Journal of Chromatographic Science, 2015, Vol. 53, No. 10, 1725–1729 doi: 10.1093/chromsci/bmv081 Advance Access Publication Date: 26 June 2015 Article
Article
Quantification of Nardosinone in Rat Plasma Using Liquid Chromatography–Tandem Mass Spectrometry and Its Pharmacokinetics Application Zhihe Lu1,†, Peng Zhou2,†, Yuzhu Zhan3, Jingrong Su4,*, and Deliang Yi5 1 Department of Pharmacy, Linyi People’s Hospital, Linyi 276003, PR China, 2Department of Assets and Equipment, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250011, China, 3Pediatric Department of Internal Medicine, Linyi People’s Hospital, Linyi 276003, PR China, 4Skill Training Room, Linyi People’s Hospital, Linyi 276003, PR China, and 5Yantai Yuhuangding Hospital, Yantai 264000, PR China
*Author to whom correspondence should be addressed. Email: [email protected] †
Zhihe Lu and Peng Zhou contributed equally to this work.
Received 26 June 2014; Revised 12 May 2015
Abstract A rapid, sensitive and high-throughput liquid chromatography–tandem mass spectrometry (LC–MS-MS) method was established and validated to assay the concentration of nardosinone, a main active compound isolated from Nardostachys chinensis, in rat plasma. Plasma samples were processed by protein precipitation with acetonitrile and separated on a Venusil MP-C18 column (50 × 2.1 mm, 5 µm) at an isocratic flow rate of 0.6 mL/min using methanol–0.1% formic acid in water (55 : 45, v/v) as mobile phase, and total run time was 2.5 min. MS–MS detection was accomplished in selected reaction monitoring mode with positive electrospray ionization. The calibration curve was linear over the concentration range of 9.60–320 ng/mL with lower limit of quantification of 9.60 ng/mL. The intra- and inter-day precisions were below 12.3% in terms of relative standard deviation, and the accuracy was within ±9.0% in terms of relative error. Extraction recovery, matrix effect and stability were also satisfactory in rat plasma. The developed method was successfully applied to a pharmacokinetic study of nardosinone following an intravenous injection at a dose of 1.04 mg/kg to Sprague-Dawley rats.
Introduction Nardostachys chinensis (Chinese name: “Gansong”) have been used in Traditional Chinese Medicine and other ethnical folk medicine in China for a long time (1). The medicine exhibited antimalarial, antinociceptive, neurotrophic and cytotoxic activities (2–4), and clinically used for treating acute gastritis, hyperlipemia and frequent premature ventricular contractions (5, 6). Moreover, N. chinensis has attracted much attention due to their effectiveness against cardiac disorders, such as myocardial ischemia-reperfusion injury and pathological cardiac hypertrophy (7, 8). N. chinensis contains guaiane, aristolane, nardosinane-type sesquiterpenoids, neolignan and lignans (9, 10). Nardosinone, belonging
to nardosinane-type sesquiterpenoids, has been chosen as a marker compound for the chemical evaluation of N. chinensis in the Chinese Pharmacopoeia (11). Nardosinone (Figure 1) could enhance staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells and was used as a potentiator of the neurite outgrowthpromoting activity of nerve growth factor (12, 13). Until now, no method has been reported to determine nardosinone in any biological matrix and no literature on its pharmacokinetics was retrieved. To better explain its action mechanism and therapeutic effect, it is very necessary to study the pharmacokinetics of nardosinone. In this article, a liquid chromatography tandem mass spectrometry (LC–MS-MS) method was developed for the determination of
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
1725
1726 nardosinone in rat plasma. The method was accurate and precise with a limit of quantification (LOQ) of 9.60 ng/mL for plasma samples. The validated method has been successfully applied to a pharmacokinetic study following intravenous bolus injection of 1.04 mg/kg nardosinone to rats.
Experimental Chemicals and reagents Nardosinone (CAS 23720-80-1) was obtained from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China), and isoalantolactone used as an internal standard (IS) was obtained from National Institutes for Food and Drug Control (Beijing, China). Other chemicals included HPLC-grade acetonitrile, HPLC-grade methanol, analytical reagent grade formic acid and deionized water through a Milli-Q system (Millipore, Bedford, MA, USA).
Instrument and analytical conditions The LC–MS-MS system consists of an Agilent 1200 HPLC system equipped with a binary pump, a degasser, an autosampler, a thermostatted column compartment (Agilent Technologies, CA, USA) and an Agilent 6460 QQQ MS-MS system (Agilent Technologies). Chromatographic separation was performed on a Venusil MP-C18 column (50 × 2.1 mm, 5 μm; Agela, DE, USA), which was protected by a Shim-pack Diol guard column (50 × 4.0 mm, 10 μm; Shimadzu, Kyoto, Japan). The mobile phase was composed of methanol–0.1% formic acid in water
Lu et al. (55 : 45, v/v) as mobile phase at a flow rate of 0.6 mL/min. The column temperature was maintained at 30°C. The injection volume was 5 μL. Mass spectrometric detection was operated in positive ionization mode using a jet stream electrospray ionization (ESI) source in selected reaction monitoring (SRM) mode. The flow of dry and sheath gas was 5 and 12 L/min, respectively. The nebulizer gas pressure was 45 psi and the capillary voltage was 4.0 kV. The optimized SRM transitions were as follows: m/z 251.1 → m/z 91.2 for nardosinone; m/z 233.2 → m/z 105.2 for IS, respectively (Figure 2). The optimal collision energies were 23 and 25 eV for nardosinone and IS, respectively.
Standard and quality control sample preparation Stock solutions of nardosinone and IS were prepared by dissolving accurate amounts of reference standards in methanol at a concentration of 0.8 and 0.5 mg/mL, respectively. A series of working solutions of nardosinone were obtained by further diluting the stock solution. Calibration standards were prepared by spiking the appropriate amounts of the working solutions into blank plasma to obtain final concentrations levels of 9.60, 16.0, 32.0, 96.0, 160 and 320 ng/mL for nardosinone, respectively. For validation, quality control (QC) samples were prepared by spiking rat plasma at three concentration levels (19.2, 64.0 and 288 ng/mL). The calibration standards and QC were stored at −60°C until analysis. The IS working solution was prepared by diluting the IS stock solution (0.5 mg/mL) with acetonitrile to produce the final concentration of 500 ng/mL and stored at 4°C.
Sample preparation The frozen plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 150 μL of the IS working solution (500 ng/mL in acetonitrile) was added to 50 μL of collected plasma sample. The tubes were vortex mixed for 1.0 min and then centrifuged at 13000 × g for 5 min. The supernatant (5 μL) was injected into the LC–MS-MS system for analysis.
Method validation The method validation assays were performed according to the currently accepted US Food and Drug Administration (FDA) bioanalytical method validation guidance (14).
Specificity and matrix effect Figure 1. Chemical structures of nardosinone and isoalantolactone (IS).
Blank plasma samples from six different rats were prepared and analyzed to evaluate potential interference from endogenous compounds.
Figure 2. Full-scan product ion spectra of [M+H]+ ions for (left profile) nardosinone and (right profile) IS. This figure is available in black and white in print and in color at JCS online.
1727
Quantification of Nardosinone in Rat Plasma A comparison study was performed on chromatograms of blank plasma, plasma spiked with the analyte and IS, as well as plasma samples after administration of the drug. Matrix effect was evaluated by comparing pretreated blank biological matrices spiked at concentration of QC samples with the direct injection of the corresponding standard solutions.
Extraction recovery The extraction recoveries were determined in six replicates by comparing the absolute peak area of pre-spiked QC samples with that of postspiked QC samples.
Precision and accuracy Precision and accuracy were investigated on 3 days by measuring the analyte in six replicates at three QC levels. To assay the QCs, different standard calibration curves were plotted and processed on each day of the three validation days. The precision at each concentration level was reported by expressing the relative standard deviation (RSD%). The criteria for the acceptability included accuracy within ±15% of the relative error (RE) from the nominal values, and a precision of within 15% of the RSD.
Linearity and LOQ Linearity of calibration curves were prepared by making serial dilutions of the working stocks and determining standard plasma samples at six concentrations of nardosinone ranging 9.60–320 ng/mL. The linearity was evaluated by plotting the peak area ratio (Y) of nardosinone to IS versus the nominal concentrations (X) of nardosinone. Calibration curves were constructed by being weighted (1/X 2) least square linear regression. The LOQ for nardosinone was assayed based on at least 10 times of the signal-to-noise (S/N) ratio and satisfactory precision (RSD ≤ 20%) and accuracy (RE ≤ ±20%).
Stability The stability protocols were performed to investigate the stability of the analytes in rat plasma under the following conditions: short-term stability at room temperature for 4 h; long-term stability at −60°C for 80 days; post-preparation stability at 4°C for 12 h in the autosampler vials and three freeze (−60°C)–thaw (room temperature) cycles. All stability experiments in plasma were evaluated by determining triplicates of QC samples at three concentration levels.
Pharmacokinetic study Ten Sprague-Dawley rats (five males and five females) weighing 200– 230 g were purchased from the Shandong Luye Pharmaceutical Company (Yantai, China). The experimental protocol (no. 20130507) is approved by the Animal Ethics Committee of Yantai Yuhuangding Hospital. The rats were housed in temperature- and humiditycontrolled conditions (temperature, 23 ± 2°C; and relative humidity, 45 ± 5%) for 1 week. The powder of nardosinone was dissolved in propylene glycol–physiologic saline (20 : 80, v/v) to be 0.104 mg/mL for dosing. After an overnight fast, the rats were intravenously administered the nardosinone solution at a single dose of 10 mL/kg (equivalent to 1.04 mg/kg body weight). Blood samples (∼250 µL) were collected into 1.5 mL of heparinized tubes via the oculi chorioideae vein at 0 ( pre-dosing), 5, 10, 30 min and 1, 1.5, 2, 3, 4, 5, 8 h after dosing. Then the blood samples were centrifuged at 4500 × g for 10 min at 4°C. The obtained plasma was stored at −60°C until LC– MS-MS analysis.
Results Method validation Specificity Blank plasma samples from six sources were investigated and no significant endogenous substance was observed in the SRM channels at the retention times of analytes. The representative SRM chromatograms of (a1, a2) a blank rat plasma sample, (b1, b2) a blank rat plasma sample spiked with nardosinone and IS and (c1, c2) a plasma sample from a rat at 30 min after an intravenous administration of 1.04 mg/kg nardosinone are illustrated in Figure 3. Extraction recovery and matrix effect The extraction recovery and matrix effect were determined by analyzing QC samples at low, medium and high concentrations. The mean recovery of the analytes and IS was within 82.1–87.3% (RSD ≤ 4.7%), and the corresponding matrix effect ranged from 95.5 to 104.8% (RSD ≤ 8.2%), which manifested that acetonitrile was an appropriate and feasible medium for the analytes and IS extraction, and moreover, there was no measurable matrix effect on the ionization efficiency of analyte and IS. Linearity and LOQ Calibration curves showed good linearity over the concentration range of 9.60–320 ng/mL for nardosinone in rat plasma. A typical linear regression equation was Y = 0.004890X + 0.007768, R 2 = 0.9962, where Y represents the ratio of peak area of the analyte to that of the IS and X is the plasma concentration of nardosinone. The LOQ value was 9.60 ng/mL, at which the intra- and inter-day accuracy was −1.0 and −2.8%, respectively, and the precision was
Article
Quantification of Nardosinone in Rat Plasma Using Liquid Chromatography–Tandem Mass Spectrometry and Its Pharmacokinetics Application Zhihe Lu1,†, Peng Zhou2,†, Yuzhu Zhan3, Jingrong Su4,*, and Deliang Yi5 1 Department of Pharmacy, Linyi People’s Hospital, Linyi 276003, PR China, 2Department of Assets and Equipment, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250011, China, 3Pediatric Department of Internal Medicine, Linyi People’s Hospital, Linyi 276003, PR China, 4Skill Training Room, Linyi People’s Hospital, Linyi 276003, PR China, and 5Yantai Yuhuangding Hospital, Yantai 264000, PR China
*Author to whom correspondence should be addressed. Email: [email protected] †
Zhihe Lu and Peng Zhou contributed equally to this work.
Received 26 June 2014; Revised 12 May 2015
Abstract A rapid, sensitive and high-throughput liquid chromatography–tandem mass spectrometry (LC–MS-MS) method was established and validated to assay the concentration of nardosinone, a main active compound isolated from Nardostachys chinensis, in rat plasma. Plasma samples were processed by protein precipitation with acetonitrile and separated on a Venusil MP-C18 column (50 × 2.1 mm, 5 µm) at an isocratic flow rate of 0.6 mL/min using methanol–0.1% formic acid in water (55 : 45, v/v) as mobile phase, and total run time was 2.5 min. MS–MS detection was accomplished in selected reaction monitoring mode with positive electrospray ionization. The calibration curve was linear over the concentration range of 9.60–320 ng/mL with lower limit of quantification of 9.60 ng/mL. The intra- and inter-day precisions were below 12.3% in terms of relative standard deviation, and the accuracy was within ±9.0% in terms of relative error. Extraction recovery, matrix effect and stability were also satisfactory in rat plasma. The developed method was successfully applied to a pharmacokinetic study of nardosinone following an intravenous injection at a dose of 1.04 mg/kg to Sprague-Dawley rats.
Introduction Nardostachys chinensis (Chinese name: “Gansong”) have been used in Traditional Chinese Medicine and other ethnical folk medicine in China for a long time (1). The medicine exhibited antimalarial, antinociceptive, neurotrophic and cytotoxic activities (2–4), and clinically used for treating acute gastritis, hyperlipemia and frequent premature ventricular contractions (5, 6). Moreover, N. chinensis has attracted much attention due to their effectiveness against cardiac disorders, such as myocardial ischemia-reperfusion injury and pathological cardiac hypertrophy (7, 8). N. chinensis contains guaiane, aristolane, nardosinane-type sesquiterpenoids, neolignan and lignans (9, 10). Nardosinone, belonging
to nardosinane-type sesquiterpenoids, has been chosen as a marker compound for the chemical evaluation of N. chinensis in the Chinese Pharmacopoeia (11). Nardosinone (Figure 1) could enhance staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells and was used as a potentiator of the neurite outgrowthpromoting activity of nerve growth factor (12, 13). Until now, no method has been reported to determine nardosinone in any biological matrix and no literature on its pharmacokinetics was retrieved. To better explain its action mechanism and therapeutic effect, it is very necessary to study the pharmacokinetics of nardosinone. In this article, a liquid chromatography tandem mass spectrometry (LC–MS-MS) method was developed for the determination of
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
1725
1726 nardosinone in rat plasma. The method was accurate and precise with a limit of quantification (LOQ) of 9.60 ng/mL for plasma samples. The validated method has been successfully applied to a pharmacokinetic study following intravenous bolus injection of 1.04 mg/kg nardosinone to rats.
Experimental Chemicals and reagents Nardosinone (CAS 23720-80-1) was obtained from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China), and isoalantolactone used as an internal standard (IS) was obtained from National Institutes for Food and Drug Control (Beijing, China). Other chemicals included HPLC-grade acetonitrile, HPLC-grade methanol, analytical reagent grade formic acid and deionized water through a Milli-Q system (Millipore, Bedford, MA, USA).
Instrument and analytical conditions The LC–MS-MS system consists of an Agilent 1200 HPLC system equipped with a binary pump, a degasser, an autosampler, a thermostatted column compartment (Agilent Technologies, CA, USA) and an Agilent 6460 QQQ MS-MS system (Agilent Technologies). Chromatographic separation was performed on a Venusil MP-C18 column (50 × 2.1 mm, 5 μm; Agela, DE, USA), which was protected by a Shim-pack Diol guard column (50 × 4.0 mm, 10 μm; Shimadzu, Kyoto, Japan). The mobile phase was composed of methanol–0.1% formic acid in water
Lu et al. (55 : 45, v/v) as mobile phase at a flow rate of 0.6 mL/min. The column temperature was maintained at 30°C. The injection volume was 5 μL. Mass spectrometric detection was operated in positive ionization mode using a jet stream electrospray ionization (ESI) source in selected reaction monitoring (SRM) mode. The flow of dry and sheath gas was 5 and 12 L/min, respectively. The nebulizer gas pressure was 45 psi and the capillary voltage was 4.0 kV. The optimized SRM transitions were as follows: m/z 251.1 → m/z 91.2 for nardosinone; m/z 233.2 → m/z 105.2 for IS, respectively (Figure 2). The optimal collision energies were 23 and 25 eV for nardosinone and IS, respectively.
Standard and quality control sample preparation Stock solutions of nardosinone and IS were prepared by dissolving accurate amounts of reference standards in methanol at a concentration of 0.8 and 0.5 mg/mL, respectively. A series of working solutions of nardosinone were obtained by further diluting the stock solution. Calibration standards were prepared by spiking the appropriate amounts of the working solutions into blank plasma to obtain final concentrations levels of 9.60, 16.0, 32.0, 96.0, 160 and 320 ng/mL for nardosinone, respectively. For validation, quality control (QC) samples were prepared by spiking rat plasma at three concentration levels (19.2, 64.0 and 288 ng/mL). The calibration standards and QC were stored at −60°C until analysis. The IS working solution was prepared by diluting the IS stock solution (0.5 mg/mL) with acetonitrile to produce the final concentration of 500 ng/mL and stored at 4°C.
Sample preparation The frozen plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 150 μL of the IS working solution (500 ng/mL in acetonitrile) was added to 50 μL of collected plasma sample. The tubes were vortex mixed for 1.0 min and then centrifuged at 13000 × g for 5 min. The supernatant (5 μL) was injected into the LC–MS-MS system for analysis.
Method validation The method validation assays were performed according to the currently accepted US Food and Drug Administration (FDA) bioanalytical method validation guidance (14).
Specificity and matrix effect Figure 1. Chemical structures of nardosinone and isoalantolactone (IS).
Blank plasma samples from six different rats were prepared and analyzed to evaluate potential interference from endogenous compounds.
Figure 2. Full-scan product ion spectra of [M+H]+ ions for (left profile) nardosinone and (right profile) IS. This figure is available in black and white in print and in color at JCS online.
1727
Quantification of Nardosinone in Rat Plasma A comparison study was performed on chromatograms of blank plasma, plasma spiked with the analyte and IS, as well as plasma samples after administration of the drug. Matrix effect was evaluated by comparing pretreated blank biological matrices spiked at concentration of QC samples with the direct injection of the corresponding standard solutions.
Extraction recovery The extraction recoveries were determined in six replicates by comparing the absolute peak area of pre-spiked QC samples with that of postspiked QC samples.
Precision and accuracy Precision and accuracy were investigated on 3 days by measuring the analyte in six replicates at three QC levels. To assay the QCs, different standard calibration curves were plotted and processed on each day of the three validation days. The precision at each concentration level was reported by expressing the relative standard deviation (RSD%). The criteria for the acceptability included accuracy within ±15% of the relative error (RE) from the nominal values, and a precision of within 15% of the RSD.
Linearity and LOQ Linearity of calibration curves were prepared by making serial dilutions of the working stocks and determining standard plasma samples at six concentrations of nardosinone ranging 9.60–320 ng/mL. The linearity was evaluated by plotting the peak area ratio (Y) of nardosinone to IS versus the nominal concentrations (X) of nardosinone. Calibration curves were constructed by being weighted (1/X 2) least square linear regression. The LOQ for nardosinone was assayed based on at least 10 times of the signal-to-noise (S/N) ratio and satisfactory precision (RSD ≤ 20%) and accuracy (RE ≤ ±20%).
Stability The stability protocols were performed to investigate the stability of the analytes in rat plasma under the following conditions: short-term stability at room temperature for 4 h; long-term stability at −60°C for 80 days; post-preparation stability at 4°C for 12 h in the autosampler vials and three freeze (−60°C)–thaw (room temperature) cycles. All stability experiments in plasma were evaluated by determining triplicates of QC samples at three concentration levels.
Pharmacokinetic study Ten Sprague-Dawley rats (five males and five females) weighing 200– 230 g were purchased from the Shandong Luye Pharmaceutical Company (Yantai, China). The experimental protocol (no. 20130507) is approved by the Animal Ethics Committee of Yantai Yuhuangding Hospital. The rats were housed in temperature- and humiditycontrolled conditions (temperature, 23 ± 2°C; and relative humidity, 45 ± 5%) for 1 week. The powder of nardosinone was dissolved in propylene glycol–physiologic saline (20 : 80, v/v) to be 0.104 mg/mL for dosing. After an overnight fast, the rats were intravenously administered the nardosinone solution at a single dose of 10 mL/kg (equivalent to 1.04 mg/kg body weight). Blood samples (∼250 µL) were collected into 1.5 mL of heparinized tubes via the oculi chorioideae vein at 0 ( pre-dosing), 5, 10, 30 min and 1, 1.5, 2, 3, 4, 5, 8 h after dosing. Then the blood samples were centrifuged at 4500 × g for 10 min at 4°C. The obtained plasma was stored at −60°C until LC– MS-MS analysis.
Results Method validation Specificity Blank plasma samples from six sources were investigated and no significant endogenous substance was observed in the SRM channels at the retention times of analytes. The representative SRM chromatograms of (a1, a2) a blank rat plasma sample, (b1, b2) a blank rat plasma sample spiked with nardosinone and IS and (c1, c2) a plasma sample from a rat at 30 min after an intravenous administration of 1.04 mg/kg nardosinone are illustrated in Figure 3. Extraction recovery and matrix effect The extraction recovery and matrix effect were determined by analyzing QC samples at low, medium and high concentrations. The mean recovery of the analytes and IS was within 82.1–87.3% (RSD ≤ 4.7%), and the corresponding matrix effect ranged from 95.5 to 104.8% (RSD ≤ 8.2%), which manifested that acetonitrile was an appropriate and feasible medium for the analytes and IS extraction, and moreover, there was no measurable matrix effect on the ionization efficiency of analyte and IS. Linearity and LOQ Calibration curves showed good linearity over the concentration range of 9.60–320 ng/mL for nardosinone in rat plasma. A typical linear regression equation was Y = 0.004890X + 0.007768, R 2 = 0.9962, where Y represents the ratio of peak area of the analyte to that of the IS and X is the plasma concentration of nardosinone. The LOQ value was 9.60 ng/mL, at which the intra- and inter-day accuracy was −1.0 and −2.8%, respectively, and the precision was
