyl acetate were added. After vortex-mixing the mixture for 3 min and centrifuging it at 16,000 rpm for 5 min, the supernatant was collected, dried with a stream of N2 , and then reconstituted in 100 L of methanol. Finally, 10 L of the methanol solution was analyzed by HPLC–ESI-QqQ-MS in the pharmacokinetics study. 2.6. Method validation In order to evaluate the method selectivity, blank rat plasma, plasma spiked with standard compounds, and urine samples were tested consecutively. The matrix effect was measured by comparing the peak responses of samples spiked post-extraction with those of standard solutions evaporated and then reconstituted in mobile phase. Analyses were performed at three QC concentration levels in six replicates. The extraction efficiency was determined by analysis of six replicates of plasma samples at three QC concentration levels. The extraction recovery was calculated by comparing the peak areas of plasma extracts spiked with analytes before extraction with those of samples spiked post-extraction. Calibration curves were obtained in duplicate on three consecutive days by plotting peak area ratio the analyte/IS versus plasma concentration using linear regression. Residual analysis, heteroskedasticity test, goodness-of-fit test, and significance test of regression coefficients were performed to monitor the accuracy of the linear calibration curves. The LLOQ was considered as the lowest concentration for which a response of at least five times that of the blank plasma could be obtained at an accuracy of ±20% and at a relative standard deviation of
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
The metabolism of YiGan San and subsequent pharmacokinetic evaluation of four metabolites in rat based on liquid chromatography with tandem mass spectrometry Yanan Gai a,b , Han Chen a , Wenyuan Liu a,c,∗ , Feng Feng d,∗∗ , Ning Xie e a
Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China The Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China c Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, China d Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China e Jiangxi Qingfeng Pharmaceutical Corporation, Ganzhou, China b
a r t i c l e
i n f o
Article history: Received 4 May 2014 Accepted 23 September 2014 Available online 30 September 2014 Keywords: YiGan San Pharmacokinetics Metabolites High performance liquid chromatography Quadrupole mass spectrometry Time-of-flight mass spectrometry
a b s t r a c t A new method based on liquid chromatography-tandem time-of-flight mass spectrometry was developed to identify the metabolites in rat urine after oral administration of YiGan San (YGS). Eighteen prototype compounds and four metabolites named 11-hydroxyhirsuteine, 19-carbonylhirsutine, 19-carbonyldihydrocorynantheine, and 18-hydroxy-geissoschizine methyl ether were identified. Subsequently, a method of high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry was established for pharmacokinetic study of YGS in rat plasma. The concentration–time curves of four prototype compounds, senkyunolide I, ajmalicine, isocorynoxeine and rhynchophylline were constructed after an oral (9.1 g YGS per kilogram of body weight) administration in rats. Method validation revealed excellent linearity over the range 220.00–0.55, 220.00–0.55, 21.40–0.05, and 19.80–0.05 ng/mL for the four prototype compounds respectively. The stabilities results indicate that all of the analytes were stable in rat plasma in the autosampler for 24 h, under freeze/thaw cycles (4 times in 24 h), and at −20 ◦ C for one week. Residual analysis, heteroskedasticity test, and goodness-of-fit test were also performed to determine the accuracy of the linear regression method. The pharmacokinetic parameters were obtained. Four hours after administration, compound 11-hydroxyhirsuteine can be detected in rat plasma. Compared with purified ligustilide, YGS required a slightly longer period to reach maximum concentration (Cmax ) in rat plasma. © 2014 Elsevier B.V. All rights reserved.
1. Introduction YiGan San (YGS), a traditional formula, is used to treat insomnia and irritability in children. An increasing number of pharmacological and clinical studies have proven its ability to improve the neuropsychiatric symptoms of Alzheimer’s disease [1] and Parkinson’s disease [2]. This ability may be associated with attenuated neuronal activity in the medial prefrontal cortex and amygdala [3], extracellular glutamate levels, and glutamate transporter-1 (GLT-1) mRNA expression in the striatum [4]. YGS is composed of
∗ Corresponding author at: Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Tel.: +86 25 83271038; fax: +86 25 83271038. ∗∗ Corresponding author. Tel.: +86 25 83271038; fax: +86 25 83271038. E-mail addresses: [email protected] (W. Liu), [email protected] (F. Feng). http://dx.doi.org/10.1016/j.jchromb.2014.09.033 1570-0232/© 2014 Elsevier B.V. All rights reserved.
seven ingredient herbals, Atractylodis rhizome, Poria sclerotium, Chuanxiong rhizome, Ramulus uncariae cum uncis, Angelicae radix, Bupleuri radix, and Glycyrrhizae radix et rhizome in the proportions of 4:4:3:3:3:2:1.5. Ramulus uncariae cum uncis, Glycyrrhizae radix et rhizome and their constituents (geissoschizine methyl ether, rhynchophylline, and isoliquiritigenin) [5–7], were found to contribute to the highly protective effects [8]. However, there are few studies on the metabolism and disposition of YGS formula, except that pharmacokinetics of four essential components (rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine) has been investigated [9]. A recent study indicated that the levels of rhynchophylline and isorhynchophylline in rat brain after administration of YGS were higher than administrating with Ramulus uncariae cum uncis singly. This difference is attributed to drug synergism, which is a beneficial effect of the traditional formula [10]. It is suggested that, administration of the formula instead of single herb may be more beneficial.
Y. Gai et al. / J. Chromatogr. B 972 (2014) 22–28
Therefore, in the present study, a high-performance liquid chromatography–electrospray ionization-hybrid quadrupole/ time-of-flight mass spectrometry (HPLC–Q-TOF-MS) method was developed to profile the metabolism of YGS formula. In vivo fingerprint chromatogram was obtained and compared with that of YGS. As a result, 18 prototype compounds and 4 metabolites were identified. Subsequently, a sensitive high-performance liquid chromatography–electrospray ionization–triple quadrupole mass spectrometry (HPLC–ESI-QqQ-MS) method with multiple reaction monitoring (MRM) mode was established to quantify senkyunolide I, ajmalicine, isocorynoxeine, and rhynchophylline in rat plasma. The quantitative method was applied in the pharmacokinetic study, and concentration–time curves of senkyunolide I, ajmalicine, isocorynoxeine, and rhynchophylline were plotted by DAS 2.0.
2. Experimental 2.1. Chemicals and reagents Reference standards of senkyunolide I, ajmalicine, rhynchophylline, isocorynoxeine, corynoxeine, and colchicine (internal standard, IS) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of the standards was >98.0%. Acetonitrile and HPLCgrade methanol were purchased from Tedia (Fair-field, OH, USA). Ultrahigh-purity water prepared by using a Milli-Q water purification system (Millipore Corporation, MA, USA) was used for HPLC. Analytically pure acetic acid, ammonium acetate, and ethyl acetate were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). 2.2. Preparation of YGS Seven medicinal herbs were purchased from local herb stores in Jiangsu Province (Jiangsu, China). 126 g herbal mixture: Atractylodis rhizome, Poria sclerotium, Chuanxiong rhizome, Ramulus uncariae cum uncis, Angelicae radix, Bupleuri radix, and Glycyrrhizae radix in the proportions of 4:4:3:3:3:2:1.5 was pulverized, soaked overnight and extracted thrice with 1 L of purified water for 60 min. 1 h of conventional heat refluxing was conducted for each extraction. The liquid was filtered, combined, and then evaporated under reduced pressure to obtain 45 g of dry extract (YGS). 1.2 g YGS was dissolved in 8 mL of purified water. The resulting mixture was centrifuged at 16,000 rpm for 5 min and the supernatant was filtered through a 0.45 m membrane. A 10 L aliquot of the supernatant was used to obtain the chemical profile by HPLC–Q-TOF-MS. 2.3. Preparation of urine samples Male Sprague–Dawley rats (200–240 g) were purchased from the Comparative Medicine Center of Yangzhou University (Jiangsu, China). The rats were housed in an environment with 25 ◦ C temperature, 45–55% humidity, and 12 h/12 h light–dark cycle. The animals had free access to food and water. All procedures used in the animal experiments complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. After 3 days of acclimatization, blank urine samples and urine samples after oral administration of YGS (9.1 g YGS per kilogram of body weight) [11] were collected by using metabolism cages. The urine was then extracted with ethyl acetate at a ratio of 1:2 (urine/ethyl acetate, v/v). The ethyl acetate extract was centrifuged at 16,000 rpm for 5 min. The supernatant was dried with a stream of N2 and then dissolved in methanol. A 10 L aliquot of the
23
methanol solution was analyzed by HPLC–Q-TOF-MS to screen for compounds that enter the systemic circulation. 2.4. Preparation of plasma samples Blood samples (500 L) were collected into heparinized 1.5 mL polyethylene centrifuge tubes before administration and at 0.08, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 20 h after administration. All of the samples were centrifuged immediately at 5000 rpm for 10 min at 4 ◦ C. Plasma from each sample was collected and frozen at −20 ◦ C until analysis. 2.5. Preparation of the calibration standards and quality control samples Standard stock solutions were prepared by separately dissolving 1.0 mg of senkyunolide I, ajmalicine, rhynchophylline, and isocorynoxeine in 10 mL of methanol to obtain a nominal concentration of 0.1 mg/mL. All of the stock solutions were kept at 4 ◦ C before use. A series of working solutions of the standard solutions were freshly prepared by diluting the standard stock solutions with methanol to appropriate concentrations. A methanolic solution of the IS (0.1 mg/mL) was prepared and then further diluted to 100 ng/mL to prepare a working solution. Calibration curves were constructed upon analysis of 200 L plasma samples spiked with 10 L of the appropriate working solution of each standard. Quality control (QC) samples were independently prepared. Each spiked sample contained all of the standards at three specified concentrations. To a 200 L aliquot of rat plasma, 10 L of IS solution and 500 L of ethyl acetate were added. After vortex-mixing the mixture for 3 min and centrifuging it at 16,000 rpm for 5 min, the supernatant was collected, dried with a stream of N2 , and then reconstituted in 100 L of methanol. Finally, 10 L of the methanol solution was analyzed by HPLC–ESI-QqQ-MS in the pharmacokinetics study. 2.6. Method validation In order to evaluate the method selectivity, blank rat plasma, plasma spiked with standard compounds, and urine samples were tested consecutively. The matrix effect was measured by comparing the peak responses of samples spiked post-extraction with those of standard solutions evaporated and then reconstituted in mobile phase. Analyses were performed at three QC concentration levels in six replicates. The extraction efficiency was determined by analysis of six replicates of plasma samples at three QC concentration levels. The extraction recovery was calculated by comparing the peak areas of plasma extracts spiked with analytes before extraction with those of samples spiked post-extraction. Calibration curves were obtained in duplicate on three consecutive days by plotting peak area ratio the analyte/IS versus plasma concentration using linear regression. Residual analysis, heteroskedasticity test, goodness-of-fit test, and significance test of regression coefficients were performed to monitor the accuracy of the linear calibration curves. The LLOQ was considered as the lowest concentration for which a response of at least five times that of the blank plasma could be obtained at an accuracy of ±20% and at a relative standard deviation of
