Research article Received: 10 October 2013,
Revised: 8 February 2014,
Accepted: 15 March 2014
Published online in Wiley Online Library: 30 April 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3210
Development and validation of a UHPLC-qTOF-MS method for quantification of fuziline in rat plasma and its application in a pharmacokinetic study Yun-xia Lia,b,c†, Xiao-hong Gonga,b,c†, Yan Lia,b,c, Ruo-qi Zhanga,b,c, Liang Xionga,b,c, Xiao-fang Xiea,b,c and Cheng Penga,b,c* ABSTRACT: A specific and sensitive UHPLC-qTOF-MS method was developed and validated for quantification of fuziline in rat plasma after oral administration of three dosages. The analyte was separated on an Acquity UPLC BEH C18 column with a total running time of 3 min using a mobile phase of 0.1% formic acid aqueous solution and methanol (80:20, v/v) at a flow-rate of 0.25 mL/min. The calibration curves for fuziline showed good linearity in the concentrations ranging from 1 to 200 ng/mL with correlation coefficients >0.997. The precision, accuracy, recovery and stability were deemed acceptable. The method was applied to a pharmacokinetics study of fuziline in rats. The mean half-life was 5.93, 6.13 and 5.12 h for 1, 2 and 4 mg/kg oral administration of fuziline, respectively. The peak concentration and area under the concentration–time curve increased linearly with the doses. The sum of these results indicated that, in the range of the doses examined, the pharmacokinetics of fuziline in rat was based on first-order kinetics. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: fuziline; UHPLC-qTOF-MS; pharmacokinetics
Introduction
Biomed. Chromatogr. 2014; 28: 1707–1713
* Correspondence to: Peng Cheng, Pharmacy College, Chengdu University of Traditional Chinese Medicine, 1166 Liutai Road, Chengdu 611137, China. Email: [email protected] †
The first two authors contributed equally to this paper.
a
Pharmacy College, Chengdu University of Traditional Chinese Medicine, 1166 Liutai Road, Chengdu 611137, China
b
The Ministry of Education Key Laboratory of Standardization of Chinese Herbal Medicine, 1166 Liutai Road, Chengdu 611137, China
c
State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources, 1166 Liutai Road, Chengdu 611137, China Abbreviations used: TCM, traditional Chinese medicine.
Copyright © 2014 John Wiley & Sons, Ltd.
1707
Currently, pharmaceutical research and development are very challenging because of high cost, high risk and lengthy research period (Fan et al., 2012). Traditional Chinese medicine (TCM) has attracted much attention owing to its effectiveness against many diseases and proven safety in clinical use over 8000 years (Xie and Leung, 2009; Pavel and Jitka, 2004). It plays an indispensable role in the prevention and treatment of diseases, especially complicated and chronic ones (Hamza, 2010; Liu et al., 2008; Lou et al., 2010). As a huge medicinal treasury, many new drugs have been discovered from TCM. For example, Artemisinin shows great success in treating malaria, and is separated from Qinghao (Artemisia species). Although many drug candidates show good activities in vitro, weak therapeutic effects in vivo are found in animal studies. The failure of in vivo studies is mostly caused by poor understanding of pharmacokinetics of the candidates (Alavijeh and Palmer, 2004). Radix Aconiti Lateralis Preparata (Fuzi) is the processed daughter roots of Aconitum carmichaeli Debx. (family Ranunculaceae; Chinese Pharmacopoeia Commission, 2010). It is widely used for the treatment of arthralgia and heart failure. Modern pharmacological studies have revealed that Fuzi plays an important role in cardiotonic, anti-arrhythmia, anti-thrombosis, anti-hypoxia, anti-shock effect and in inhibiting thrombosis (Zhou and Liu, 2013). Pharmacologically active components of Fuzi are diterpenoid alkaloids, including hetisine, fuziline, benzoylmesaconine, aconitine, hypaconitine and mesaconitine (Wang and Zhu, 2010). Some research has been carried out on aconitine, hypaconitine and mesoconitine using various analytical methods, including highperformance liquid chromatography (HPLC; Xiang et al., 2006), liquid chromatography–mass spectrometry (LC-MS; Zhang et al., 2009),
LC-MS/MS (Yue et al., 2009) and Ultra high performance liquid chromatography (HPLC)-quadrupole time-of-flight tandem mass spectrometry (TOF/MS) (Tang et al., 2012). Compared with the numbers of studies on pharmacological activities of other chemical components in Fuzi, only one report was found on investigation of the pharmacokinetics of fuziline using a hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometric method (Sun et al., 2013). Owing to hydroxyl and tertiary ammonium groups in fuziline, the mobile phase consists of 0.05% formic acid aqueous solution and acetonitrile at a 60:40 (v/v) ratio. However an obvious interference peak is found in the blank plasma with (M + H)+ ions of fuziline in the separation condition. In our laboratory, the effect of fuziline (10, 1 and 0.1 μM) on neonatal rat cardiomyocytes was investigated by methyl thiazolyl
Y.-x. Li et al. tetrazolium assay (MTT) method (Xiong et al., 2012). Fuziline showed activity against pentobarbital sodium-induced cardiomyocytes damage by recovering the beating rhythm and increasing the cell viability. However, many phytochemicals isolated by activity-guided isolation from herbs or plants exhibit poor pharmacokinetic characteristics. For further preclinical research, a sensitive UHPLC-qTOF-MS method was developed to perform the pharmacokinetic study of fuziline in rats.
A stock solution of fuziline was prepared at a concentration of 20 μg/mL in methanol and was diluted to 0.01–2 μg/mL as working standard solutions. Neoline, an internal standard (IS), was prepared at a concentration of 2 μg/mL with methanol and diluted into 200 ng/mL before the experiment. All solutions were stored at 4°C before use for no longer than 2 weeks. Preparation of standard samples
Materials and methods Reagents and materials Fuziline (purity >98%) and neoline (purity >98% ) were gifts from Associate Professor Xiong Liang, Department of Chinese Medicine Chemistry of Chengdu University of Traditional Chinese Medicine. HPLC-grade methanol was purchased from Merck (Darmstadt, Germany). HPLC-grade formic acid was purchased from Sigma–Aldrich (St Louis, MO, USA). Ultrapure water used throughout the experiments was prepared using a Milli-Q Ultrapure water purification system (Millipore, Bedford, MA, USA). Other chemicals and solvents were of analytical grade and obtained from Nanjing Chemical Reagent Company (Nanjing, China). Instrumentation and operating conditions
1708
The MS parameters, such as capillary voltage, cone voltage, extraction voltage, source temperature, desolvation temperature, speed of desolvation gas and cone gas, were optimized by the responses of fuziline. In order to analyze fuziline with satisfactory peak shape and response, several different solvents, such as methanol, acetonitrile, pure water, 0.1% formic acid aqueous solution and 5 mmol/L ammonium acetate, were taken into account as mobile phases. Fuziline was analyzed on an Acquity UPLC-Q-TOF/MS system (Waters Corp., Milford, MA, USA) using an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm, Waters, USA). A 5 μL sample or standard solution was injected into the system and the column temperature was maintained at 40°C. The mobile phase was composed of 0.1% formic acid aqueous solution (A) and methanol (B) with a ratio of 80:20 and run at a flow rate of 0.25 mL/min. The MS instrument was equipped with an ESI ion source operating in positive ion mode. A mass range of 100–900 Da was set with a 0.14 s scan time. The main working parameters for mass spectrometry were set as follows: the capillary voltage, 3200 V; desolvation temperature, 400°C; sample cone voltage, 35 V; extraction cone voltage, 4 V; source temperature, 120°C; cone gas flow, 50 L/h; desolvation gas flow, 700 L/h; collision energy, 3 eV; and collision gas pressure, 2.8 × 103 mbar (argon). Mass was corrected during acquisition using an external reference (LockSpray) consisting of a 0.2 ng/mL solution of leucine enkephalin infused at a flow rate of 100 μL/min via a lockspray interface, generating a reference ion for positive ion mode [(M + H)+ m/z 556.2771) to ensure mass accuracy during the MS analysis. The Lock-Spray scan time was set at 0.5 s with the interval of 15 s, each having a dwell time of 10 ms. The (M + H)+ions of fuziline (m/z 454.35) and neoline (an internal standard m/z 438.32) were detected in analyser mode of sensitivity with Enhanced Analog-to-Digital Converter (EDC) set as 454.35 for best determination of fuziline in positive ionization mode. The acquisition and processing of data were performed using Masslynx (V4.1) software.
wileyonlinelibrary.com/journal/bmc
Preparation of stock and working standard solutions
Fuziline standard samples (1, 2, 5, 10, 20, 50, 100 and 200 ng/mL) were prepared by spiking control rat plasma with 10 μL of the working standard solutions prepared above. Quality control (QC) samples for investigating the accuracy and precision of the method were independently prepared at low (2 ng/mL), medium (20 ng/mL) and high (100 ng/mL) concentrations in the same manner. The IS was added to each standard plasma sample just prior to sample processing. Treatment of plasma samples The plasma samples were extracted using a liquid–liquid extraction technique. A 100 μL plasma sample was spiked with 10 μL IS solution (200 ng/mL) and mixed for 15 s. After being alkalified with 100 μL of 0.1 mol/L ammonia water, 3 mL ethyl acetate was added to each tube for drug extraction. The tubes were vortexed for 5 min and left to stand for 1 min at room temperature to allow complete extraction. The extracts were centrifuged at 8000 g for 5 min. The supernatants were then transferred to clean plastic tubes. The ethyl acetate extracts were evaporated to dryness under a gentle stream of nitrogen at 37°C. The extract residues were reconstituted in 100 μL methanol with vortexmixing for 3 min, and then centrifuged at 8000 g for 15 min. Standard and QC samples were prepared following the above method. A 5 μL aliquot of the supernatant was injected for analysis. Method validation The method was validated in terms of specificity, recovery, matrix effect, linearity, accuracy, precision and stability according to the US Food and Drug Administration (2001) guidelines for validation of bioanalytical method. The specificity was evaluated by analyzing six different batches of blank rat plasma with or without fuziline and neoline (IS) by comparison of corresponding peaks to exclude potential endogenous interference. The recovery for fuziline and matrix effect from rat plasma extract were determined at 2, 20 and 100 ng/mL (n = 6) by comparing three sets of samples: (A) fuziline spiked into plasma before extraction but IS spiked into dry residue; (B) both fuziline and IS spiked into the residue after extraction of blank plasma; and (C) fuziline and IS spiked directly into methanol. Recovery was calculated as percentage of the peak area ratio (fuziline/IS) of set A compared with that of set B. Similarly, matrix effect was calculated as percentage of the peak area ratio (fuziline/IS) of set B compared with that of set C. The recovery of IS (20 ng/mL) was calculated by comparing the peak area in plasma and in methanol. Calibration standard samples were prepared by spiking 10 μL working solutions into 90 μL drug-free rat plasma to achieve final concentrations of 1–200 ng/mL for fuziline. Eight-point calibration curves were constructed by plotting peak area ratio
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 1707–1713
A novel analytical UHPLC-qTOF method for fuziline of fuziline to internal standard vs nominated concentrations. The lower limit of quantification (LLOQ), which was taken as the
lowest concentration on the calibration curve, could fulfill the analytical requirement of signal-to-noise ratio > 10 with acceptable accuracy and precision. The accuracy and precision of the established method were evaluated by QC samples at low, medium and high concentrations (2, 20 and 100 ng/mL). Accuracy was defined as percentage of the observed concentration value relative to the spiked concentration value. Precision was evaluated as the relative standard deviation (RSD, %). The intra-day accuracy was demonstrated by analyzing six replicates at each concentration level on 1 day, and inter-day accuracy by analyzing six replicates of each concentration over three consecutive validation days. QC samples were used to evaluate the stability of the analyte in rat plasma under a variety of storage and handling conditions: short-term stability, at room temperature for 10 h, long-term stability, at 80°C for 30 days, postpreparative stability, in the autosampler (4°C) for 24 h, and after three freeze–thaw cycles.
Pharmacokinetic studies
Figure 1. Chromatograms of blank plasma: (a) neoline; (b) fuziline; and (c) base peak intensity (BPI) chromatograms.
Sprague–Dawley rats, weighing 250 ± 20 g (certificate no. SYXK 2008-049) were provided by the Animal Center of Chengdu University of Traditional Chinese Medicine. Animals were housed in a temperature-controlled facility with a 12 h light/dark cycle, and had unlimited access to food and water for 7 days. The animal experiments were conducted in accordance with the US guidelines (NIH publication no. 85-23, revised in 1985).
Biomed. Chromatogr. 2014; 28: 1707–1713
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
1709
Figure 2. Chromatograms of blank plasma spiked with fuziline (5 ng/mL in plasma) and neoline (20 ng/mL in plamsa): (a) neoline; (b) fuziline; and (c) BPI chromatograms.
Y.-x. Li et al. Eighteen male rats were randomly divided into three dosage groups. The animal was placed in metabolic cage, fasted overnight but with free access to water before dosing. On the day of experiment, 4, 2 and 1 mg/kg fuziline in saline were intragastrically administered to rats. Serial blood samples (0.25 mL) were drawn at 0, 10, 20 and 40 min, and 1, 2, 3, 4, 6, 8, 10, 12 and 24 h into heparinized tubes. Plasma samples were obtained following centrifugation at 1500 g for 10 min and kept frozen at 80°C until analysis. The pharmacokinetic analysis of fuziline was performed by a noncompartmental approach using the WinNonlin software package (version 6.1, Pharsight, USA) to calculate area under the concentration–time curve (AUC) and half-life. The maximum value of concentration (Cmax) and time to reach Cmax (Tmax) were obtained directly from the mean plasma concentration–time curve. Statistical analysis among the three groups was performed using SPSS 16.0 (Statistical Package for the Social Science) . A p-value < 0.05 was considered statistically significant for all the tests. All data were expressed as mean ± standard deviation (SD).
Results and discussion As we knew, effect-guided detection of biologically active natural products plays a strategic role in the phytochemical investigation of TCM. Many phytochemicals isolated by activityguided isolation from herbs or plants show good pharmacology activity in vitro. However, poor understanding of their pharmacokinetic characteristics has limited the applications in clinic.
With its capability of accurate mass measurements and high resolution, time-of-flight (TOF) MS is good analytical tool for qualitative identification of organic molecules. To avoid the endogenous interference mentioned in the reported study on fuziline, a sensitive UHPLC-q/TOF-MS method was developed to investigate the pharmacokinetic characteristic of fuziline after intragastric administration to rats. The use of the hyphenated techniques allowed rapid separation and quantification of the analyte in plasma extracts within a few minutes.
Method validation Specificity. Specificity of the method was investigated by analyzing rat plasma from different sources. Typical chromatograms of blank plasma sample, blank plasma sample spiked with fuziline and IS, and rat plasma sample after fuziline administration are shown in Figs 1–3. Each figure has three panels representing the chromatograms of fuziline, neoline and BPI (base peak intensity). As shown in the figures, the retention times for fuziline and neoline were 1.01 min under the described conditions. The total running time was set as 3 min to elute the peak in 2.79 min so that it did not interfere with the next sample. Chromatograms showed no endogenous peaks interfering with the analyte and internal standard detection, which proved the specificity of the method. Recovery and matrix effect. The results of recovery and matrix effect of fuziline and IS are summarized in Table 1. The recoveries of fuziline at three concentration levels were
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Figure 3. Chromatograms of plasma sample at 6 h after oral administration of fuziline (2 mg/kg): : (a) neoline; (b) fuziline; and (c) BPI chromatograms.
wileyonlinelibrary.com/journal/bmc
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 1707–1713
A novel analytical UHPLC-qTOF method for fuziline detected with an intra-day RSD ≤20%. At this LLOQ, signal-to-noise ratio was >10, and the accuracy and precision were 93.26 and 11.82%, respectively. The LLOQ was sufficient for a rat pharmacokinetic study following intragastric administration of fuziline.
Table 1. Recovery of fuziline from rat plasma (n = 6) Spiked concentration (ng/mL)
Recovery (%)
Fuziline 2 20 100 Neoline 20
Matrix effect (%)
86.06 83.29 81.76
97.86 99.73 102.32
85.74
98.73
Accuracy and precision. Accuracy and precision data for fuziline at three different concentration levels (2, 20 and 100 ng/mL) are presented in Table 2. The intra-assay and interassay results were reproducible with an intra-day RSD
Revised: 8 February 2014,
Accepted: 15 March 2014
Published online in Wiley Online Library: 30 April 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3210
Development and validation of a UHPLC-qTOF-MS method for quantification of fuziline in rat plasma and its application in a pharmacokinetic study Yun-xia Lia,b,c†, Xiao-hong Gonga,b,c†, Yan Lia,b,c, Ruo-qi Zhanga,b,c, Liang Xionga,b,c, Xiao-fang Xiea,b,c and Cheng Penga,b,c* ABSTRACT: A specific and sensitive UHPLC-qTOF-MS method was developed and validated for quantification of fuziline in rat plasma after oral administration of three dosages. The analyte was separated on an Acquity UPLC BEH C18 column with a total running time of 3 min using a mobile phase of 0.1% formic acid aqueous solution and methanol (80:20, v/v) at a flow-rate of 0.25 mL/min. The calibration curves for fuziline showed good linearity in the concentrations ranging from 1 to 200 ng/mL with correlation coefficients >0.997. The precision, accuracy, recovery and stability were deemed acceptable. The method was applied to a pharmacokinetics study of fuziline in rats. The mean half-life was 5.93, 6.13 and 5.12 h for 1, 2 and 4 mg/kg oral administration of fuziline, respectively. The peak concentration and area under the concentration–time curve increased linearly with the doses. The sum of these results indicated that, in the range of the doses examined, the pharmacokinetics of fuziline in rat was based on first-order kinetics. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: fuziline; UHPLC-qTOF-MS; pharmacokinetics
Introduction
Biomed. Chromatogr. 2014; 28: 1707–1713
* Correspondence to: Peng Cheng, Pharmacy College, Chengdu University of Traditional Chinese Medicine, 1166 Liutai Road, Chengdu 611137, China. Email: [email protected] †
The first two authors contributed equally to this paper.
a
Pharmacy College, Chengdu University of Traditional Chinese Medicine, 1166 Liutai Road, Chengdu 611137, China
b
The Ministry of Education Key Laboratory of Standardization of Chinese Herbal Medicine, 1166 Liutai Road, Chengdu 611137, China
c
State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources, 1166 Liutai Road, Chengdu 611137, China Abbreviations used: TCM, traditional Chinese medicine.
Copyright © 2014 John Wiley & Sons, Ltd.
1707
Currently, pharmaceutical research and development are very challenging because of high cost, high risk and lengthy research period (Fan et al., 2012). Traditional Chinese medicine (TCM) has attracted much attention owing to its effectiveness against many diseases and proven safety in clinical use over 8000 years (Xie and Leung, 2009; Pavel and Jitka, 2004). It plays an indispensable role in the prevention and treatment of diseases, especially complicated and chronic ones (Hamza, 2010; Liu et al., 2008; Lou et al., 2010). As a huge medicinal treasury, many new drugs have been discovered from TCM. For example, Artemisinin shows great success in treating malaria, and is separated from Qinghao (Artemisia species). Although many drug candidates show good activities in vitro, weak therapeutic effects in vivo are found in animal studies. The failure of in vivo studies is mostly caused by poor understanding of pharmacokinetics of the candidates (Alavijeh and Palmer, 2004). Radix Aconiti Lateralis Preparata (Fuzi) is the processed daughter roots of Aconitum carmichaeli Debx. (family Ranunculaceae; Chinese Pharmacopoeia Commission, 2010). It is widely used for the treatment of arthralgia and heart failure. Modern pharmacological studies have revealed that Fuzi plays an important role in cardiotonic, anti-arrhythmia, anti-thrombosis, anti-hypoxia, anti-shock effect and in inhibiting thrombosis (Zhou and Liu, 2013). Pharmacologically active components of Fuzi are diterpenoid alkaloids, including hetisine, fuziline, benzoylmesaconine, aconitine, hypaconitine and mesaconitine (Wang and Zhu, 2010). Some research has been carried out on aconitine, hypaconitine and mesoconitine using various analytical methods, including highperformance liquid chromatography (HPLC; Xiang et al., 2006), liquid chromatography–mass spectrometry (LC-MS; Zhang et al., 2009),
LC-MS/MS (Yue et al., 2009) and Ultra high performance liquid chromatography (HPLC)-quadrupole time-of-flight tandem mass spectrometry (TOF/MS) (Tang et al., 2012). Compared with the numbers of studies on pharmacological activities of other chemical components in Fuzi, only one report was found on investigation of the pharmacokinetics of fuziline using a hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometric method (Sun et al., 2013). Owing to hydroxyl and tertiary ammonium groups in fuziline, the mobile phase consists of 0.05% formic acid aqueous solution and acetonitrile at a 60:40 (v/v) ratio. However an obvious interference peak is found in the blank plasma with (M + H)+ ions of fuziline in the separation condition. In our laboratory, the effect of fuziline (10, 1 and 0.1 μM) on neonatal rat cardiomyocytes was investigated by methyl thiazolyl
Y.-x. Li et al. tetrazolium assay (MTT) method (Xiong et al., 2012). Fuziline showed activity against pentobarbital sodium-induced cardiomyocytes damage by recovering the beating rhythm and increasing the cell viability. However, many phytochemicals isolated by activity-guided isolation from herbs or plants exhibit poor pharmacokinetic characteristics. For further preclinical research, a sensitive UHPLC-qTOF-MS method was developed to perform the pharmacokinetic study of fuziline in rats.
A stock solution of fuziline was prepared at a concentration of 20 μg/mL in methanol and was diluted to 0.01–2 μg/mL as working standard solutions. Neoline, an internal standard (IS), was prepared at a concentration of 2 μg/mL with methanol and diluted into 200 ng/mL before the experiment. All solutions were stored at 4°C before use for no longer than 2 weeks. Preparation of standard samples
Materials and methods Reagents and materials Fuziline (purity >98%) and neoline (purity >98% ) were gifts from Associate Professor Xiong Liang, Department of Chinese Medicine Chemistry of Chengdu University of Traditional Chinese Medicine. HPLC-grade methanol was purchased from Merck (Darmstadt, Germany). HPLC-grade formic acid was purchased from Sigma–Aldrich (St Louis, MO, USA). Ultrapure water used throughout the experiments was prepared using a Milli-Q Ultrapure water purification system (Millipore, Bedford, MA, USA). Other chemicals and solvents were of analytical grade and obtained from Nanjing Chemical Reagent Company (Nanjing, China). Instrumentation and operating conditions
1708
The MS parameters, such as capillary voltage, cone voltage, extraction voltage, source temperature, desolvation temperature, speed of desolvation gas and cone gas, were optimized by the responses of fuziline. In order to analyze fuziline with satisfactory peak shape and response, several different solvents, such as methanol, acetonitrile, pure water, 0.1% formic acid aqueous solution and 5 mmol/L ammonium acetate, were taken into account as mobile phases. Fuziline was analyzed on an Acquity UPLC-Q-TOF/MS system (Waters Corp., Milford, MA, USA) using an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm, Waters, USA). A 5 μL sample or standard solution was injected into the system and the column temperature was maintained at 40°C. The mobile phase was composed of 0.1% formic acid aqueous solution (A) and methanol (B) with a ratio of 80:20 and run at a flow rate of 0.25 mL/min. The MS instrument was equipped with an ESI ion source operating in positive ion mode. A mass range of 100–900 Da was set with a 0.14 s scan time. The main working parameters for mass spectrometry were set as follows: the capillary voltage, 3200 V; desolvation temperature, 400°C; sample cone voltage, 35 V; extraction cone voltage, 4 V; source temperature, 120°C; cone gas flow, 50 L/h; desolvation gas flow, 700 L/h; collision energy, 3 eV; and collision gas pressure, 2.8 × 103 mbar (argon). Mass was corrected during acquisition using an external reference (LockSpray) consisting of a 0.2 ng/mL solution of leucine enkephalin infused at a flow rate of 100 μL/min via a lockspray interface, generating a reference ion for positive ion mode [(M + H)+ m/z 556.2771) to ensure mass accuracy during the MS analysis. The Lock-Spray scan time was set at 0.5 s with the interval of 15 s, each having a dwell time of 10 ms. The (M + H)+ions of fuziline (m/z 454.35) and neoline (an internal standard m/z 438.32) were detected in analyser mode of sensitivity with Enhanced Analog-to-Digital Converter (EDC) set as 454.35 for best determination of fuziline in positive ionization mode. The acquisition and processing of data were performed using Masslynx (V4.1) software.
wileyonlinelibrary.com/journal/bmc
Preparation of stock and working standard solutions
Fuziline standard samples (1, 2, 5, 10, 20, 50, 100 and 200 ng/mL) were prepared by spiking control rat plasma with 10 μL of the working standard solutions prepared above. Quality control (QC) samples for investigating the accuracy and precision of the method were independently prepared at low (2 ng/mL), medium (20 ng/mL) and high (100 ng/mL) concentrations in the same manner. The IS was added to each standard plasma sample just prior to sample processing. Treatment of plasma samples The plasma samples were extracted using a liquid–liquid extraction technique. A 100 μL plasma sample was spiked with 10 μL IS solution (200 ng/mL) and mixed for 15 s. After being alkalified with 100 μL of 0.1 mol/L ammonia water, 3 mL ethyl acetate was added to each tube for drug extraction. The tubes were vortexed for 5 min and left to stand for 1 min at room temperature to allow complete extraction. The extracts were centrifuged at 8000 g for 5 min. The supernatants were then transferred to clean plastic tubes. The ethyl acetate extracts were evaporated to dryness under a gentle stream of nitrogen at 37°C. The extract residues were reconstituted in 100 μL methanol with vortexmixing for 3 min, and then centrifuged at 8000 g for 15 min. Standard and QC samples were prepared following the above method. A 5 μL aliquot of the supernatant was injected for analysis. Method validation The method was validated in terms of specificity, recovery, matrix effect, linearity, accuracy, precision and stability according to the US Food and Drug Administration (2001) guidelines for validation of bioanalytical method. The specificity was evaluated by analyzing six different batches of blank rat plasma with or without fuziline and neoline (IS) by comparison of corresponding peaks to exclude potential endogenous interference. The recovery for fuziline and matrix effect from rat plasma extract were determined at 2, 20 and 100 ng/mL (n = 6) by comparing three sets of samples: (A) fuziline spiked into plasma before extraction but IS spiked into dry residue; (B) both fuziline and IS spiked into the residue after extraction of blank plasma; and (C) fuziline and IS spiked directly into methanol. Recovery was calculated as percentage of the peak area ratio (fuziline/IS) of set A compared with that of set B. Similarly, matrix effect was calculated as percentage of the peak area ratio (fuziline/IS) of set B compared with that of set C. The recovery of IS (20 ng/mL) was calculated by comparing the peak area in plasma and in methanol. Calibration standard samples were prepared by spiking 10 μL working solutions into 90 μL drug-free rat plasma to achieve final concentrations of 1–200 ng/mL for fuziline. Eight-point calibration curves were constructed by plotting peak area ratio
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 1707–1713
A novel analytical UHPLC-qTOF method for fuziline of fuziline to internal standard vs nominated concentrations. The lower limit of quantification (LLOQ), which was taken as the
lowest concentration on the calibration curve, could fulfill the analytical requirement of signal-to-noise ratio > 10 with acceptable accuracy and precision. The accuracy and precision of the established method were evaluated by QC samples at low, medium and high concentrations (2, 20 and 100 ng/mL). Accuracy was defined as percentage of the observed concentration value relative to the spiked concentration value. Precision was evaluated as the relative standard deviation (RSD, %). The intra-day accuracy was demonstrated by analyzing six replicates at each concentration level on 1 day, and inter-day accuracy by analyzing six replicates of each concentration over three consecutive validation days. QC samples were used to evaluate the stability of the analyte in rat plasma under a variety of storage and handling conditions: short-term stability, at room temperature for 10 h, long-term stability, at 80°C for 30 days, postpreparative stability, in the autosampler (4°C) for 24 h, and after three freeze–thaw cycles.
Pharmacokinetic studies
Figure 1. Chromatograms of blank plasma: (a) neoline; (b) fuziline; and (c) base peak intensity (BPI) chromatograms.
Sprague–Dawley rats, weighing 250 ± 20 g (certificate no. SYXK 2008-049) were provided by the Animal Center of Chengdu University of Traditional Chinese Medicine. Animals were housed in a temperature-controlled facility with a 12 h light/dark cycle, and had unlimited access to food and water for 7 days. The animal experiments were conducted in accordance with the US guidelines (NIH publication no. 85-23, revised in 1985).
Biomed. Chromatogr. 2014; 28: 1707–1713
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
1709
Figure 2. Chromatograms of blank plasma spiked with fuziline (5 ng/mL in plasma) and neoline (20 ng/mL in plamsa): (a) neoline; (b) fuziline; and (c) BPI chromatograms.
Y.-x. Li et al. Eighteen male rats were randomly divided into three dosage groups. The animal was placed in metabolic cage, fasted overnight but with free access to water before dosing. On the day of experiment, 4, 2 and 1 mg/kg fuziline in saline were intragastrically administered to rats. Serial blood samples (0.25 mL) were drawn at 0, 10, 20 and 40 min, and 1, 2, 3, 4, 6, 8, 10, 12 and 24 h into heparinized tubes. Plasma samples were obtained following centrifugation at 1500 g for 10 min and kept frozen at 80°C until analysis. The pharmacokinetic analysis of fuziline was performed by a noncompartmental approach using the WinNonlin software package (version 6.1, Pharsight, USA) to calculate area under the concentration–time curve (AUC) and half-life. The maximum value of concentration (Cmax) and time to reach Cmax (Tmax) were obtained directly from the mean plasma concentration–time curve. Statistical analysis among the three groups was performed using SPSS 16.0 (Statistical Package for the Social Science) . A p-value < 0.05 was considered statistically significant for all the tests. All data were expressed as mean ± standard deviation (SD).
Results and discussion As we knew, effect-guided detection of biologically active natural products plays a strategic role in the phytochemical investigation of TCM. Many phytochemicals isolated by activityguided isolation from herbs or plants show good pharmacology activity in vitro. However, poor understanding of their pharmacokinetic characteristics has limited the applications in clinic.
With its capability of accurate mass measurements and high resolution, time-of-flight (TOF) MS is good analytical tool for qualitative identification of organic molecules. To avoid the endogenous interference mentioned in the reported study on fuziline, a sensitive UHPLC-q/TOF-MS method was developed to investigate the pharmacokinetic characteristic of fuziline after intragastric administration to rats. The use of the hyphenated techniques allowed rapid separation and quantification of the analyte in plasma extracts within a few minutes.
Method validation Specificity. Specificity of the method was investigated by analyzing rat plasma from different sources. Typical chromatograms of blank plasma sample, blank plasma sample spiked with fuziline and IS, and rat plasma sample after fuziline administration are shown in Figs 1–3. Each figure has three panels representing the chromatograms of fuziline, neoline and BPI (base peak intensity). As shown in the figures, the retention times for fuziline and neoline were 1.01 min under the described conditions. The total running time was set as 3 min to elute the peak in 2.79 min so that it did not interfere with the next sample. Chromatograms showed no endogenous peaks interfering with the analyte and internal standard detection, which proved the specificity of the method. Recovery and matrix effect. The results of recovery and matrix effect of fuziline and IS are summarized in Table 1. The recoveries of fuziline at three concentration levels were
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Figure 3. Chromatograms of plasma sample at 6 h after oral administration of fuziline (2 mg/kg): : (a) neoline; (b) fuziline; and (c) BPI chromatograms.
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Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 1707–1713
A novel analytical UHPLC-qTOF method for fuziline detected with an intra-day RSD ≤20%. At this LLOQ, signal-to-noise ratio was >10, and the accuracy and precision were 93.26 and 11.82%, respectively. The LLOQ was sufficient for a rat pharmacokinetic study following intragastric administration of fuziline.
Table 1. Recovery of fuziline from rat plasma (n = 6) Spiked concentration (ng/mL)
Recovery (%)
Fuziline 2 20 100 Neoline 20
Matrix effect (%)
86.06 83.29 81.76
97.86 99.73 102.32
85.74
98.73
Accuracy and precision. Accuracy and precision data for fuziline at three different concentration levels (2, 20 and 100 ng/mL) are presented in Table 2. The intra-assay and interassay results were reproducible with an intra-day RSD
