Research article Received: 17 October 2013,
Revised: 12 May 2014,
Accepted: 13 May 2014
Published online in Wiley Online Library: 24 June 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3274
Tissue distribution and excretion study of neopanaxadiol in rats by ultra-performance liquid chromatography quadrupole time-offlight mass spectrometry Cong Genga, Jian-yuan Yina, Xiu-hua Yua,b, Jing-yan Liua, Yu-xia Yanga, De-ya Suna, Qin Menga, Zhong-lin Weic and Ji-hua Liua* ABSTRACT: Neopanaxadiol (NPD), a major ginsenoside in Panax ginseng C. A. Meyer (Araliaceae), was reported to have neuroprotective effect. In this study, a method of ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/QTOF-MS) was developed and validated for quantitative analysis of NPD in tissues, urine and feces, using liquid–liquid extraction (LLE) to isolate NPD from different biological samples, and chromatographic separation was performed on an Agilent Zorbax Stable Bond C18 (2.1 × 50 mm, 1.8 μm) column with 0.1% formic acid in water and acetonitrile. All standard calibration curves were linear (all r2 > 0.995) within the test range. After oral administration, NPD was extensively distributed to most of the tissues without long-term accumulation. The higher levels were observed in stomach and intestine, followed by kidney and liver. Approximately 64.56 ± 20.32% of administered dose in feces and 0.0233 ± 0.0356% in urine were found within 96 h, which indicated that the major elimination route was fecal excretion. This analytical method was applied to the study of NPD distribution and excretion in rats after oral intake for the first time. The results we found here are helpful for us to understand the pharmacological effects of NPD, as well as its toxicity. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: UPLC-QTOF/MS; neopanaxadiol; ginsenoside; distribution; excretion
Introduction
Biomed. Chromatogr. 2015; 29: 333–340
* Correspondence to: J.-h. Liu, Department of Natural products Chemistry, College of Pharmacy, Jilin University, 1266 Fujin Road, Changchun 130021, People’s Republic of China. Email: [email protected] a
Department of Natural products Chemistry, College of Pharmacy, Jilin University, 1266 Fujin Road, Changchun 130021, People’s Republic of China
b
Chinese Medicine Research Center, The Affiliated Hospital To Changchun University of Chinese Medicine, 1478 Gongnong Road, Changchun 130021, People’s Republic of China
c
College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, People’s Republic of China Abbreviations used: NPD, neopanaxadiol; ESI, electrospray ionization; MRM, multiple reaction monitoring; PD, panaxadiol; PK, pharmacokinetic.
Copyright © 2014 John Wiley & Sons, Ltd.
333
Ginsenosides are major active ingredients of ginseng because of various therapeutic and pharmacological applications (Lee et al., 2012; Leung et al., 2011; Liu et al., 2012). Neopanaxadiol [dammar-(E)-20(22)-ene-3β, 12β, 25-triol, NPD, structure shown in Fig. 1] is a new panaxadiol component extracted from acid degradation of total ginsenosides and suggested to be a drug candidate against Alzheimer’s disease (Tao et al., 2009). With interest in potential beneficial role of NPD for healthcare, it is necessary to make clear the detailed pharmacokinetics and disposition in vivo; the reason for this is that pharmacokinetic studies are playing an increasingly important role in drug development processes to further support toxicity and clinical studies (Lan and Jia, 2010). Meanwhile, absorption, distribution, metabolism and excretion studies are components of drug discovery and development pivotal to understanding the optimal balance between the safety and efficacy of drugs (Xie et al., 2012). Earlier pharmacokinetic (PK) studies have focused on the development of bioanalytical assays for ginsenosides and their application to preliminary PK assessments (Li et al., 2005; Patel et al., 2012; Sun et al., 2005). The systemic exposure and disposition of ginsenosides remain to be unknown. Our previous study documenting the pharmacokinetic of NPD after a single dose has shown that NPD is rapidly absorbed and slowly eliminated, and the value of the corresponding area under the plasma
concentration–time curve (AUC) is very low (Geng et al., 2013). In order to explore the reason for this, the tissue distribution and excretion of NPD are required to be conducted in a satisfying manner. Recently, ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) has become a powerful tool in biomedical research, particularly for the analysis of biological samples because of its efficient separation, rapid analysis and high sensitivity (Fang et al., 2011; Goldwirt et al., 2012; Yin et al., 2012). Owing to its simple operation and high throughput, UPLC-MS
C. Geng et al.
Figure 1. Chemical structures of (A) neopanaxadiol (NPD) and (B) panaxadiol (PD; internal standard, IS).
is popular for the analysis of ginseng saponins, involved in metabolomic fingerprinting (Dan et al., 2008; Xie et al., 2008a, 2008b), quality control (Guan et al., 2007) and biological samples assay (Deng et al., 2009; Wang et al., 2007). Quadrupole time-offlight (QTOF) instruments also have several advantages for quantitative analysis of NPD in biological samples. One advantage is that the ion chromatograms can be extracted using a narrow mass window, which increases the probability that the chromatographic peaks of NPD are free from background interferences. Another is that the time of method development with QTOF instruments may be shorter than with triple quadrupole instruments because precursor-to-product ion transitions do not have to be determined and optimized (Williamson and Bartlett, 2007). Accordingly, a rapid and specifical UPLC-QTOF/ MS method was developed to explore tissue distribution and excretion patterns of NPD after oral administration in rats in the present study.
Experimental Chemicals and reagents Neopanaxadiol was isolated from total ginsenosides acid degradated composition in our laboratory. Its molecular structure was elucidated 1 13 based on spectroscopic data (IR, MS, H NMR, C NMR and X-ray; Tao et al., 2009) and its purity was >99%. Panaxadiol (PD; purity >99%, internal standard, IS) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile (Chromasolv) was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid (Chromasolv) was purchased from SigmaAldrich (St Louis, MO, USA). Deionized water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA). All other reagents were analytical grade.
operated at 30°C. The mobile phase consisted of 0.1% formic acid water (A) and acetonitrile (B), formed by a gradient elution of 70–70% (v/v) B at 0–0.5 min, 70–90% B at 0.5–2 min, and 95–95% B at 2–5 min. The flow rate was 0.4 mL/min. The mass spectrometer was operated in the positive ionization mode using multiple reaction monitoring mode (MRM) to monitor the mass transitions. The precursor-to-product ion pairs of NPD and IS (PD) were all m/z 461.4 → 425.4. Optimized tandem MS parameters of capillary and endplate voltage were 4.5 kV and 500 V, respectively. Nitrogen was used as the dry gas and nebulizer gas at a rate of 6.0 L/min and pressure 0.8 bar. The dry gas temperature was set at 200°C. Argon was used as the collision gas and collision energy was set to 8 eV for all of the transitions. Data were acquired and analyzed with Bruker DataAnalysis (version 4.0 SP 1) and QuantAnalysis software (version 2.0 SP 1).
Preparation of stock and working solutions For preparation the calibration standards, the primary stock solutions of NPD and IS were prepared individually in methanol, and then serially diluted to achieve different concentrations in tissue homogenates (50, 100, 200, 500, 1000, 4000 and 8000 ng/mL), urine (80, 160, 320, 480, 640, 960 and 1280 ng/mL) and fecal homogenate (100, 200, 500, 1000, 2000, 4000 and 8000 ng/mL). Three levels of quality control (QC) samples were prepared in the same manner (100, 500 and 4000 ng/mL in tissue homogenates; 160, 480 and 960 ng/mL in urine; and 200, 1000 and 4000 ng/mL in fecal homogenate). All solutions were stored at 4°C and brought to room temperature before processing.
Sample preparation Extraction of NPD from tissue samples. The tissues were ground with 0.9% NaCl injectable solution (1:3, w/v), then the homogenates were centrifuged at 10,000 rpm for 10 min at 4°C, and each supernatant was collected and frozen at 20°C until analysis. When determining NPD level in the tissues, IS solution (100 μL, 150 ng/mL) and 4 mL of extraction solvent (Et2O:CH2Cl2, 3:2, v/v) were added to an Eppendorf tube containing 500 μL biological supernatant, vortexed for 15 min and centrifuged for 10 min at 10,000 rpm, then the organic layer was collected and evaporated to dryness at 40°C under a stream of nitrogen. The residues were resolved in 150 μL mobile phase and transferred to an autosampler vial until the UPLC-QTOF/MS assay. Extraction of NPD from urine and feces samples. A 100 μL aliquot of IS solution (90 ng/mL) and 4–6 mL extraction solvent (Et2O:CH2Cl2 = 3:2, v/v) were added in an Eppendorf tube containing 1000 μL urine or 200 μL fecal homogenate, which were ground with methanol (1:2, w/v) to homogenate. The samples were then processed using the same procedures as for tissue samples.
Animals Sprague–Dawley rats weighing 200–220 g were obtained from the Laboratory Animal Center of Jilin University (Changchun, China). The animals were housed under a controlled temperature of 25 ± 1°C, relative humidity 40–70% and a 12 h light/dark cycle, and fed with food and water ad libitum. All the procedures were in strict accordance with Chinese legislation on the use and care of laboratory animals and the guidelines established by the Institutional Animal Ethics Committee and Committee for the Purpose of Control and Supervision of Experiments on Animals in China.
Instrumentation and chromatographic conditions
334
The UPLC-MS/MS system consisted of an Agilent 1290 system (Aglient Technologies, USA) and a Bruker micrOTOF QII (Bruker Daltonics, USA) mass spectrometer equipped with an electrospray ionization (ESI) source. Chromatographic separation was achieved on a Zorbax Stable Bond C18 analytical column (2.1 × 50 mm, 1.8 μm; Aglient Technologies, USA)
wileyonlinelibrary.com/journal/bmc
Method validation The method was validated according to the US Food and Drug Administration (2001). Specificity was tested inspecting drug-free rat urine, fecal and tissue homogenates in each validation run for interfering peaks. Calibration curves were determined by plotting the peak area ratios of the NPD to the IS, vs the corresponding concentrations of injected standards. The lower limit of quantitation (LLOQ) was the concentration giving the analyte peak (response) identifiable and reproducible with a precision of 20% and accuracy of 80–120%, and analyte response at the LLOQ should be at least five times the blank response. Intra- and inter-day precision and accuracy were measured from six replicates of QC samples analysed on the same and three different days. At each concentration, acceptable precision and accuracy were defined by a relative standard deviation (RSD) 0.997. This method also provided an LLOQ around 50 ng/mL in tissue homogenates, 80 ng/mL in urine, and 100 ng/mL in fecal homogenate, and the detailed data are listed in the Supporting Information, Table S2. The overall reproducibility of the method was defined by examing both intra- and inter-day accuracy and precision. The results are summarized in Table 1. The intra- and inter-day relative errors varied from 7.02 to 6.41% and from 1.61 to 7.06%, respectively, while precision values were constantly better than
12.78 and 13.26%, respectively. These informations indicated a creditable performance of the quantitative analysis of NPD. Influence of dilution was validated by diluting fecal homogenate at 1/40, 1/160 and 1/320. The results indicated that the precision (RSD) was
Revised: 12 May 2014,
Accepted: 13 May 2014
Published online in Wiley Online Library: 24 June 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3274
Tissue distribution and excretion study of neopanaxadiol in rats by ultra-performance liquid chromatography quadrupole time-offlight mass spectrometry Cong Genga, Jian-yuan Yina, Xiu-hua Yua,b, Jing-yan Liua, Yu-xia Yanga, De-ya Suna, Qin Menga, Zhong-lin Weic and Ji-hua Liua* ABSTRACT: Neopanaxadiol (NPD), a major ginsenoside in Panax ginseng C. A. Meyer (Araliaceae), was reported to have neuroprotective effect. In this study, a method of ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/QTOF-MS) was developed and validated for quantitative analysis of NPD in tissues, urine and feces, using liquid–liquid extraction (LLE) to isolate NPD from different biological samples, and chromatographic separation was performed on an Agilent Zorbax Stable Bond C18 (2.1 × 50 mm, 1.8 μm) column with 0.1% formic acid in water and acetonitrile. All standard calibration curves were linear (all r2 > 0.995) within the test range. After oral administration, NPD was extensively distributed to most of the tissues without long-term accumulation. The higher levels were observed in stomach and intestine, followed by kidney and liver. Approximately 64.56 ± 20.32% of administered dose in feces and 0.0233 ± 0.0356% in urine were found within 96 h, which indicated that the major elimination route was fecal excretion. This analytical method was applied to the study of NPD distribution and excretion in rats after oral intake for the first time. The results we found here are helpful for us to understand the pharmacological effects of NPD, as well as its toxicity. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: UPLC-QTOF/MS; neopanaxadiol; ginsenoside; distribution; excretion
Introduction
Biomed. Chromatogr. 2015; 29: 333–340
* Correspondence to: J.-h. Liu, Department of Natural products Chemistry, College of Pharmacy, Jilin University, 1266 Fujin Road, Changchun 130021, People’s Republic of China. Email: [email protected] a
Department of Natural products Chemistry, College of Pharmacy, Jilin University, 1266 Fujin Road, Changchun 130021, People’s Republic of China
b
Chinese Medicine Research Center, The Affiliated Hospital To Changchun University of Chinese Medicine, 1478 Gongnong Road, Changchun 130021, People’s Republic of China
c
College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, People’s Republic of China Abbreviations used: NPD, neopanaxadiol; ESI, electrospray ionization; MRM, multiple reaction monitoring; PD, panaxadiol; PK, pharmacokinetic.
Copyright © 2014 John Wiley & Sons, Ltd.
333
Ginsenosides are major active ingredients of ginseng because of various therapeutic and pharmacological applications (Lee et al., 2012; Leung et al., 2011; Liu et al., 2012). Neopanaxadiol [dammar-(E)-20(22)-ene-3β, 12β, 25-triol, NPD, structure shown in Fig. 1] is a new panaxadiol component extracted from acid degradation of total ginsenosides and suggested to be a drug candidate against Alzheimer’s disease (Tao et al., 2009). With interest in potential beneficial role of NPD for healthcare, it is necessary to make clear the detailed pharmacokinetics and disposition in vivo; the reason for this is that pharmacokinetic studies are playing an increasingly important role in drug development processes to further support toxicity and clinical studies (Lan and Jia, 2010). Meanwhile, absorption, distribution, metabolism and excretion studies are components of drug discovery and development pivotal to understanding the optimal balance between the safety and efficacy of drugs (Xie et al., 2012). Earlier pharmacokinetic (PK) studies have focused on the development of bioanalytical assays for ginsenosides and their application to preliminary PK assessments (Li et al., 2005; Patel et al., 2012; Sun et al., 2005). The systemic exposure and disposition of ginsenosides remain to be unknown. Our previous study documenting the pharmacokinetic of NPD after a single dose has shown that NPD is rapidly absorbed and slowly eliminated, and the value of the corresponding area under the plasma
concentration–time curve (AUC) is very low (Geng et al., 2013). In order to explore the reason for this, the tissue distribution and excretion of NPD are required to be conducted in a satisfying manner. Recently, ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) has become a powerful tool in biomedical research, particularly for the analysis of biological samples because of its efficient separation, rapid analysis and high sensitivity (Fang et al., 2011; Goldwirt et al., 2012; Yin et al., 2012). Owing to its simple operation and high throughput, UPLC-MS
C. Geng et al.
Figure 1. Chemical structures of (A) neopanaxadiol (NPD) and (B) panaxadiol (PD; internal standard, IS).
is popular for the analysis of ginseng saponins, involved in metabolomic fingerprinting (Dan et al., 2008; Xie et al., 2008a, 2008b), quality control (Guan et al., 2007) and biological samples assay (Deng et al., 2009; Wang et al., 2007). Quadrupole time-offlight (QTOF) instruments also have several advantages for quantitative analysis of NPD in biological samples. One advantage is that the ion chromatograms can be extracted using a narrow mass window, which increases the probability that the chromatographic peaks of NPD are free from background interferences. Another is that the time of method development with QTOF instruments may be shorter than with triple quadrupole instruments because precursor-to-product ion transitions do not have to be determined and optimized (Williamson and Bartlett, 2007). Accordingly, a rapid and specifical UPLC-QTOF/ MS method was developed to explore tissue distribution and excretion patterns of NPD after oral administration in rats in the present study.
Experimental Chemicals and reagents Neopanaxadiol was isolated from total ginsenosides acid degradated composition in our laboratory. Its molecular structure was elucidated 1 13 based on spectroscopic data (IR, MS, H NMR, C NMR and X-ray; Tao et al., 2009) and its purity was >99%. Panaxadiol (PD; purity >99%, internal standard, IS) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile (Chromasolv) was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid (Chromasolv) was purchased from SigmaAldrich (St Louis, MO, USA). Deionized water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA). All other reagents were analytical grade.
operated at 30°C. The mobile phase consisted of 0.1% formic acid water (A) and acetonitrile (B), formed by a gradient elution of 70–70% (v/v) B at 0–0.5 min, 70–90% B at 0.5–2 min, and 95–95% B at 2–5 min. The flow rate was 0.4 mL/min. The mass spectrometer was operated in the positive ionization mode using multiple reaction monitoring mode (MRM) to monitor the mass transitions. The precursor-to-product ion pairs of NPD and IS (PD) were all m/z 461.4 → 425.4. Optimized tandem MS parameters of capillary and endplate voltage were 4.5 kV and 500 V, respectively. Nitrogen was used as the dry gas and nebulizer gas at a rate of 6.0 L/min and pressure 0.8 bar. The dry gas temperature was set at 200°C. Argon was used as the collision gas and collision energy was set to 8 eV for all of the transitions. Data were acquired and analyzed with Bruker DataAnalysis (version 4.0 SP 1) and QuantAnalysis software (version 2.0 SP 1).
Preparation of stock and working solutions For preparation the calibration standards, the primary stock solutions of NPD and IS were prepared individually in methanol, and then serially diluted to achieve different concentrations in tissue homogenates (50, 100, 200, 500, 1000, 4000 and 8000 ng/mL), urine (80, 160, 320, 480, 640, 960 and 1280 ng/mL) and fecal homogenate (100, 200, 500, 1000, 2000, 4000 and 8000 ng/mL). Three levels of quality control (QC) samples were prepared in the same manner (100, 500 and 4000 ng/mL in tissue homogenates; 160, 480 and 960 ng/mL in urine; and 200, 1000 and 4000 ng/mL in fecal homogenate). All solutions were stored at 4°C and brought to room temperature before processing.
Sample preparation Extraction of NPD from tissue samples. The tissues were ground with 0.9% NaCl injectable solution (1:3, w/v), then the homogenates were centrifuged at 10,000 rpm for 10 min at 4°C, and each supernatant was collected and frozen at 20°C until analysis. When determining NPD level in the tissues, IS solution (100 μL, 150 ng/mL) and 4 mL of extraction solvent (Et2O:CH2Cl2, 3:2, v/v) were added to an Eppendorf tube containing 500 μL biological supernatant, vortexed for 15 min and centrifuged for 10 min at 10,000 rpm, then the organic layer was collected and evaporated to dryness at 40°C under a stream of nitrogen. The residues were resolved in 150 μL mobile phase and transferred to an autosampler vial until the UPLC-QTOF/MS assay. Extraction of NPD from urine and feces samples. A 100 μL aliquot of IS solution (90 ng/mL) and 4–6 mL extraction solvent (Et2O:CH2Cl2 = 3:2, v/v) were added in an Eppendorf tube containing 1000 μL urine or 200 μL fecal homogenate, which were ground with methanol (1:2, w/v) to homogenate. The samples were then processed using the same procedures as for tissue samples.
Animals Sprague–Dawley rats weighing 200–220 g were obtained from the Laboratory Animal Center of Jilin University (Changchun, China). The animals were housed under a controlled temperature of 25 ± 1°C, relative humidity 40–70% and a 12 h light/dark cycle, and fed with food and water ad libitum. All the procedures were in strict accordance with Chinese legislation on the use and care of laboratory animals and the guidelines established by the Institutional Animal Ethics Committee and Committee for the Purpose of Control and Supervision of Experiments on Animals in China.
Instrumentation and chromatographic conditions
334
The UPLC-MS/MS system consisted of an Agilent 1290 system (Aglient Technologies, USA) and a Bruker micrOTOF QII (Bruker Daltonics, USA) mass spectrometer equipped with an electrospray ionization (ESI) source. Chromatographic separation was achieved on a Zorbax Stable Bond C18 analytical column (2.1 × 50 mm, 1.8 μm; Aglient Technologies, USA)
wileyonlinelibrary.com/journal/bmc
Method validation The method was validated according to the US Food and Drug Administration (2001). Specificity was tested inspecting drug-free rat urine, fecal and tissue homogenates in each validation run for interfering peaks. Calibration curves were determined by plotting the peak area ratios of the NPD to the IS, vs the corresponding concentrations of injected standards. The lower limit of quantitation (LLOQ) was the concentration giving the analyte peak (response) identifiable and reproducible with a precision of 20% and accuracy of 80–120%, and analyte response at the LLOQ should be at least five times the blank response. Intra- and inter-day precision and accuracy were measured from six replicates of QC samples analysed on the same and three different days. At each concentration, acceptable precision and accuracy were defined by a relative standard deviation (RSD) 0.997. This method also provided an LLOQ around 50 ng/mL in tissue homogenates, 80 ng/mL in urine, and 100 ng/mL in fecal homogenate, and the detailed data are listed in the Supporting Information, Table S2. The overall reproducibility of the method was defined by examing both intra- and inter-day accuracy and precision. The results are summarized in Table 1. The intra- and inter-day relative errors varied from 7.02 to 6.41% and from 1.61 to 7.06%, respectively, while precision values were constantly better than
12.78 and 13.26%, respectively. These informations indicated a creditable performance of the quantitative analysis of NPD. Influence of dilution was validated by diluting fecal homogenate at 1/40, 1/160 and 1/320. The results indicated that the precision (RSD) was
