Short communication Received: 30 March 2014,
Revised: 12 June 2014,
Accepted: 27 June 2014
Published online in Wiley Online Library: 26 July 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3299
Development of a liquid chromatography– mass spectrometry method for determination of agrimol B in rat plasma: application to preclinical pharmacokinetics Zhenglin Wang*, Peng Liu, Pin Liang and Xiang Hu ABSTRACT: A sensitive and accurate liquid chromatography coupled with mass spectrometry (LC-MS) method was developed for the determination of agrimol B, a main active ingredient isolated from Agrimonia pilosa Ledeb., in rat plasma. Chromatographic separation was achieved on a Zorbax CN column (150 × 4.6 mm, 5 μm), with isocratic elution consisting of acetonitrile and water (15:85, v/v) at a flow rate of 0.6 mL/min. Agrimol B and dryocrassin ABBA, an internal standard (IS), were analyzed by selected ion monitoring at m/z transitions of 681.3 and 819.4, respectively. This assay exhibited a good linearity with a correlation coefficient >0.99 and showed no endogenous interference with the analyte and IS. The limit of quantification of agrimol B was 8.025 ng/mL with acceptable precision and accuracy. The method was successfully applied in the pharmacokinetic study of agrimol B in rats after intravenous (1 mg/kg) and oral (2, 5 and 10 mg/kg) doses of agrimol B. The absolute bioavailability of agrimol B was 16.4–18.0% in rat. Our study clarifies the pharmacokinetic behavior of agrimol B in animals. 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: Agrimonia pilosa Ledeb; agrimol B; LC-MS; rat plasma; pharmacokinetics
Introduction
Biomed. Chromatogr. 2015; 29: 481–484
Experimental Chemicals and materials Agrimol B (purity, 95.5%) and dryocrassin ABBA (purity 97.2%) used as the internal standard (IS) were purchased from Shanxi Han-Ming Biotechnology (Xi’an, China). HPLC-grade methanol and acetonitrile were purchased from Buridick & Jackson (Muskegon, USA). Ultra-pure water (18.2 MΩ) was obtained using a Milli-Q water purification system (MA, USA).
Instruments and LC-MS conditions The LC-MS system consisted of an Agilent 1200 high-performance liquid chromatographic (HPLC) system and an Agilent 6130 single-quadrupole
* Correspondence to: Z. Wang, Department of Gastroenterological Surgery, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, China. Email: [email protected] Department of Gastroenterological Surgery, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, China Abbreviations used: ESI, electrospray ionization; SIM, selected ion monitoring; SPE, solid-phase extraction.
Copyright © 2014 John Wiley & Sons, Ltd.
481
Agrimonia pilosa Ledeb. is a well-known traditional Chinese medicinal plant used for its anti-oxidant, anti-inflammatory, anti-viral, anti-hyperglycemic, anti-tumor and analgesic effects (Hah et al., 2009; Jung et al., 2010; Shin et al., 2010), and clinically used for treating hemorrhage, chronic-fatigue syndrome, liver disorders and abdominal pain (Park et al., 2004; Kim et al., 2012). In recent years, A. pilosa has attracted much attention owing to its effectiveness against several cancers including gastric, intestinal, lung and liver (Choi et al., 2009; Nho et al., 2011). A. pilosa has a high polyphenol content, such as phenolic glycosides, flavonoids, 3,4-dihydroisocoumarins, triterpenoids and isoflavonoids (Kato et al., 2010; Taira et al., 2012). Agrimol B, belonging to the polyphenol type, has a functional group and various pharmacological activities, and is the main active ingredients of A. pilosa. Therefore, agrimol B has been chosen as a marker compound for the chemical evaluation or quality control of A. pilosa in the Chinese Pharmacopoeia (The State Pharmacopoeia Commission of the P.R. China, 2010). Agrimol B is now being evaluated in preclinical studies as an anti-gastric/intestinal tumor agent using nude mice as an animal model in our group, suggesting that the compound is a novel promising anti-tumor drug candidate. Until now, no method has been reported to determine agrimol B in biological samples and no published literature on the pharmacokinetics of agrimol B was retrieved. As a promising anti-tumor agent, a better understanding of the pharmacokinetics of agrimol B is necessary to explain its action mechanism and therapeutic effect. In this paper, a liquid chromatography coupled with mass spectrometry (LC-MS) method was developed for the
determination of agrimol B in rat plasma samples. The method was accurate and precise with a limit of quantification (LOQ) of 8.025 ng/mL for plasma samples. The validated method has been successfully applied to a pharmacokinetic studies of single intravenous (1 mg/kg) and oral administration (2, 5 and 10 mg/kg) of agrimol B to rats.
Z. Wang et al. mass spectrometric detector (Agilent Technologies, USA). The separation of agrimol B and IS from endogenous substances was performed on a Zorbax SB-CN (150 × 4.6 mm, 5 μm; Agilent Technologies) column protected by a short guard column SecurityGuard CN (4 × 3 mm, 5 μm; Phenomenex Inc.) column. The mobile phase consisted of acetonitrile and water (15:85, v/v) at a flow rate of 0.6 mL/min. Mass spectrometric detection was performed with an electrospray ionization (ESI) source in negative-ion scan mode. The settings of the mass spectrometer for agrimol B and IS were 4.0 kV for capillary voltage, 11 L/min for drying gas flow, 35 psi for nebulizer gas pressure, 300 °C for source temperature, and 145 and 165 V for the fragment electric voltages for agrimol B and IS, respectively. In this present study, the pseudomolecular ions [M – H] for agrimol B and IS were monitored simultaneously at m/z 681.3 and 819.4, respectively.
Preparation of standards and quality control samples Stock solutions of agrimol B and IS were both prepared in methanol at concentrations of 0.321 and 0.157 mg/mL, respectively. Stock solution of agrimol B was then serially diluted with pure methanol to prepare working standard solutions. The IS solution was freshly prepared by diluting IS stock solution in methanol to 1570 ng/mL. Calibration standards were prepared by spiking 20 μL of the appropriate standard solutions to 180 μL of blank plasma to yield the final concentrations of 8.025, 16.05, 32.10, 80.25, 160.5, 321, 802.5 and 3210 ng/mL. Quality control (QC) samples were prepared with the same procedure as the calibration standards, to give low, medium and high concentrations of 20.06, 128.4 and 2889 ng/mL. All the samples were stored at 80 °C.
Plasma sample preparation Satisfactory recovery of agrimol B and IS was obtained with a solid-phase 3 extraction (SPE) procedure with an Oasis HLB cartridge (1 cm /30 mg) for isolation of the drugs from plasma samples. Before analysis, plasma samples (100 μL) spiked with 50 μL of IS (1570 ng/mL) were accurately diluted to 1 mL with water. The HLB cartridge was conditioned sequentially by 1 mL of methanol, followed by equilibration with 1 mL of water. The plasma sample (1 mL) was introduced into the cartridges. The loaded samples were rinsed in two steps; first, 1 mL of 5% methanol was used followed by 1 mL of 50% methanol. Finally, the analytes were eluted with 1 mL of methanol. The eluate thus obtained was evaporated to dryness under a gentle stream of nitrogen at 40 °C. The dry residue was reconstituted in 100 μL of mobile phase, and 40 μL was injected for LC-MS analysis.
Linearity and sensitivity
and 2889 ng/mL) with those obtained from direct injection of the analyte dissolved in the supernatant of the processed blank plasma.
Pharmacokinetic study Twenty-four Sprague–Dawley male rats, weighing 200–230 g, were supplied by the Animal Center of Dalian Medical University (Dalian, China). The experimental protocols were approved by the Animal Ethics Committee of Dalian Medical University. Twenty-four rats were divided into four groups at random. Group 1 was given a single dose of agrimol B solution at 1.0 mg/kg by intravenous administration. Groups 2–4 were given a single oral administration of agrimol B at a dose of 2.0, 5.0 and 10.0 mg/kg by gavage, respectively. Agrimol B solution for intravenous administration was prepared by dissolving agrimol B with isotonic sodium chloride containing 5% ethanol. Agrimol B solution for oral administration was prepared by mixing agrimol B with 0.5% carboxymethyl cellulose sodium (CMC-Na) aqueous solution. After a single dose was administrated, about 0.3 mL of blood samples was collected in heparinized tubes via the orbital vein at 0, 0.083, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and 24 h. All blood samples were centrifuged at 3000 g for 10 min at 4 °C and the harvested plasma was immediately processed for LC-MS analysis.
Results and discussion LC-MS condition and IS selection According to the polyphenol groups of agrimol B and IS, the amounts of the analytes were analyzed in an ESI negative ion mode, and their full-scan mass spectra after direct injection were obtained (see Fig. S1). The results indicated that the ions of the analytes were both [M – H] ions, and the responses were very stable and showed good linearity in selected ion monitoring (SIM) mode. The quantitative analysis was carried out in SIM mode as follows: [M – H] m/z 681.3 for agrimol B, [M – H] m/z 819.4 for the IS. An optimized chromatographic run time of 4.2 min per sample yielded symmetric peaks for both agrimol B and IS with adequate separation. The representative LC-MS/MS chromatograms of plasma samples for both compounds are shown in Fig. 1. Dryocrassin ABBA was selected as an appropriate IS owing to the fact that its chemical structure, ionization mode, chromatographic behavior and extraction recovery were similar to those of agrimol B. Linearity and sensitivity
482
Calibration curves were prepared by spiking blank rat plasma with the agrimol B standard solution to the concentrations: 8.025, 16.05, 32.10, 80.25, 160.5, 321, 802.5 and 3210 ng/mL. The calibration curves of agrimol B were constructed by plotting the peak-area ratios of agrimol B/IS vs concentrations of agrimol B in plasma, and the linearity was assessed using linear regression analysis. The LOQ was determined as the lowest plasma concentration that produced a signal-to-noise ratio ≥10.
The calibration curves for agrimol B were found to be linear over the concentration range of 8.025–3210 ng/mL (r > 0.99). Typical equations for the calibration curves were y = (0.0021 ± 0.0002) x + (0.0032 ± 0.0008). The LOQ of this method was 8.025 ng/mL, and the precision and accuracy of LOQ were
Revised: 12 June 2014,
Accepted: 27 June 2014
Published online in Wiley Online Library: 26 July 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3299
Development of a liquid chromatography– mass spectrometry method for determination of agrimol B in rat plasma: application to preclinical pharmacokinetics Zhenglin Wang*, Peng Liu, Pin Liang and Xiang Hu ABSTRACT: A sensitive and accurate liquid chromatography coupled with mass spectrometry (LC-MS) method was developed for the determination of agrimol B, a main active ingredient isolated from Agrimonia pilosa Ledeb., in rat plasma. Chromatographic separation was achieved on a Zorbax CN column (150 × 4.6 mm, 5 μm), with isocratic elution consisting of acetonitrile and water (15:85, v/v) at a flow rate of 0.6 mL/min. Agrimol B and dryocrassin ABBA, an internal standard (IS), were analyzed by selected ion monitoring at m/z transitions of 681.3 and 819.4, respectively. This assay exhibited a good linearity with a correlation coefficient >0.99 and showed no endogenous interference with the analyte and IS. The limit of quantification of agrimol B was 8.025 ng/mL with acceptable precision and accuracy. The method was successfully applied in the pharmacokinetic study of agrimol B in rats after intravenous (1 mg/kg) and oral (2, 5 and 10 mg/kg) doses of agrimol B. The absolute bioavailability of agrimol B was 16.4–18.0% in rat. Our study clarifies the pharmacokinetic behavior of agrimol B in animals. 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: Agrimonia pilosa Ledeb; agrimol B; LC-MS; rat plasma; pharmacokinetics
Introduction
Biomed. Chromatogr. 2015; 29: 481–484
Experimental Chemicals and materials Agrimol B (purity, 95.5%) and dryocrassin ABBA (purity 97.2%) used as the internal standard (IS) were purchased from Shanxi Han-Ming Biotechnology (Xi’an, China). HPLC-grade methanol and acetonitrile were purchased from Buridick & Jackson (Muskegon, USA). Ultra-pure water (18.2 MΩ) was obtained using a Milli-Q water purification system (MA, USA).
Instruments and LC-MS conditions The LC-MS system consisted of an Agilent 1200 high-performance liquid chromatographic (HPLC) system and an Agilent 6130 single-quadrupole
* Correspondence to: Z. Wang, Department of Gastroenterological Surgery, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, China. Email: [email protected] Department of Gastroenterological Surgery, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, China Abbreviations used: ESI, electrospray ionization; SIM, selected ion monitoring; SPE, solid-phase extraction.
Copyright © 2014 John Wiley & Sons, Ltd.
481
Agrimonia pilosa Ledeb. is a well-known traditional Chinese medicinal plant used for its anti-oxidant, anti-inflammatory, anti-viral, anti-hyperglycemic, anti-tumor and analgesic effects (Hah et al., 2009; Jung et al., 2010; Shin et al., 2010), and clinically used for treating hemorrhage, chronic-fatigue syndrome, liver disorders and abdominal pain (Park et al., 2004; Kim et al., 2012). In recent years, A. pilosa has attracted much attention owing to its effectiveness against several cancers including gastric, intestinal, lung and liver (Choi et al., 2009; Nho et al., 2011). A. pilosa has a high polyphenol content, such as phenolic glycosides, flavonoids, 3,4-dihydroisocoumarins, triterpenoids and isoflavonoids (Kato et al., 2010; Taira et al., 2012). Agrimol B, belonging to the polyphenol type, has a functional group and various pharmacological activities, and is the main active ingredients of A. pilosa. Therefore, agrimol B has been chosen as a marker compound for the chemical evaluation or quality control of A. pilosa in the Chinese Pharmacopoeia (The State Pharmacopoeia Commission of the P.R. China, 2010). Agrimol B is now being evaluated in preclinical studies as an anti-gastric/intestinal tumor agent using nude mice as an animal model in our group, suggesting that the compound is a novel promising anti-tumor drug candidate. Until now, no method has been reported to determine agrimol B in biological samples and no published literature on the pharmacokinetics of agrimol B was retrieved. As a promising anti-tumor agent, a better understanding of the pharmacokinetics of agrimol B is necessary to explain its action mechanism and therapeutic effect. In this paper, a liquid chromatography coupled with mass spectrometry (LC-MS) method was developed for the
determination of agrimol B in rat plasma samples. The method was accurate and precise with a limit of quantification (LOQ) of 8.025 ng/mL for plasma samples. The validated method has been successfully applied to a pharmacokinetic studies of single intravenous (1 mg/kg) and oral administration (2, 5 and 10 mg/kg) of agrimol B to rats.
Z. Wang et al. mass spectrometric detector (Agilent Technologies, USA). The separation of agrimol B and IS from endogenous substances was performed on a Zorbax SB-CN (150 × 4.6 mm, 5 μm; Agilent Technologies) column protected by a short guard column SecurityGuard CN (4 × 3 mm, 5 μm; Phenomenex Inc.) column. The mobile phase consisted of acetonitrile and water (15:85, v/v) at a flow rate of 0.6 mL/min. Mass spectrometric detection was performed with an electrospray ionization (ESI) source in negative-ion scan mode. The settings of the mass spectrometer for agrimol B and IS were 4.0 kV for capillary voltage, 11 L/min for drying gas flow, 35 psi for nebulizer gas pressure, 300 °C for source temperature, and 145 and 165 V for the fragment electric voltages for agrimol B and IS, respectively. In this present study, the pseudomolecular ions [M – H] for agrimol B and IS were monitored simultaneously at m/z 681.3 and 819.4, respectively.
Preparation of standards and quality control samples Stock solutions of agrimol B and IS were both prepared in methanol at concentrations of 0.321 and 0.157 mg/mL, respectively. Stock solution of agrimol B was then serially diluted with pure methanol to prepare working standard solutions. The IS solution was freshly prepared by diluting IS stock solution in methanol to 1570 ng/mL. Calibration standards were prepared by spiking 20 μL of the appropriate standard solutions to 180 μL of blank plasma to yield the final concentrations of 8.025, 16.05, 32.10, 80.25, 160.5, 321, 802.5 and 3210 ng/mL. Quality control (QC) samples were prepared with the same procedure as the calibration standards, to give low, medium and high concentrations of 20.06, 128.4 and 2889 ng/mL. All the samples were stored at 80 °C.
Plasma sample preparation Satisfactory recovery of agrimol B and IS was obtained with a solid-phase 3 extraction (SPE) procedure with an Oasis HLB cartridge (1 cm /30 mg) for isolation of the drugs from plasma samples. Before analysis, plasma samples (100 μL) spiked with 50 μL of IS (1570 ng/mL) were accurately diluted to 1 mL with water. The HLB cartridge was conditioned sequentially by 1 mL of methanol, followed by equilibration with 1 mL of water. The plasma sample (1 mL) was introduced into the cartridges. The loaded samples were rinsed in two steps; first, 1 mL of 5% methanol was used followed by 1 mL of 50% methanol. Finally, the analytes were eluted with 1 mL of methanol. The eluate thus obtained was evaporated to dryness under a gentle stream of nitrogen at 40 °C. The dry residue was reconstituted in 100 μL of mobile phase, and 40 μL was injected for LC-MS analysis.
Linearity and sensitivity
and 2889 ng/mL) with those obtained from direct injection of the analyte dissolved in the supernatant of the processed blank plasma.
Pharmacokinetic study Twenty-four Sprague–Dawley male rats, weighing 200–230 g, were supplied by the Animal Center of Dalian Medical University (Dalian, China). The experimental protocols were approved by the Animal Ethics Committee of Dalian Medical University. Twenty-four rats were divided into four groups at random. Group 1 was given a single dose of agrimol B solution at 1.0 mg/kg by intravenous administration. Groups 2–4 were given a single oral administration of agrimol B at a dose of 2.0, 5.0 and 10.0 mg/kg by gavage, respectively. Agrimol B solution for intravenous administration was prepared by dissolving agrimol B with isotonic sodium chloride containing 5% ethanol. Agrimol B solution for oral administration was prepared by mixing agrimol B with 0.5% carboxymethyl cellulose sodium (CMC-Na) aqueous solution. After a single dose was administrated, about 0.3 mL of blood samples was collected in heparinized tubes via the orbital vein at 0, 0.083, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and 24 h. All blood samples were centrifuged at 3000 g for 10 min at 4 °C and the harvested plasma was immediately processed for LC-MS analysis.
Results and discussion LC-MS condition and IS selection According to the polyphenol groups of agrimol B and IS, the amounts of the analytes were analyzed in an ESI negative ion mode, and their full-scan mass spectra after direct injection were obtained (see Fig. S1). The results indicated that the ions of the analytes were both [M – H] ions, and the responses were very stable and showed good linearity in selected ion monitoring (SIM) mode. The quantitative analysis was carried out in SIM mode as follows: [M – H] m/z 681.3 for agrimol B, [M – H] m/z 819.4 for the IS. An optimized chromatographic run time of 4.2 min per sample yielded symmetric peaks for both agrimol B and IS with adequate separation. The representative LC-MS/MS chromatograms of plasma samples for both compounds are shown in Fig. 1. Dryocrassin ABBA was selected as an appropriate IS owing to the fact that its chemical structure, ionization mode, chromatographic behavior and extraction recovery were similar to those of agrimol B. Linearity and sensitivity
482
Calibration curves were prepared by spiking blank rat plasma with the agrimol B standard solution to the concentrations: 8.025, 16.05, 32.10, 80.25, 160.5, 321, 802.5 and 3210 ng/mL. The calibration curves of agrimol B were constructed by plotting the peak-area ratios of agrimol B/IS vs concentrations of agrimol B in plasma, and the linearity was assessed using linear regression analysis. The LOQ was determined as the lowest plasma concentration that produced a signal-to-noise ratio ≥10.
The calibration curves for agrimol B were found to be linear over the concentration range of 8.025–3210 ng/mL (r > 0.99). Typical equations for the calibration curves were y = (0.0021 ± 0.0002) x + (0.0032 ± 0.0008). The LOQ of this method was 8.025 ng/mL, and the precision and accuracy of LOQ were
