Research article Received: 17 October 2014,
Revised: 8 April 2015,
Accepted: 16 April 2015
Published online in Wiley Online Library: 1 June 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3497
Simultaneous determination of five triterpene acids in rat plasma by liquid chromatography– mass spectrometry and its application in pharmacokinetic study after oral administration of Folium Eriobotryae effective fraction Zhen-Hua Lia,b†, He Zhub†, Xue-Ping Caia,b, Dan-Dan Hea, Jun-Lei Huaa,b, Jian-Ming Jub*, Han Lvc, Li Mac and Wei-Lin Lic ABSTRACT: Folium Eriobotryae effective fraction (FEA), the extract of Folium Eriobotryae, had been used as anti-hyperglycemia and anti-hyperlipemia medicine in China. A previous study indicated that euscaphic acid, maslinic acid, corosolic acid, oleanolic acid and ursolic acid, the five structurally similar triterpene acids (containing two groups of structural isomers), are the major components of FEA. In the present study, we developed a specific and reliable LC-MS method for simultaneous determination of the five triterpene acids in rat plasma, and further investigated their pharmacokinetic properties after oral administration of FEA. Following a simple sample preparation, chromatographic separation was achieved on a C18 column with a mobile phase composed of methanol–0.1% ammonium acetate (80:20, v/v). Quantification was achieved by monitoring the selected ions at m/z 487.6 for euscaphic acid, m/z 471.5 for maslinic acid and corosolic acid, m/z 455.5 for oleanolic acid and ursolic acid and m/z 469.5 for internal standard. The method was validated to be specific, accurate and precise over the concentration ranges of 10–3000 ng/mL with limits of detections of 5 ng/mL for the five triterpene acids. Finally, the method was successfully applied to the pharmacokinetic study of the five structurally similar triterpene acids in rats after oral administration of FEA. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Folium Eriobotryae; triterpene acids; pharmacokinetic; quantitative analysis; liquid chromatography–mass spectrometry
Introduction
Biomed. Chromatogr. 2015; 29: 1791–1797
* Correspondence to: J.-M. Ju, Department of Pharmaceutical Analysis, Metabolomics, Jiangsu Province Academy of Traditional Chinese Medicine, Jiangsu Branch of China Academy of Chinese Medical Sciences, Nanjing 210028, People’s Republic of China. Email: [email protected] †
†Zhen-Hua Li and He Zhu contributed equally to this work.
a
Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing 210046, People’s Republic of China
b
Jiangsu Province Academy of Traditional Chinese Medicine and Jiangsu Branch of China Academy of Chinese Medical Sciences, Nanjing 210028, People’s Republic of China
c
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China Abbreviations used: ESI, electrospray ionization; FEA, Folium Eriobotryae effective fraction; FDA, US Food and Drug Administration; SIR, selected ion recording.
Copyright © 2015 John Wiley & Sons, Ltd.
1791
Folium Eriobotryae, the dried leaves of Eriobotrya japonica (Thunb.) Lindl., has been commonly used for clearing lung-heat, eliminating phlegm, relieving cough and regulating the stomach to restrain vomiting in China for many years (Committee of the Pharmacopoeia of P. R. China, 2010). From the folk usage of Traditional Chinese herbs, Folium Eriobotryae effective fraction (FEA), the extract of Folium Eriobotryae, has been used to treat diabetes in Jiangsu province, the eastern part of China (Li et al., 2004; Lü et al., 2009). Our previous study found that the main chemical components of FEA were five triterpene acids, which were euscaphic acid, maslinic acid, corosolic acid, oleanolic acid and ursolic acid (Lü et al., 2011a; Cai et al., 2013a). In addition, the pharmacological effects of FEA were further verified and the results proved that it possessed hypoglycemic and hypolipidemic effects for mice with a dose-dependent response ( Ju et al., 2003; Li et al., 2007; Chen et al., 2008). In order to understand the pharmacological effects and further enhance the clinical usage of FEA, pharmacokinetic study of the FEA is needed. However, at present, the information on the pharmacokinetic study of the FEA is very limited. The reason for this might be that the simultaneous determination method in plasma
is very difficult to validate, since the structures of the five detected triterpene acids are similar. In particular, two groups of structural isomers were existed in the five analytes, which were oleanolic
Z.-H. Li et al. acid and ursolic acid, and maslinic acid and corosolic acid, respectively. Therefore, to study the pharmacokinetic profiles of the FEA, it is necessary to develop and validate a highly sensitive and selective method for determination of the structurally similar triterpene acids in biological samples. In this study, we present a simple LC-MS method in selected ion recording (SIR) mode for the simultaneous determination of structurally similar triterpene acids, which were euscaphic acid, maslinic acid, corosolic acid, oleanolic acid and ursolic acid, in rat plasma using glycyrrhetic acid as the internal standard. This method was successfully used to evaluate the pharmacokinetic profiles of the five analytes in rats after oral administration of FEA at a dose of 0.1 g/kg.
as m/z 471.5 for maslinic acid and corosolic acid, m/z 487.6 for euscaphic acid, m/z 455.5 for oleanolic acid and ursolic acid, and m/z 469.5 for IS, respectively.
Preparation of standard solutions The standard stock solutions of euscaphic acid, maslinic acid, corosolic acid, oleanolic acid, ursolic acid and IS were prepared in methanol at the concentrations of 0.109, 0.123, 0.108, 0.099, 0.103 and 0.106 mg/mL, respectively. The working standards were prepared by dilution of the stock solutions in methanol to obtain the desired concentrations. All of the standard solutions were kept at 20°C away from light for further use.
Sample pretreatment
Experimental Chemicals and reagents Reference standards of maslinic acid, corosolic acid, euscaphic acid and tormentic acid were provided by the Institute of Botany, Jiangsu Province, China and Chinese Academy of Sciences (Nanjing, China). Oleanolic acid and ursolic acid standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Glycyrrhetic acid (internal standard, IS) was purchased from Nanjing Zelang pharmaceutical Co. Ltd (Nanjing, China). The purity of all reference standards was >98%. Ammonium acetate (analytical grade) was purchased from Nanjing Chemical Reagents Co. Ltd (Nanjing, China). Folium Eriobotryae total triterpene acid effective fraction [FEA; the contents of the five triterpene acids in FEA were measured quantitatively by a high-performance liquid chromatography method, which was published by our team (Cai et al., 2013b); the results revealed that the percentage of euscaphic acid in FEA was 13.71%, that of maslinic acid was 13.69%, that of corosolic acid was 18.26%, that of oleanolic acid was 6.02% and that of ursolic acid was 18.95%] was provided by the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing, China). Methanol (HPLC grade) was purchased from Merck KGaA (Darmstadt, Germany), and all other reagents were of analytical grade and obtained from commercial sources. Deionized water was produced using Milli-Q Reagent Water System (Millipore China Co., USA).
Instrument The liquid chromatography was performed on a Waters 2695-Micromass Quattro micro (Waters, Milford, MA, USA). The signal acquisition and peak integration were performed using the Masslynx 4.0 software and Quanlynx V4.1 (Micromass, Manchester, UK). All centrifugations were performed using a Microfuge 16 (Beckman Coulter, USA).
Chromatographic conditions Chromatographic separations were performed on a reverse-phase C18 column (150 × 4.6 mm, 3 μm, Alltima) equipped with an Alltima guard column, eluting with an isocratic mobile phase system, which consisted of methanol and 0.1% ammonium acetate (80:20, v:v). The flow rate was at 1.0 mL/min. Twenty percent of eluate was introduced to an electrospray ionization (ESI) mass spectrometer. The sample injection volume was 20 μL and the column temperature was maintained at 35 °C.
MS conditions
1792
Analytes were detected by MS with ESI interface in negative SIR mode. The operational parameters of ESI-MS were as follows: nitrogen was used as the desolvation and cone gas with flow rates of 500 and 50 L/h; source and desolvation gas temperatures were set at 120 and 400°C; capillary voltage was set as 3.5 kV and the cone voltage was 30 V. The MS ions were recorded
wileyonlinelibrary.com/journal/bmc
The protein precipitation procedure was applied in the sample pretreatment. All of the frozen standards and plasma samples were allowed to thaw at room temperature. After 10 μL IS working solution (1.0 μg/mL) was added, 100 μL plasma sample was treated with 300 μL methanol. The sample mixture was vortex-mixed for 3 min and centrifuged at 10,625g for 10 min. The supernatant was transferred into a vial and 20 μL was injected for LC-MS analysis.
Assay validation A thorough and complete method validation for assaying the five triterpene acids in biological matrices was performed under the guidelines of the US Food and Drug Administration (2001). The method was validated for selectivity, accuracy, linearity and precision, recovery, matrix effect and stability.
Selectivity The selectivity was assessed by comparing the chromatograms of six different sources of blank rat plasma with the corresponding spiked plasma. Each blank plasma was tested using the proposed extraction procedure and LC-MS conditions to ensure no interference with the five triterpene acids and the IS from plasma.
Linearity of calibration curves, LLOQ and LOD Calibration standard samples were prepared by spiking appropriate volume of mixing working standard solutions at different concentrations in blank plasma (100 μL) to yield final concentrations ranging from 10.65 to 3195 ng/mL for euscaphic acid; from 10.80 to 3240 ng/mL for corosolic acid; from 9.996 to 2970 ng/mL for oleanolic acid; from 12.31 to 3690 ng/mL for maslinic acid; and from 10.33 to 3090 ng/mL for ursolic acids. The calibration curves were constructed by linear regression of the peak area ratio of the five triterpene acids to the is (y-axis) and the final concen2 trations (x-axis) with a 1/x weighting factor, described as y = a + bx. Concentrations of quality controls (QCs) and samples were calculated using the regression equation of the calibration curve. The lower limit of quantitation (LLOQ) was determined as the lowest concentration of each calibration curve (signal-to-noise ratio >10). The limit of detection (LOD) was based on a signal-to-noise ratio (S/N>6), and the results of LLOQ and LOD are listed in Table 1.
The intra-, inter-day precisions and accuracies The intra-day and inter-day precisions and accuracies of the method were evaluated by analyzing six replicates of QC samples at low, medium and high concentrations on the same day and five consecutive days. The standard deviation (SD) was calculated using one-way-ANOVA and the relative standard deviation (RSD) was calculated by the ratio of SD and average; the intra- and inter-day precisions are required to be 10 was taken as the LLOQ and was determined to be 5 ng/mL, which was sufficient to perform pharmacokinetic studies of the triterpene acids in rats. The LLOQs for
1794
Figure 1. The mass spectra of euscaphic acid (A), maslinic acid (B), corosolic acid (C), oleanolic acid (D), ursolic acid (E) and IS (F).
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1791–1797
Pharmacokinetic study of Folium Eriobotryae effective fraction in rats
Figure 2. (A) Selected ion recording (SIR) chromatogram of blank plasma. (B) SIR chromatogram of sample plasma (I, euscaphic acid; II, maslinic acid; III, corosolic acid; IV, oleanolic acid; V, ursolic acid). (C) SIR chromatogram of standard-spiked plasma sample.
euscaphic acid, maslinic acid, corosolic acid, oleanolic acid and ursolic acid were 10.65, 12.31, 10.80, 9.996 and 10.33 ng/mL, respectively, indicating that the method is sensitive for quantitative evaluation of the five compounds. The intra- and inter-day precisions and accuracies. The results showed that, for all five triterpene acids, the intra- and inter-day precision values (RSD %) were both
Revised: 8 April 2015,
Accepted: 16 April 2015
Published online in Wiley Online Library: 1 June 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3497
Simultaneous determination of five triterpene acids in rat plasma by liquid chromatography– mass spectrometry and its application in pharmacokinetic study after oral administration of Folium Eriobotryae effective fraction Zhen-Hua Lia,b†, He Zhub†, Xue-Ping Caia,b, Dan-Dan Hea, Jun-Lei Huaa,b, Jian-Ming Jub*, Han Lvc, Li Mac and Wei-Lin Lic ABSTRACT: Folium Eriobotryae effective fraction (FEA), the extract of Folium Eriobotryae, had been used as anti-hyperglycemia and anti-hyperlipemia medicine in China. A previous study indicated that euscaphic acid, maslinic acid, corosolic acid, oleanolic acid and ursolic acid, the five structurally similar triterpene acids (containing two groups of structural isomers), are the major components of FEA. In the present study, we developed a specific and reliable LC-MS method for simultaneous determination of the five triterpene acids in rat plasma, and further investigated their pharmacokinetic properties after oral administration of FEA. Following a simple sample preparation, chromatographic separation was achieved on a C18 column with a mobile phase composed of methanol–0.1% ammonium acetate (80:20, v/v). Quantification was achieved by monitoring the selected ions at m/z 487.6 for euscaphic acid, m/z 471.5 for maslinic acid and corosolic acid, m/z 455.5 for oleanolic acid and ursolic acid and m/z 469.5 for internal standard. The method was validated to be specific, accurate and precise over the concentration ranges of 10–3000 ng/mL with limits of detections of 5 ng/mL for the five triterpene acids. Finally, the method was successfully applied to the pharmacokinetic study of the five structurally similar triterpene acids in rats after oral administration of FEA. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Folium Eriobotryae; triterpene acids; pharmacokinetic; quantitative analysis; liquid chromatography–mass spectrometry
Introduction
Biomed. Chromatogr. 2015; 29: 1791–1797
* Correspondence to: J.-M. Ju, Department of Pharmaceutical Analysis, Metabolomics, Jiangsu Province Academy of Traditional Chinese Medicine, Jiangsu Branch of China Academy of Chinese Medical Sciences, Nanjing 210028, People’s Republic of China. Email: [email protected] †
†Zhen-Hua Li and He Zhu contributed equally to this work.
a
Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing 210046, People’s Republic of China
b
Jiangsu Province Academy of Traditional Chinese Medicine and Jiangsu Branch of China Academy of Chinese Medical Sciences, Nanjing 210028, People’s Republic of China
c
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China Abbreviations used: ESI, electrospray ionization; FEA, Folium Eriobotryae effective fraction; FDA, US Food and Drug Administration; SIR, selected ion recording.
Copyright © 2015 John Wiley & Sons, Ltd.
1791
Folium Eriobotryae, the dried leaves of Eriobotrya japonica (Thunb.) Lindl., has been commonly used for clearing lung-heat, eliminating phlegm, relieving cough and regulating the stomach to restrain vomiting in China for many years (Committee of the Pharmacopoeia of P. R. China, 2010). From the folk usage of Traditional Chinese herbs, Folium Eriobotryae effective fraction (FEA), the extract of Folium Eriobotryae, has been used to treat diabetes in Jiangsu province, the eastern part of China (Li et al., 2004; Lü et al., 2009). Our previous study found that the main chemical components of FEA were five triterpene acids, which were euscaphic acid, maslinic acid, corosolic acid, oleanolic acid and ursolic acid (Lü et al., 2011a; Cai et al., 2013a). In addition, the pharmacological effects of FEA were further verified and the results proved that it possessed hypoglycemic and hypolipidemic effects for mice with a dose-dependent response ( Ju et al., 2003; Li et al., 2007; Chen et al., 2008). In order to understand the pharmacological effects and further enhance the clinical usage of FEA, pharmacokinetic study of the FEA is needed. However, at present, the information on the pharmacokinetic study of the FEA is very limited. The reason for this might be that the simultaneous determination method in plasma
is very difficult to validate, since the structures of the five detected triterpene acids are similar. In particular, two groups of structural isomers were existed in the five analytes, which were oleanolic
Z.-H. Li et al. acid and ursolic acid, and maslinic acid and corosolic acid, respectively. Therefore, to study the pharmacokinetic profiles of the FEA, it is necessary to develop and validate a highly sensitive and selective method for determination of the structurally similar triterpene acids in biological samples. In this study, we present a simple LC-MS method in selected ion recording (SIR) mode for the simultaneous determination of structurally similar triterpene acids, which were euscaphic acid, maslinic acid, corosolic acid, oleanolic acid and ursolic acid, in rat plasma using glycyrrhetic acid as the internal standard. This method was successfully used to evaluate the pharmacokinetic profiles of the five analytes in rats after oral administration of FEA at a dose of 0.1 g/kg.
as m/z 471.5 for maslinic acid and corosolic acid, m/z 487.6 for euscaphic acid, m/z 455.5 for oleanolic acid and ursolic acid, and m/z 469.5 for IS, respectively.
Preparation of standard solutions The standard stock solutions of euscaphic acid, maslinic acid, corosolic acid, oleanolic acid, ursolic acid and IS were prepared in methanol at the concentrations of 0.109, 0.123, 0.108, 0.099, 0.103 and 0.106 mg/mL, respectively. The working standards were prepared by dilution of the stock solutions in methanol to obtain the desired concentrations. All of the standard solutions were kept at 20°C away from light for further use.
Sample pretreatment
Experimental Chemicals and reagents Reference standards of maslinic acid, corosolic acid, euscaphic acid and tormentic acid were provided by the Institute of Botany, Jiangsu Province, China and Chinese Academy of Sciences (Nanjing, China). Oleanolic acid and ursolic acid standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Glycyrrhetic acid (internal standard, IS) was purchased from Nanjing Zelang pharmaceutical Co. Ltd (Nanjing, China). The purity of all reference standards was >98%. Ammonium acetate (analytical grade) was purchased from Nanjing Chemical Reagents Co. Ltd (Nanjing, China). Folium Eriobotryae total triterpene acid effective fraction [FEA; the contents of the five triterpene acids in FEA were measured quantitatively by a high-performance liquid chromatography method, which was published by our team (Cai et al., 2013b); the results revealed that the percentage of euscaphic acid in FEA was 13.71%, that of maslinic acid was 13.69%, that of corosolic acid was 18.26%, that of oleanolic acid was 6.02% and that of ursolic acid was 18.95%] was provided by the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing, China). Methanol (HPLC grade) was purchased from Merck KGaA (Darmstadt, Germany), and all other reagents were of analytical grade and obtained from commercial sources. Deionized water was produced using Milli-Q Reagent Water System (Millipore China Co., USA).
Instrument The liquid chromatography was performed on a Waters 2695-Micromass Quattro micro (Waters, Milford, MA, USA). The signal acquisition and peak integration were performed using the Masslynx 4.0 software and Quanlynx V4.1 (Micromass, Manchester, UK). All centrifugations were performed using a Microfuge 16 (Beckman Coulter, USA).
Chromatographic conditions Chromatographic separations were performed on a reverse-phase C18 column (150 × 4.6 mm, 3 μm, Alltima) equipped with an Alltima guard column, eluting with an isocratic mobile phase system, which consisted of methanol and 0.1% ammonium acetate (80:20, v:v). The flow rate was at 1.0 mL/min. Twenty percent of eluate was introduced to an electrospray ionization (ESI) mass spectrometer. The sample injection volume was 20 μL and the column temperature was maintained at 35 °C.
MS conditions
1792
Analytes were detected by MS with ESI interface in negative SIR mode. The operational parameters of ESI-MS were as follows: nitrogen was used as the desolvation and cone gas with flow rates of 500 and 50 L/h; source and desolvation gas temperatures were set at 120 and 400°C; capillary voltage was set as 3.5 kV and the cone voltage was 30 V. The MS ions were recorded
wileyonlinelibrary.com/journal/bmc
The protein precipitation procedure was applied in the sample pretreatment. All of the frozen standards and plasma samples were allowed to thaw at room temperature. After 10 μL IS working solution (1.0 μg/mL) was added, 100 μL plasma sample was treated with 300 μL methanol. The sample mixture was vortex-mixed for 3 min and centrifuged at 10,625g for 10 min. The supernatant was transferred into a vial and 20 μL was injected for LC-MS analysis.
Assay validation A thorough and complete method validation for assaying the five triterpene acids in biological matrices was performed under the guidelines of the US Food and Drug Administration (2001). The method was validated for selectivity, accuracy, linearity and precision, recovery, matrix effect and stability.
Selectivity The selectivity was assessed by comparing the chromatograms of six different sources of blank rat plasma with the corresponding spiked plasma. Each blank plasma was tested using the proposed extraction procedure and LC-MS conditions to ensure no interference with the five triterpene acids and the IS from plasma.
Linearity of calibration curves, LLOQ and LOD Calibration standard samples were prepared by spiking appropriate volume of mixing working standard solutions at different concentrations in blank plasma (100 μL) to yield final concentrations ranging from 10.65 to 3195 ng/mL for euscaphic acid; from 10.80 to 3240 ng/mL for corosolic acid; from 9.996 to 2970 ng/mL for oleanolic acid; from 12.31 to 3690 ng/mL for maslinic acid; and from 10.33 to 3090 ng/mL for ursolic acids. The calibration curves were constructed by linear regression of the peak area ratio of the five triterpene acids to the is (y-axis) and the final concen2 trations (x-axis) with a 1/x weighting factor, described as y = a + bx. Concentrations of quality controls (QCs) and samples were calculated using the regression equation of the calibration curve. The lower limit of quantitation (LLOQ) was determined as the lowest concentration of each calibration curve (signal-to-noise ratio >10). The limit of detection (LOD) was based on a signal-to-noise ratio (S/N>6), and the results of LLOQ and LOD are listed in Table 1.
The intra-, inter-day precisions and accuracies The intra-day and inter-day precisions and accuracies of the method were evaluated by analyzing six replicates of QC samples at low, medium and high concentrations on the same day and five consecutive days. The standard deviation (SD) was calculated using one-way-ANOVA and the relative standard deviation (RSD) was calculated by the ratio of SD and average; the intra- and inter-day precisions are required to be 10 was taken as the LLOQ and was determined to be 5 ng/mL, which was sufficient to perform pharmacokinetic studies of the triterpene acids in rats. The LLOQs for
1794
Figure 1. The mass spectra of euscaphic acid (A), maslinic acid (B), corosolic acid (C), oleanolic acid (D), ursolic acid (E) and IS (F).
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1791–1797
Pharmacokinetic study of Folium Eriobotryae effective fraction in rats
Figure 2. (A) Selected ion recording (SIR) chromatogram of blank plasma. (B) SIR chromatogram of sample plasma (I, euscaphic acid; II, maslinic acid; III, corosolic acid; IV, oleanolic acid; V, ursolic acid). (C) SIR chromatogram of standard-spiked plasma sample.
euscaphic acid, maslinic acid, corosolic acid, oleanolic acid and ursolic acid were 10.65, 12.31, 10.80, 9.996 and 10.33 ng/mL, respectively, indicating that the method is sensitive for quantitative evaluation of the five compounds. The intra- and inter-day precisions and accuracies. The results showed that, for all five triterpene acids, the intra- and inter-day precision values (RSD %) were both
